Littérature scientifique sur le sujet « Phagemid vectors »

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.

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.

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.

2007/2008
Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system (CNS) which typical feature is the plaques of demyelination. The factor triggering the disease is still unknown, but the most diffuse opinion is that the autoimmune reaction is due to a new contact with environmental factors, like virus, that mimic self molecules (molecular mimicry). MS is a T- cell mediated disease. These cells activated by myelin antigens activate, in turn, B lymphocytes, macrophages and cytotoxic lymphocytes which strongly damage oligodendrocytes and neurons, both directly and through complement activation, leading to an inhibition of nervous conductivity in CNS. In this project, MS is studied through an “epitomic” approach by means of the construction of epitope phage display libraries, obtained by cloning cDNAs derived from CNS tissue. These collections of billions of different phages displaying different polypeptides on their surface give the total representation of the CNS expression pattern and are useful for the selection against MS serum and CSFs, in order to identify potential autoimmune epitopes and the relatives auto-antibodies. The choice of phage display technology as tool to investigate MS autoimmune response depends on the possibility to couple the phenotype to genotype in a selectable format. Selection of the phages on the basis of the binding properties of the polypeptide displayed on the surface, simultaneously results in the isolation of the gene encoding the polypeptide. In order to obtain the epitope phage display libraries, two different versions of a new vector, called pEP1 and pEP2 respectively, for the selection of the cDNA fragments cloned in the correct open reading frame (ORF) were designed. A well characterized vector for the ORF selection, previously described by Zacchi and colleagues (2003), was used as backbone and modified in two alternative ways on its original polylinker to improve the cloning efficiency. In addition, for its oriented cloning system, pEP2 was able to give a greater ratio of “in frame”/ “out of frame” clones (1/9 rather than 1/18) than pEP1. After their validation, the new vectors were employed to generate two kinds of cDNA phage display libraries: pEP1 was tested with the construction of single myelin genes (myelin basic protein, MBP; and myelin oligodendrocyte glycoprotein, MOG) mini-libraries; while the more advanced pEP2 was used to create a total human brain library. A first question was related to the kind of epitope recognized by autoantibodies in MS. Generally, autoantibodies recognize both linear and conformational epitope, but at present the conformational epitopes of myelin proteins have not been identified. We have set a system to make minilibraries from single gene through the fragmentation of the gene of interest with nuclease protection by means of archibacteria histones. In this way it’s possible to obtain phage display libraries of overlapping fragments of the same gene useful to define the precise epitope recognized by a biological sample. For the project, human MBP and MOG mini-libraries were obtained and characterized through PCR analysis and enzymatic fingerprinting. By sequencing of 30 randomly picked clones for both mini-libraries, a set of overlapping fragments which gave a total representation of mbp and mog gene, was respectively found. MBP mini-library was subjected to two selections: against the serum and the CSF of a patient with a Clinically Isolated Syndrome (CIS), diagnosed after a first clinically episode of MS symptoms. Instead, MOG mini-library was selected against a Relapsing Remitting (RR) serum of a MS patient. To identify the more reactive clones, 95 clones from each last round of selection were randomly picked and subjected to a phage-ELISA test. Panning the hMBP mini-library with CIS serum and CSF, some distinct epitopes of the protein were identified, suggesting a specific pattern of recognition with some overlapping regions in both the CIS samples. Among the epitopes recognized, was identified the more immunogenic region of hMBP (a.a. 85-96). In the case of hMOG mini-library against RR serum, selected phage clones shared the epitope corresponding to a.a. 33-104 of the protein, which is localized inside the more immunogenic region of hMOG. A cDNA phage display library which should contain all the potential auto-antigens of the CNS is a useful tool to perform a serological analysis of MS sera and CSFs, in order to identify the epitopes involved in the immune response and to identify subclinical conditions of patients. With these purposes, a human brain phage display library was produced. Starting from human brain mRNA, a new protocol for the cDNA production was set. Employing the “OrientExpress Random Primers” (Novagen), an orientated synthesis of the cDNA was obtained. Following the ligation to the vector (digested pEP2), a human brain library of 1.35x10^5 individual clones selected for the presence of ORF inserts was produced. Through PCR analysis, enzymatic fingerprinting and sequencing of randomly picked clones a library diversity of 90% was estimated. The library was employed for three distinct selections: 1). against IgGs purified from a pool of RRMS sera; 2). against a pool of Secondary Progressive (SP) MS sera; and 3). against a pool of MS CSFs. From the last round of each selection test were respectively picked 190, 95 and 50 clones. These randomly picked clones were checked by phage-ELISA to identify those more reactive. The positive clones were respectively 34%, 16% and 50% on the total. Interestingly, some clones were shared among the selections and it was identified less clone diversity in the selection against CSFs pool. The reactivity of some selected clones, as phages, as recombinant protein or as synthetic peptides, was tested in ELISA in order to establish the sensibility and specificity in discriminating MS and other neurological diseases (OND). The ELISAs on "omissis patent pending" peptide resulted particularly predictive (LR+ 12.2) with a sensitivity and specificity of discrimination of 31.7% and 97.4%, respectively. The final aim of the project was in providing new tools to investigate the antibody response in MS and in the improvement of the currently used diagnostic tests through the detection of new potential markers of the disease. At the moment there are not validated biomarkers of MS. The new candidate autoantigens identified in this study could be one of those and integrate the current diagnostic method of MS.
XXI Ciclo
1980