Articles de revues : « Phagemid vectors »

1

Schüller, Carolin, Julia C. Wiebe, Antje Pegel, Karl Kramer, Arne Skerra et Bertold Hock. « A System for Repertoire Cloning and Phage Display of Murine and Leporid Antibody Fragments ». Journal of AOAC INTERNATIONAL 93, n^o 1 (1 janvier 2010) : 66–79. http://dx.doi.org/10.1093/jaoac/93.1.66.

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Abstract Even though rabbit antibodies (Abs) are known to exceed murine Abs with respect to specificity, affinity, and stability, cloned leporid immune repertoires have been rarely considered in recombinant Ab preparation for environmental analysis. We have developed a set of four tetp/o-based phasmid vectors that allow the efficient cloning of both murine and leporid Ab repertoires. These vectors differ in the design of the cloning sites, choice of signal peptides, and antibiotic selection markers. A set of 39 primer oligodeoxynucleotides has been developed for the PCR amplification of rabbit Ab genes, representing the most exhaustive coverage of the leporid immune repertoire described so far. The atrazine-specific murine Fab fragment K411B and a cloned V-gene repertoire from sulfonamide-immunized rabbits were used to compare these phasmids with respect to expression of Fab fragments, phagemid titers, and number of Fab displaying phagemid particles. Our results show that the ratio of recombinant phagemids could be increased up to 65% of total phage titer by utilizing the appropriate phasmid. Based on this system, the selection of two sulfonamide-specific rabbit Abs, SA2 23 and SA2 90, was accomplished after a single phagemid panning round.

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Sambrook, Joseph, et David W. Russell. « Producing Single-stranded DNA with Phagemid Vectors ». Cold Spring Harbor Protocols 2006, n^o 1 (juin 2006) : pdb.prot4019. http://dx.doi.org/10.1101/pdb.prot4019.

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Krawetz, Stephen A., Daniel Sellos, Norman C. W. Wong et Gordon H. Dixon. « Phagemid VPCS vectors for priming, cloning and sequencing ». Gene 82, n^o 2 (octobre 1989) : 317–20. http://dx.doi.org/10.1016/0378-1119(89)90057-7.

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Qi, Huan, Haiqin Lu, Hua-Ji Qiu, Valery Petrenko et Aihua Liu. « Phagemid Vectors for Phage Display : Properties, Characteristics and Construction ». Journal of Molecular Biology 417, n^o 3 (mars 2012) : 129–43. http://dx.doi.org/10.1016/j.jmb.2012.01.038.

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Alting-Mees, Michelle A., et Jay M. Short. « Polycos vectors : a system for packaging filamentous phage and phagemid vectors using lambda phage packaging extracts ». Gene 137, n^o 1 (décembre 1993) : 93–100. http://dx.doi.org/10.1016/0378-1119(93)90256-3.

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Soderlind, Eskil, Ann Catrin Simonsson et Carl A. K. Borrebaeck. « Phage Display Technology in Antibody Engineering : Design of Phagemid Vectors and in vitro Maturation Systems ». Immunological Reviews 130, n^o 1 (décembre 1992) : 109–24. http://dx.doi.org/10.1111/j.1600-065x.1992.tb01523.x.

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Xu, Fu Yong, Ren Rong Liu, Ling Xu, Xue Mei Qiu et Li Xin Zhu. « Two Expression Vectors, Designated as pGEX-CDON and pC89S4-CDON, for Producing GST-CDON and pVIII-CDON Fusion Proteins ». Advanced Materials Research 726-731 (août 2013) : 505–10. http://dx.doi.org/10.4028/www.scientific.net/amr.726-731.505.

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Deoxynivalenol (DON) mimotope, designated as CDON, is an epitope (CMRPWLQ) immunoscreened from a phage-displayed random peptide library. In order to replace the conjugated toxin with non-toxic recombinant proteins in ELISA, two novel expression vectors, which were designated as plasmid pGEX-CDON and phagemid pC89S4-CDON for producing GST-CDON and pVIII-CDON fusion proteins in E.coli were constructed. After purification, both GST-CDON and pVIII-CDON fusion proteins show good reactogenicity with an anti-DON antibody in a competitive inhibition ELISA test. When GST-CDON was used as coating antigen, the linear range of the competitive inhibition ELISA is from 62ng/ml to 410ng/ml, the linear equation is Y= 186.6-23.87Ln (X), IC50 is 194ng/ml. For pVIII-CDON as coating protein, the linear range of the competitive inhibition ELISA is from 20ng/ml to 470ng/ml, the linear equation is Y = 161.3-25.49Ln (X), R2=0.9962, IC50 is 94ng/ml. ELISA analysis and comparison show the reactogenicity and specificity of pVIII-CDON binding to anti-DON antibody are better than GST-CDON fusion protein. The pVIII-CDON is promising in establishing an ELISA without the use of the toxic mycotoxin conjugate.

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Jeung, Ji Ung, Sung Ki Cho, Kyu Suk Shim, Sung Han Ok, Dae Sik Lim et Jeong Sheop Shin. « Construction of two pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites for direct cloning of PCR products ». Plasmid 48, n^o 2 (septembre 2002) : 160–63. http://dx.doi.org/10.1016/s0147-619x(02)00122-1.

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Teo, Michelle Yee Mun, Jeremy Jeack Ceen Ng, Jung Yin Fong, Jung Shan Hwang, Adelene Ai-Lian Song, Renee Lay Hong Lim et Lionel Lian Aun In. « Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells ». PeerJ 9 (15 avril 2021) : e11063. http://dx.doi.org/10.7717/peerj.11063.

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Background KRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins. Methods This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells. Results The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells. Discussion These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.

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Kuba, Hiroyoshi, Atsuko Furukawa, Toshihide Okajima et Koji Furukawa. « Efficient bacterial production of functional antibody fragments using a phagemid vector ». Protein Expression and Purification 58, n^o 2 (avril 2008) : 292–300. http://dx.doi.org/10.1016/j.pep.2007.10.019.

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Rosander, Anna, Lars Frykberg, Nora Ausmees et Peter Müller. « Identification of Extracytoplasmic Proteins in Bradyrhizobium japonicum Using Phage Display ». Molecular Plant-Microbe Interactions® 16, n^o 8 (août 2003) : 727–37. http://dx.doi.org/10.1094/mpmi.2003.16.8.727.

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A novel gene bank of Bradyrhizobium japonicum USDA110spc4 was constructed using pG3DSS, a phagemid vector designed for detecting genes encoding secreted proteins. In this phagemid, the phage protein III lacks its indigenous signal peptide required for protein secretion, thus recombinant fusion proteins are displayed on the phage surface only if a functional signal peptide is provided by an inserted DNA fragment. In addition, the N-terminal half of protein III has been replaced by a short linker region (the E-tag) that is recognized by a monoclonal antibody, which enables isolation of phages displaying a fusion protein. The expression library described here, therefore, provides a powerful means to affinity select for B. japonicum genes encoding extracytoplasmic proteins. In total, 182 DNA sequences were analyzed, among which 132 different putative extracytoplasmic proteins could be identified. The function of most proteins could be predicted and support an extracytoplasmic localization. In addition, genes encoding novel extracytoplasmic proteins were found. In particular, a novel family of small proteins has been identified that is characterized by a conserved pattern of four cysteine residues.

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Akamatsu, Y., M. S. Cole, J. Y. Tso et N. Tsurushita. « Construction of a human Ig combinatorial library from genomic V segments and synthetic CDR3 fragments. » Journal of Immunology 151, n^o 9 (1 novembre 1993) : 4651–59. http://dx.doi.org/10.4049/jimmunol.151.9.4651.

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Abstract A naive combinatorial Ig library was constructed from semi-synthetic V genes consisting of human genomic V segments and synthetic CDR3 fragments. VH and V kappa segments were amplified from human genomic DNA by polymerase chain reaction using V subgroup-specific primers. The amplified VH and V kappa segments were combined with synthetic oligonucleotides containing a J region and CDR3 with amino acid sequence variations, resulting in complete V genes. These V genes were cloned into a phagemid expression vector in a single-chain form fused to the carboxyl-terminus of the M13 minor coat protein III. Phagemid particles displaying the single chain hybrid proteins on their surface were screened with Con A as Ag. Several clones showing specific binding to Con A were obtained after four rounds of selection and were further analyzed for their binding properties and DNA sequences. This method provides a novel way to create a naive combinatorial library without using mRNA from B lymphocytes as template. The method should be useful to isolate human antibodies that react with self-Ag.

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Lah, Maria, Alanna Goldstraw, Jacinta F. White, Olan Dolezal, Robyn Malby et Peter J. Hudson. « Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector ». Human Antibodies 5, n^o 1-2 (1 mars 1994) : 48–56. http://dx.doi.org/10.3233/hab-1994-51-207.

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Lin, Nien-Tsung, et Yi-Hsiung Tseng. « A phagemid shuttle vector based on filamentous phage φLf and theE. coli vector pOK12 for use inXanthomonas campestris ». Biotechnology Techniques 11, n^o 1 (janvier 1997) : 43–45. http://dx.doi.org/10.1007/bf02764450.

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Pershad, Kritika, Mark A. Sullivan et Brian K. Kay. « Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats ». Analytical Biochemistry 412, n^o 2 (mai 2011) : 210–16. http://dx.doi.org/10.1016/j.ab.2011.02.006.

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Waye, Mary M. Y., Florence Mui, Kathy Hodge et Vincent K. C. Li. « A phagemid vector library for cloning DNA with four-nucleotide 5′ or 3′ overhangs ». Plasmid 26, n^o 1 (juillet 1991) : 74–77. http://dx.doi.org/10.1016/0147-619x(91)90038-x.

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Healy, Judith M., Mitsuru Haruki et Masakazu Kikuchi. « Preferred Motif for Integrin Binding Identified Using a Library of Randomized RGD Peptides Displayed on Phage ». Protein & ; Peptide Letters 3, n^o 1 (février 1996) : 23–30. http://dx.doi.org/10.2174/092986650301220608153153.

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Abstract: We have selected RGD peptides recognized efficiently by integrin av3 through randomization of amino acids flanking the RGD motif. Degenerate RGD peptides were displayed on phage as fusions to the N-terminus of major coat protein Vill of M13 using a specially constructed phagemid vector. Identification of an apparent consensus sequence among ligands preferred by av3 supports the view that amino acids in the vicinity of RGD can influence receptor recognition of phage-peptides. In addition, several phage isolates encoded a potentially cyclic peptide which bound to av3 with superior affinity. Binding assays with cyclic and linear forms of synthetic RGD peptides confirmed the capacity of conformational constraint of RGD to confer high-affinity integrin binding.

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Yazynin, Sergey, Hans Lange, Thilo Mokros, Sergey Deyev et Hilmar Lemke. « A new phagemid vector for positive selection of recombinants based on a conditionally lethal barnase gene ». FEBS Letters 452, n^o 3 (11 juin 1999) : 351–54. http://dx.doi.org/10.1016/s0014-5793(99)00661-4.

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Beekwilder, J., J. Rakonjac, M. Jongsma et D. Bosch. « A phagemid vector using the E. coli phage shock promoter facilitates phage display of toxic proteins ». Gene 228, n^o 1-2 (mars 1999) : 23–31. http://dx.doi.org/10.1016/s0378-1119(99)00013-x.

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Huang, Wanzhi, Joseph Petrosino et Timothy Palzkill. « Display of Functional β-Lactamase Inhibitory Protein on the Surface of M13 Bacteriophage ». Antimicrobial Agents and Chemotherapy 42, n^o 11 (1 novembre 1998) : 2893–97. http://dx.doi.org/10.1128/aac.42.11.2893.

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ABSTRACT The display of proteins on the surface of filamentous phage has been shown to be a powerful method to select variants of a protein with altered binding properties from large combinatorial libraries of mutants. The β-lactamase inhibitory protein (BLIP) is a 165-amino-acid protein that binds and inhibits TEM-1 β-lactamase-catalyzed hydrolysis of the penicillin and cephalosporin antibiotics. Here we describe the construction of a new phagemid vector and the use of this vector to display BLIP on the surface of filamentous phage. It is shown that BLIP-displaying phage bind to immobilized β-lactamase and that the binding can be competed off by the addition of soluble β-lactamase. In addition, a two-step phage enzyme-linked immunosorbent assay procedure was used to demonstrate that the BLIP-displaying phage bind β-lactamase with a 50% inhibitory concentration of 1 nM, which compares favorably with a previously published Ki of 0.6 nM. A system has therefore been established for protein engineering of BLIP to expand its range of binding to other β-lactamases and penicillin-binding proteins.

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Zhao, Qi, Yin-Wah Chan, Susanna Sau-Tuen Lee et Wing-Tai Cheung. « One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector ». Protein Expression and Purification 68, n^o 2 (décembre 2009) : 190–95. http://dx.doi.org/10.1016/j.pep.2009.08.004.

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EL-MATBOULI, M., et H. SOLIMAN. « Construction and screening of a cDNA library from the triactinomyxon spores ofMyxobolus cerebralis, the causative agent of salmonid Whirling Diseases ». Parasitology 132, n^o 4 (3 janvier 2006) : 467–77. http://dx.doi.org/10.1017/s0031182005009522.

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The ZAP Express cDNA library was constructed using mRNA extracted from the triactinomyxon spores ofMyxobolus cerebralis. First-strand cDNA was synthesized using Moloney Murine leukaemia virus reverse transcriptase. Following second-strand cDNA synthesis, the double-stranded cDNA was digested withXhoI restriction enzyme, cDNA fragments less than 400 bp were removed and the remaining cDNA was ligated with the lambda ZAP Express vector. The recombinants were packagedin vitrousing Gigapack III gold packaging extract. The primary cDNA library titre contained 0·5×106clones, with 97% recombinant and only 3% non-recombinant clones. The cDNA library was then screened using the anti-triactinomyxon antibodies. Positive clones were selected and re-screened twice more to give a final selection of 526 clones. One clone (46-5) was selected randomly and subjected toin vivo excision of the pBK-CMV phagemid from the ZAP express vector. The sequence of the entire clone was obtained using rapid amplification of the cDNA ends. A search of the clone sequence against GenBank revealed that it related to ribosomal protein L23 and it had a high percentage similarity to this protein from different species. A conserved domain for ribosomal protein L23 was also identified in the clone sequence.

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Wall, Torun, Stefan Roos, Karin Jacobsson, Anna Rosander et Hans Jonsson. « Phage display reveals 52 novel extracellular and transmembrane proteins from Lactobacillus reuteri DSM 20016T ». Microbiology 149, n^o 12 (1 décembre 2003) : 3493–505. http://dx.doi.org/10.1099/mic.0.26530-0.

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Extracellular and transmembrane proteins are important for the binding of bacteria to intestinal surfaces and for their interaction with the host. The aim of this study was to identify genes encoding extracellular and transmembrane proteins from the probiotic bacterium Lactobacillus reuteri by construction and screening of a phage display library. This library was constructed by insertion of randomly fragmented DNA from L. reuteri into the phagemid vector pG3DSS, which was previously developed for screening for extracellular proteins. After affinity selection of the library, the L. reuteri inserts were sequenced and analysed with bioinformatic tools. The screening resulted in the identification of 52 novel genes encoding extracellular and transmembrane proteins. These proteins were classified as: transport proteins; enzymes; sensor–regulator proteins; proteins involved in host/microbial interactions; conserved hypothetical proteins; and unconserved hypothetical proteins. Further characterization of the extracellular and transmembrane proteins identified should contribute to the understanding of the probiotic properties of L. reuteri.

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Ossysek, Karolina, Tomasz Uchański, Małgorzata Kulesza, Monika Bzowska, Tomasz Klaus, Klaudia Woś, Mariusz Madej et Joanna Bereta. « A new expression vector facilitating production and functional analysis of scFv antibody fragments selected from Tomlinson I + J phagemid libraries ». Immunology Letters 167, n^o 2 (octobre 2015) : 95–102. http://dx.doi.org/10.1016/j.imlet.2015.07.005.

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Halum, Stacey L., Christy B. Erbe, David R. Friedland et Phillip A. Wackym. « Gene Discovery Using a Human Vestibular Schwannoma cDNA Library Constructed from a Patient with Neurofibromatosis Type 2 (NF2) ». Otolaryngology–Head and Neck Surgery 128, n^o 3 (mars 2003) : 364–71. http://dx.doi.org/10.1067/mhn.2003.99.

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BACKGROUND: Despite a strong association of schwannomin/merlin gene mutations with vestibular schwannoma formation, the regulatory mechanisms and biologic pathways involved are still largely unknown. The hypothesis of this study is that the genesis and growth characteristics of neurofibromatosis type 2 (NF2)-associated vestibular schwannomas are determined by genetic alterations that vary in gene transcript expression; this transcript expression includes oncogenic gene products that may be identified by construction and sequencing of a cDNA library from NF2-associated vestibular schwannoma. METHODS: Approximately 3 mL of fresh tumor was obtained during resection of a 4-cm vestibular schwannoma from a patient with NF2. Poly(A)+ mRNA was isolated, synthesized into double-stranded cDNA, and unidirectionally inserted into Uni-Zap XR (Stratagene, La Jolla, CA) bacteriophage vectors. Bacteriophage vectors containing cDNA inserts were processed into phagemids according to Uni-Zap XR protocol, and inserted vectors were sequenced and analyzed using BLAST software (National Institutes of Health, Bethesda, MD) with GenBank, EMBL, DDBJ, and PBD databases. RESULTS: The cDNA library contained 2.4 million primary plaques. Inserts averaged 1.8 kilobases (kb) in length, with a range of 0.8 to 3.0 kb. BLAST multi-database comparison of the sequence data obtained from 50 randomly selected clones yielded identification of 13 sequences representing known human genes and 17 sequences representing cloned sequences with unknown function. Three clones represented sequences not previously described in vestibular schwannomas but strongly implicated in oncogenesis within other tissues. CONCLUSIONS: These data have implications for understanding the molecular mechanisms of vestibular schwannoma tumor biology. Identified genes may provide future diagnostic/prognostic markers and therapeutic targets.

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Björling, Ewa, Eva von Garrelts, Andreas Mörner, Mariethe Ehnlund et Mats A. A. Persson. « Human neutralizing human immunodeficiency virustype 2-specific Fab molecules generated by phage display ». Journal of General Virology 80, n^o 8 (1 août 1999) : 1987–93. http://dx.doi.org/10.1099/0022-1317-80-8-1987.

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A panel of human immunodeficiency virus type 2 (HIV-2)-neutralizing, recombinant Fab fragments was generated by using the phage display technique. The combinatorial library was derived from an asymptomatic, HIV-2-seropositive individual and constructed on the surface of filamentous phage by using the pComb3 phagemid vector and then screened against native HIV-2 envelope glycoprotein (gp125). Ten of 30 Fab fragments generated displayed strong reactivity in an ELISA and were therefore selected for further study. Six of these possessed neutralizing capacity, with titres varying from 20 to 80 against the homologous HIV-2 strain, and one also had a weak neutralizing capacity against a heterologous HIV-2 isolate, K135. Sequencing of the heavy chain CDR3 regions showed that the gp125-specific Fabs represented individual clones. These reagents may be useful for studies on the conformational structures of the HIV-2 envelope antigens and their immunogenicity, which may help in vaccine design. Furthermore, the cloned Fab genes may be transformed into whole IgG for eukaryotic expression, and as such used for therapeutic and immunoprophylactic studies in HIV-2-infected macaques and, possibly, for human immunoprophylaxis against HIV-2.

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Manosroi, Jiradej, Chatchai Tayapiwatana, Friedrich Götz, Rolf G. Werner et Aranya Manosroi. « Secretion of Active Recombinant Human Tissue Plasminogen Activator Derivatives in Escherichia coli ». Applied and Environmental Microbiology 67, n^o 6 (1 juin 2001) : 2657–64. http://dx.doi.org/10.1128/aem.67.6.2657-2664.2001.

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ABSTRACT The DNA fragment coding for kringle 2 plus serine protease domains (K2S) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS. In the recombinant vector, pComb3H-K2S, the K2S gene was fused togpIII of ΦM13 and linked to the OmpA signal sequence. The resulting gene, rK2S-gpIII, was inducibly expressed in Escherichia coli XL-1 Blue. The protein was presented on the phage particle. To stop the expression of gpIII,a stop codon between K2S and the gpIIIgene was inserted by site-directed mutagenesis. This mutated vector, MpComb3H-K2S, was transformed in XL-1 Blue. After induction with IPTG (isopropyl-β-d-thiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation. The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B). Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/μg. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes. This finding is an important technological advance in the development of large-scale, bacterium-based tPA production systems.

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Inglis, Stephen C., J. Guy Guillemette, Jeanette A. Johnson et Michael Smith. « Analysis of the invariant Phe82 residue of yeast iso-1-cytochrome c by site-directed mutagenesis using a phagemid yeast shuttle vector ». "Protein Engineering, Design and Selection" 4, n^o 5 (1991) : 569–74. http://dx.doi.org/10.1093/protein/4.5.569.

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Soltes, Glenn, Heather Barker, Kristine Marmai, Elaine Pun, Amy Yuen et Erik J. Wiersma. « A new helper phage and phagemid vector system improves viral display of antibody Fab fragments and avoids propagation of insert-less virions ». Journal of Immunological Methods 274, n^o 1-2 (mars 2003) : 233–44. http://dx.doi.org/10.1016/s0022-1759(02)00294-6.

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Thuong, Ho Thi, Nguyen Thi Thom, Nguyen Thi Tra, Trinh Thai Vy, Pham Bich Ngoc, Le Van Son, Nguyen Thi Bich Ngoc et Ha Hoang Chu. « Production of monoclonal antibody against Ompa protein of Candidatus Liberibacter asiaticus causing citrus greening disease ». Vietnam Journal of Biotechnology 18, n^o 3 (28 novembre 2020) : 529–41. http://dx.doi.org/10.15625/1811-4989/18/3/14634.

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Citrus Greening, also known as HuangLongbing (HLB), is considered one of the most dangerous citrus diseases, and limiting the production of citrus trees all over the world. Production of antibodies against Ompa protein of Candidatus Liberibacter asiaticus (CLas) for detection of citrus greening disease is considered as promising research direction. In this study, for the purpose of producting antibodies against Ompa of CLas, we firstly used the camel VHH antibody library for screening VHH antibodies against Ompa using phage-display technique. Next, phages which had strong interaction with Ompa as shown in ELISA were selected for phagemid isolation and the DNA fragments encoding VHH antibodies were sequenced. The DNA fragment encoding the best VHH antibody was then selected and inserted into the expression vector pET-21a (+), then cloned in Ecoli DH5α strain and expressed in BL21 (DE3) strain. The expression of VHH antibodies against Ompa was optimized at different temperatures with an inductive concentration of 0.1 M IPTG. Anti-Ompa VHH antibodies were purified under denatured conditions then re-folded. The biological activity of the VHH antibody with Ompa antigen was assessed by indirect-ELISA reaction. Results indicated that the VHH antibody had a very strong interaction with the Ompa antigen. This opens up the prospect of applying VHH antibody in the detection of citrus greening disease.

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Singh, Pawan Kumar, Ranu Agrawal, Dev Vrat Kamboj, Garima Gupta, M. Boopathi, Ajay Kumar Goel et Lokendra Singh. « Construction of a Single-Chain Variable-Fragment Antibody against the Superantigen Staphylococcal Enterotoxin B ». Applied and Environmental Microbiology 76, n^o 24 (15 octobre 2010) : 8184–91. http://dx.doi.org/10.1128/aem.01441-10.

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ABSTRACT Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.

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Maenaka, Katsumi, Masaru Furuta, Kouhei Tsumoto, Kimitsuna Watanabe, Yoshitaka Ueda et Izumi Kumagai. « A Stable Phage-Display System Using a Phagemid Vector : Phage Display of Hen Egg-White Lysozyme (HEL),Escherichia coliAlkaline, Phosphatase, and Anti-HEL Monoclonal Antibody, HyHEL10 ». Biochemical and Biophysical Research Communications 218, n^o 3 (janvier 1996) : 682–87. http://dx.doi.org/10.1006/bbrc.1996.0122.

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LEIRO, J., M. I. G. SISO, A. PARAMÁ, F. M. UBEIRA et M. L. SANMARTÍN. « DNA probes for detection of the fish microsporidians Microgemma caulleryi and Tetramicra brevifilum ». Parasitology 119, n^o 3 (septembre 1999) : 267–72. http://dx.doi.org/10.1017/s0031182099004758.

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DNA probes were developed for the detection and identification of 2 microsporidian parasites of marine fishes, Microgemma caulleryi (infecting the liver of the greater sand-eel, Hyperoplus lanceolatus) and Tetramicra brevifilum (infecting muscle, intestine and liver of the turbot, Scophthalmus maximus, a commercially important species). The probe-development procedure used is fast and straightforward, and readily applicable to the development of probes for other microsporidian species. First, genomic DNA of microsporidian spores was isolated and digested with the restriction enzyme Hind III. The fragments obtained were ligated into the vector pBluescript SK(+) and cloned in Escherichia coli. Appropriate inserts were identified and then amplified by PCR, using primers specific for regions adjacent to the Hind III restriction site in the vector sequence (and thus avoiding the need to develop primers specific for the inserts themselves). The copies were labelled with digoxigenin, for subsequent use as probes, during PCR itself. The specificity of candidate probes was tested in dot–blot hybridization assays, with the target DNA being (a) genomic DNA of the microsporidian from which the probe had been obtained, or of another species, (b) the corresponding genomic DNA in the phagemid, or (c) DNA from the corresponding host tissue. These assays identified a ca 1180 bp probe for M. caulleryi, denominated C38, and a ca 1363 bp probe for T. brevifilum, denominated F9. Similar assays designed to assess sensitivity indicated that F9 showed detectable binding to as little as 500 ng of T. brevifilum genomic DNA, and C38 to as little as 125 ng of M. caulleryi DNA; these results were obtained with detection of DIG by enzyme immunoassay (i.e. using a phosphatase-coupled anti-DIG antibody), and could no doubt be improved if a radioactive labelling and detection system were used. The probes developed in this study will greatly facilitate detection and identification of M. caulleryi and T. brevifilum in fish tissues, and may prove useful for identifying possible intermediate hosts used by these species.

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Kearns-Jonker, Mary, Joyce Swensson, Cristina Ghiuzeli, Wilson Chu, Yuka Osame, Vaughn Starnes et Donald V. Cramer. « The Human Antibody Response to Porcine Xenoantigens Is Encoded by IGHV3-11 and IGHV3-74 IgVH Germline Progenitors ». Journal of Immunology 163, n^o 8 (15 octobre 1999) : 4399–412. http://dx.doi.org/10.4049/jimmunol.163.8.4399.

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Abstract Preformed and induced Ab responses present a major immunological barrier to the use of pig organs for human xenotransplantation. We generated IgM and IgG gene libraries established from lymphocytes of patients treated with a bioartificial liver (BAL) containing pig hepatocytes and used these libraries to identify IgVH genes that encode human Ab responses to pig xenoantigens. Genes encoded by the VH3 family are increased in expression in patients following BAL treatment. cDNA libraries representing the VH3 gene family were generated, and the relative frequency of expression of genes used to encode the Ab response was determined at days 0, 10, and 21. Ig genes derived from the IGHV3-11 and IGHV3-74 germline progenitors increase in frequency post-BAL. The IGHV3-11 gene encodes 12% of VH3 cDNA clones expressed as IgM Abs at day 0 and 32.4–39.0% of cDNA clones encoding IgM Abs in two patients at day 10. IGHV3-11 and IGHV3-74 genes encoding IgM Abs in these patients are expressed without evidence of somatic mutation. By day 21, an isotype switch occurs and IGHV3-11 IgVH progenitors encode IgG Abs that demonstrate somatic mutation. We cloned these genes into a phagemid vector, expressed these clones as single-chain Abs, and demonstrated that the IGHV3-11 gene encodes Abs with the ability to bind to the gal α (1,3) gal epitope. Our results demonstrate that the xenoantibody response in humans is encoded by IgVH genes restricted to IGHV3-11 and IGHV3-74 germline progenitors. IgM Abs are expressed in germline configuration and IgG Abs demonstrate somatic mutations by day 21.

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Soltes, Glenn, Heather Barker, Kristine Marmai, Elaine Pun, Amy Yuen et Erik J. Wiersma. « Corrigendum to “A new helper phage and phagemid vector system improves viral display of antibody Fab fragments and avoids propagation of insert-less virions” [Journal of Immunological Methods 274 (2003) 233–244] ». Journal of Immunological Methods 311, n^o 1-2 (avril 2006) : 226. http://dx.doi.org/10.1016/j.jim.2006.01.013.

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Austin, RC, RA Rachubinski, F. Fernandez-Rachubinski et MA Blajchman. « Expression in a cell-free system of normal and variant forms of human antithrombin III. Ability to bind heparin and react with alpha-thrombin ». Blood 76, n^o 8 (15 octobre 1990) : 1521–29. http://dx.doi.org/10.1182/blood.v76.8.1521.1521.

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Abstract Human antithrombin III (AT-III) cDNA was cloned into the cell-free expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was used to remove nucleotides encoding the signal peptide. AT-III messenger RNA (mRNA) transcripts derived from this construct were translated in an mRNA-dependent rabbit reticulocyte lysate (RRL) system containing (35S)methionine. Immunoprecipitation of the cell-free translation mixture with rabbit polyclonal antibodies to AT-III showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), a 47-Kd polypeptide which is the non-glycosylated mature form of plasma AT-III. Densitometric scanning showed that this polypeptide constitutes greater than 90% of the radiolabeled polypeptides produced in this system. Heparin-Sepharose chromatography resulted in the elution of cell-free derived AT-III as a broad peak between 0.2 and 0.7 mol/L NaCl. The cell-free derived AT-III also reacted with human alpha- thrombin. In 2 minutes approximately 20% of the AT-III was found associated with a higher molecular weight species, consistent with the formation of a 1:1 stoichiometric covalent complex between alpha- thrombin and AT-III. Unfractionated heparin accelerated the rate of formation of such complexes. When Ser394 was mutated to Leu to form the AT-III Denver mutant, the cell-free translation product of this mutation did not show any significant complex formation when reacted with alpha-thrombin. A truncated form of AT-III (Met251-Lys432), containing only the putative thrombin-binding domain, was synthesized independently. This 21-Kd polypeptide did not bind heparin; however, it was cleaved by alpha-thrombin presumably at the reactive center Arg393- Ser394. When Ser394 was mutated to Leu the cell-free translation product of this truncated AT-III mutation did not react with alpha- thrombin at the reactive center. This simple cell-free approach, along with site-directed mutagenesis, should allow for the rapid and accurate mapping of the functional domains of human AT-III.

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Austin, RC, RA Rachubinski, F. Fernandez-Rachubinski et MA Blajchman. « Expression in a cell-free system of normal and variant forms of human antithrombin III. Ability to bind heparin and react with alpha-thrombin ». Blood 76, n^o 8 (15 octobre 1990) : 1521–29. http://dx.doi.org/10.1182/blood.v76.8.1521.bloodjournal7681521.

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Human antithrombin III (AT-III) cDNA was cloned into the cell-free expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was used to remove nucleotides encoding the signal peptide. AT-III messenger RNA (mRNA) transcripts derived from this construct were translated in an mRNA-dependent rabbit reticulocyte lysate (RRL) system containing (35S)methionine. Immunoprecipitation of the cell-free translation mixture with rabbit polyclonal antibodies to AT-III showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), a 47-Kd polypeptide which is the non-glycosylated mature form of plasma AT-III. Densitometric scanning showed that this polypeptide constitutes greater than 90% of the radiolabeled polypeptides produced in this system. Heparin-Sepharose chromatography resulted in the elution of cell-free derived AT-III as a broad peak between 0.2 and 0.7 mol/L NaCl. The cell-free derived AT-III also reacted with human alpha- thrombin. In 2 minutes approximately 20% of the AT-III was found associated with a higher molecular weight species, consistent with the formation of a 1:1 stoichiometric covalent complex between alpha- thrombin and AT-III. Unfractionated heparin accelerated the rate of formation of such complexes. When Ser394 was mutated to Leu to form the AT-III Denver mutant, the cell-free translation product of this mutation did not show any significant complex formation when reacted with alpha-thrombin. A truncated form of AT-III (Met251-Lys432), containing only the putative thrombin-binding domain, was synthesized independently. This 21-Kd polypeptide did not bind heparin; however, it was cleaved by alpha-thrombin presumably at the reactive center Arg393- Ser394. When Ser394 was mutated to Leu the cell-free translation product of this truncated AT-III mutation did not react with alpha- thrombin at the reactive center. This simple cell-free approach, along with site-directed mutagenesis, should allow for the rapid and accurate mapping of the functional domains of human AT-III.

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Everard, John, Rebecca Grumet et Wayne Loescher. « Cloning of Mannose 6-phosphate Reductase from Celery. » HortScience 30, n^o 4 (juillet 1995) : 898F—898. http://dx.doi.org/10.21273/hortsci.30.4.898f.

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In celery, photosynthetic carbon partitioning between mannitol and sucrose is highly dependent on developmental (leaf age) and environmental (salt stress) factors. Mannose 6-phosphate reductase (M6PR) mediates a key step in mannitol biosynthesis and may regulate partitioning between sucrose and mannitol. We have constructed a cDNA library and have isolated M6PR-specific clones. Before library construction, poly(A)+ RNA, extracted from newly fully expanded leaves, was translated in vitro. A single polypeptide (35.1 kD), immunoprecipitated with M6PR-specific antisera, accounted for ≈5% of the total 35S incorporated into TCA-precipitated products. Parity between the molecular masses of the immunoprecipitated product and authentic M6PR indicated minimal posttranslational modification. The unidirectional primary library, constructed in UniZap XR vector (Stratagene), consisted of 1.53 million plaque forming-units (pfus) of which <0.4% were nonrecombinant, as estimated by “blue/white”' screening. After a single amplification, ≈0.14% of the 200,000 pfus screened with M6PR-specific antisera were identified as putative M6PR clones. Following two further rounds of screening and in vivo excision of the pBluescript phagemids their identity as full length M6PR clones was confirmed as follows: 1) IPTG-induced expression of M6PR activity in crude extracts; 2) IPTG-induced expression of a polypeptide that specifically interacted with M6PR antisera and with identical mobility (on SDS gels) to authentic M6PR; 3) 100% sequence homology to an internal peptide from a tryptic digest of purified M6PR. Based on these criteria, we conclude that we successfully cloned M6PR. The sequence is similar to several reductases from both plants and animals including an aldose 6-phosphate reductase from apple. Supported by USDA-NRI grant 940-1439.

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Yang, Shihong, Xin An, Ross D. Brown, Joy Ho, John Gibson, Douglas E. Joshua, Winfried Wels et Daniel M. Sze. « Development of Retargeted CD38-Specific NK-92 Cell Line for Potential Anti-Myeloma Immunotherapy. » Blood 106, n^o 11 (16 novembre 2005) : 5104. http://dx.doi.org/10.1182/blood.v106.11.5104.5104.

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Abstract Multiple Myeloma (MM) is a B cell neoplasm in which malignant plasma cells accumulate in the bone marrow and produce excessive amounts of a monoclonal immunoglobulin. Among the large number of antigens expressed by plasma cells, CD38 is one of the highest expressed surface molecules, suggesting an alternative public tumour target. For instance, the myeloma cell line 8226 has a high surface expression of CD38. We plan to introduce genetically recombinant single-chain variable fragments (scFv) of anti-CD38 antibody, along with CD3 chain, into NK-92 a natural killer cell line which is highly cytotoxic against a variety of malignant cells (the only natural killer cell line being used in phase I/II clinical trials). It is likely that the retargeted NK-92 cells may specifically and efficiently kill CD38-expressing myeloma cells. By using a series of degenerate primer sets for mouse immunoglobulin variable region genes, VH and VL regions were isolated through reverse transcription PCR from OKT10 cell line. Further sequencing analysis provided novel information of the OKT10 immunoglobulin heavy and light chains. A PCR-based assembling strategy was employed to construct the scFv with FLAG sequence integrated as a tag. VH and VL domains were joined together with a hinge region of (Gly4Ser)3. The phagemid construct has been formed by subcloning the scFv fragment into the Sfi I/Not I cloning sites of the expression vector pHEN2, and subsequently transformed into the E. Coli HB2151. Five out of 7 transformed bacterial colonies screened did not contain the scFv fragment. For the remaining two clones with the correct-sized insert, we tested the specificity of the scFv fragments that were prepared through intracellular antibody capture as periplasmic proteins. We performed a competitive flow assay using periplasmic proteins with commercially available anti-CD38 PE conjugated antibodies on 8226 myeloma cell line. We found that one of these two clones (clone 6) showed a reduction in CD38 staining and presented as double peaks; while this pattern was not seen in the clone 7. Subsequent DNA sequencing revealed that there was a 100-bp truncation in the heavy chain region in clone 7; while the clone 6 has a complete productive heavy chain and light chain spanned by the linker region. We are going to transfect NK-92 cells with retrovial vector pL-scFv(clone 6) construct as the basis of an efficient approach for anti-myelome immunotherapy. (Dr. Daniel Sze is supported by the International Myeloma Foundation Senior Research Grant).

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Liu, Yanlong, Kexin Ao, Fuxiang Bao, Yi Cheng, Yanxia Hao, Huimin Zhang, Shan Fu, Jiaqi Xu et Qiyao Wu. « Development of a Bispecific Nanobody Targeting CD20 on B-Cell Lymphoma Cells and CD3 on T Cells ». Vaccines 10, n^o 8 (17 août 2022) : 1335. http://dx.doi.org/10.3390/vaccines10081335.

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B-cell lymphoma is a group of malignant proliferative diseases originating from lymphoid tissue with different clinical manifestations and biological characteristics. It can occur in any part of the body, accounting for more than 80% of all lymphomas. The present study aimed to construct bispecific single-domain antibodies against CD20 and CD3 and to evaluate their function in killing tumor cells in vitro. A Bactrian camel was immunized with a human CD20 extracellular peptide, and the VHH gene was cloned and ligated into a phagemid vector to construct the phage antibody display library. A phage antibody library with a size of 1.2 × 108 was successfully constructed, and the VHH gene insertion rate was 91.7%. Ninety-two individual clones were randomly picked and screened by phage ELISA. Six strains with the high binding ability to human CD20 were named 11, 30, 71, 72, 83, and 92, and induced expression and purification were performed to obtain soluble CD20 single-domain antibodies. The obtained single-domain antibodies could specifically bind to human CD20 polypeptide and cell surface-expressed CD20 molecules in ELISA, Western blot, and cell immunofluorescence assays. The anti-CD20/CD3 bispecific nanobody (BsNb) was successfully constructed by fusing the anti-CD20 VHH gene with the anti-CD3 VHH and the bispecific single-domain antibody was expressed, purified, and validated. Anti-CD20/CD3 BsNb can specifically bind CD20 molecules on the surface of human lymphoma Raji cells and CD3 molecules on the surface of T cells in flow cytometry analysis and effectively mediate peripheral blood mononuclear cells (PBMCs) target Raji cells with a killing efficiency of up to 30.4%, as measured by the lactate dehydrogenase (LDH) method. The release of hIFN-γ from PBMCs during incubation with anti-CD20/CD3 BsNb was significantly higher than that of the control group (p < 0.01). The anti-CD20/CD3 BsNb could maintain 80% binding activity after incubation with human serum at 37 °C for 48 h. These results indicated the strong antitumor effect of the constructed anti-CD20/CD3 BsNb and laid the foundation for the further development of antitumor agents and the clinical application of anti-CD20/CD3 BsNb.

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Balcha, Fikre Birhanu, et Sultan Abda Neja. « CRISPR-Cas9 mediated phage therapy as an alternative to antibiotics ». Animal Diseases 3, n^o 1 (27 février 2023). http://dx.doi.org/10.1186/s44149-023-00065-z.

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AbstractInappropriate use of antibiotics is globally creating public health hazards associated with antibiotic resistance. Bacteria often acquire antibiotic resistance by altering their genes through mutation or acquisition of plasmid-encoding resistance genes. To treat drug-resistant strains of bacteria, the recently developed CRISPR-Cas9 system might be an alternative molecular tool to conventional antibiotics. It disables antibiotic-resistance genes (plasmids) or deactivates bacterial virulence factors and sensitizes drug-resistant bacteria through site-specific cleavage of crucial domains of their genome. This molecular tool uses phages as vehicles for CRISPR-cas9 delivery into bacteria. Since phages are species-specific and natural predators of bacteria, they are capable of easily injecting their DNA to target bacteria. The CRISPR system is packaged into phagemid vectors, in such a way that the bacteria containing the antibiotic-resistance plasmid sequence or that containing specific DNA sequences were made to be targeted. Upon CRISPR delivery, Cas9 is programmed to recognize target sequences through the guide RNA thereby causing double-strand cleavage of targeted bacterial DNA or loss of drug resistance plasmid, which results in cell death. Remarkably, the safety and efficacy of this newly developed biotechnology tool and the biocontrol product need to be further refined for its usage in clinical translation.

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« Development of Specific Nano-Antibody for Application in Selective and Rapid Environmental Diagnoses of Salmonella arizonae ». Biointerface Research in Applied Chemistry 10, n^o 6 (7 juin 2020) : 7198–208. http://dx.doi.org/10.33263/briac106.71987208.

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Treatment of human and animals for protection from pathogens infection has significant economic value, especially with the harmful Salmonella arizonae. Beneficial cameloid heavy-chain antibodies as single-domain antigen-binding fragments known as VHHs or nano-bodies may be the acceptable option for producing the treatment and/or diagnostic agents. In the current study, we developed a sandwich ELISA based nano-body towards S. arizonae as the first report for the treatment of S. arizonae. using the cDNA synthesized from immunized camels RNA to isolate 700 bp DNA fragment, which contains all VH domains of IgG2 and IgG3 isotypes followed by the second amplification VHH PCR with amplified fragments at 450 bp. The final PCR products were cloned into the phagemid vector pMECS then via phage display technique. Nano-antibodies protein was purified and separated under non-denaturing conditions by SDS-PAGE. The reactivity of each VHH of the selected clones was analyzed by Western blot assay. The isolated nano-antibodies showed binding not only to S. arizonae, but also to other bacterial strains, indicating that these nano-antibodies can be used in treatment but cannot use in diagnostic.

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Moreno, Ernesto, Mario S. Valdés-Tresanco, Andrea Molina-Zapata et Oliberto Sánchez-Ramos. « Structure-based design and construction of a synthetic phage display nanobody library ». BMC Research Notes 15, n^o 1 (29 mars 2022). http://dx.doi.org/10.1186/s13104-022-06001-7.

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Abstract Objective To design and construct a new synthetic nanobody library using a structure-based approach that seeks to maintain high protein stability and increase the number of functional variants within the combinatorial space of mutations. Results Synthetic nanobody (Nb) libraries are emerging as an attractive alternative to animal immunization for the selection of stable, high affinity Nbs. Two key features define a synthetic Nb library: framework selection and CDR design. We selected the universal VHH framework from the cAbBCII10 Nb. CDR1 and CDR2 were designed with the same fixed length as in cAbBCII10, while for CDR3 we chose a 14-long loop, which creates a convex binding site topology. Based on the analysis of the cAbBCII10 crystal structure, we carefully selected the positions to be randomized and tailored the codon usage at each position, keeping at particular places amino acids that guarantee stability, favoring properties like polarity at solvent-exposed positions and avoiding destabilizing amino acids. Gene synthesis and library construction were carried out by GenScript, using our own phagemid vector. The constructed library has an estimated size of 1.75 × 108. NGS showed that the amino acid diversity and frequency at each randomized position are the expected from the codon usage.

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Sawicka, Róza, Paweł Siedlecki, Barbara Kalenik, Jan P. Radomski, Violetta Sączyńska, Anna Porębska, Bogusław Szewczyk, Agnieszka Sirko et Anna Góra-Sochacka. « Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody ». Acta Biochimica Polonica 64, n^o 1 (7 décembre 2016). http://dx.doi.org/10.18388/abp.2016_1292.

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Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitated epitope mapping. Based on the sequences of the affinity-selected polypeptides and the structural model of HA the epitope has been located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibition activity and its antigenic specificity. Additionally, total RNA from hybridoma cells secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed slight differences in specificity are discussed.

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Pursey, Elizabeth, Tatiana Dimitriu, Fernanda L. Paganelli, Edze R. Westra et Stineke van Houte. « CRISPR-Cas is associated with fewer antibiotic resistance genes in bacterial pathogens ». Philosophical Transactions of the Royal Society B : Biological Sciences 377, n^o 1842 (29 novembre 2021). http://dx.doi.org/10.1098/rstb.2020.0464.

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The acquisition of antibiotic resistance (ABR) genes via horizontal gene transfer (HGT) is a key driver of the rise in multidrug resistance amongst bacterial pathogens. Bacterial defence systems per definition restrict the influx of foreign genetic material, and may therefore limit the acquisition of ABR. CRISPR-Cas adaptive immune systems are one of the most prevalent defences in bacteria, found in roughly half of bacterial genomes, but it has remained unclear if and how much they contribute to restricting the spread of ABR. We analysed approximately 40 000 whole genomes comprising the full RefSeq dataset for 11 species of clinically important genera of human pathogens, including Enterococcus , Staphylococcus , Acinetobacter and Pseudomonas . We modelled the association between CRISPR-Cas and indicators of HGT, and found that pathogens with a CRISPR-Cas system were less likely to carry ABR genes than those lacking this defence system. Analysis of the mobile genetic elements (MGEs) targeted by CRISPR-Cas supports a model where this host defence system blocks important vectors of ABR. These results suggest a potential ‘immunocompromised’ state for multidrug-resistant strains that may be exploited in tailored interventions that rely on MGEs, such as phages or phagemids, to treat infections caused by bacterial pathogens. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

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Yasmin, Mohamad, Laura J. Rojas, Steven H. Marshall, Andrea M. Hujer, Anna Cmolik, Emma Marshall, Helen W. Boucher et al. « Characterization of a Novel Pathogen in Immunocompromised Patients : Elizabethkingia anophelis - Exploring the Scope of Resistance to Contemporary Antimicrobial Agents and β-lactamase Inhibitors ». Open Forum Infectious Diseases, 31 janvier 2023. http://dx.doi.org/10.1093/ofid/ofad014.

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Abstract Objective Elizabethkingia anophelis is an emerging Gram-negative non-lactose fermenter in the healthcare setting where it causes life-threatening infections in immunocompromised patients. We aimed to characterize the molecular mechanisms of antimicrobial resistance and evaluate the utility of contemporary antibiotics with the intent to offer targeted therapy against an uncommonly encountered pathogen. Methods Whole genome sequencing (WGS) was conducted to accurately identify isolate species and elucidate the determinants of β-lactam resistance. Antimicrobial susceptibility testing (AST) was performed using broth microdilution (BMD) and disk diffusion assays. To assess the functional contribution of the major metallo-β-lactamase (MBL) encoding genes to the resistance profile, blaBlaB was cloned into pBC SK (-) phagemid vector and transformed into Escherichia coli DH10B. Results WGS identified the organism as E. anophelis. MBL genes blaBlaB-1 and blaGOB-26 were identified, in addition to blaCME-2 which encodes for an extended-spectrum β-lactamase (ESBL). Plasmids were not detected. The isolate was non-susceptible to all commonly available β-lactams, carbapenems, newer β-lactam β-lactamase inhibitor (BL-BLI) combinations, and to the combination of aztreonam (ATM) with ceftazidime-avibactam (CAZ-AVI). Susceptibility to the novel siderophore cephalosporin cefiderocol (FDC) was determined. A BlaB-1 transformant E. coli DH10B isolate was obtained and demonstrated increased MICs to cephalosporins, carbapenems, and CAZ-AVI, but not ATM. Conclusion Using WGS, we accurately identified and characterized an extensively drug resistant E. anophelis in an immunocompromised patient. Rapid evaluation of the genetic background can guide accurate susceptibility testing to better inform antimicrobial therapy selection.

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Lauretti-Ferreira, Fabiana, André Azevedo Reis Teixeira, Ricardo José Giordano, Josefa Bezerra da Silva, Patricia Antonia Estima Abreu, Angela Silva Barbosa, Milena Apetito Akamatsu et Paulo Lee Ho. « Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage display ». Frontiers in Microbiology 13 (28 novembre 2022). http://dx.doi.org/10.3389/fmicb.2022.1051698.

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Pathogenic species of Leptospira are etiologic agents of leptospirosis, an emerging zoonotic disease of worldwide extent and endemic in tropical regions. The growing number of identified leptospiral species sheds light to their genetic diversity and unique virulence mechanisms, many of them still remain unknown. Toxins and adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines with cross-protection and long-lasting effect against leptospirosis. For this aim, we used the shotgun phage display technique to unravel new proteins with adhesive properties. A shotgun library was constructed using fragmented genomic DNA from Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 and pG8SAET phagemid vector. Selection of phages bearing new possible cell-binding antigens was performed against VERO cells, using BRASIL biopanning methodology. Analysis of selected clones revealed the hypothetical protein LIC10778, a potentially exposed virulence factor that belongs to the virulence-modifying (VM) protein family (PF07598), composed of 13 members in the leptospiral strain Fiocruz L1-130. Prediction of LIC10778 tertiary structure indicates that the protein contains a cellular-binding domain (N-terminal portion) and an unknown domain of no assigned activity (C-terminal portion). The predicted N-terminal domain shared structural similarities with the cell-binding and internalization domain of toxins like Ricin and Abrin, as well as to the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin in Mycoplasma pneumoniae. Interestingly, recombinant portions of the N-terminal region of LIC10778 protein showed binding to laminin, collagens I and IV, vitronectin, and plasma and cell fibronectins using overlay blotting technique, especially regarding the binding site identified by phage display. These data validate our preliminary phage display biopanning and support the predicted three-dimensional models of LIC10778 protein and other members of PF07598 protein family, confirming the identification of the N-terminal cell-binding domains that are similar to ricin-like toxins. Moreover, fluorescent fused proteins also confirmed that N-terminal region of LIC10778 is capable of binding to VERO and A549 cell lines, further highlighting its virulence role during host-pathogen interaction in leptospirosis probably mediated by its C-terminal domain. Indeed, recent results in the literature confirmed this assumption by demonstrating the cytotoxicity of a closely related PF07598 member.

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Articles de revues sur le sujet « Phagemid vectors »

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