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Construction of Phage Display Single-chain Fv Antibody Library Against UV-attenuated Schistosoma Japonicum Cercariae and the Screening of the Sj SCA66-68kDa ScFv

Author: ZhouShuaiFeng
Tutor: WangShiPing
School: Central South University
Course: Microbiology
Keywords: Schistosoma japonicum Attenuated cercariae Passive transfer Protection force Single-chain antibody Phage display antibody library Protein N- sequencing
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 27
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Abstract

Research purposes, based on the characteristics of attenuated cercariae vaccine and natural molecular vaccine high protection force, the use of phage display technology many advantages, build efficient Japanese schistosome UV-induced the weak the cercariae single chain antibody (ScFv) expression library, and as a high The flux source library screened for the potentially protective natural molecular antigen SjSCA66-68kDA single chain antibody, to lay the foundation for further access to the natural molecules of Schistosoma japonicum candidate vaccine SjSCA66-68kDA coding genes, to speed up the anti-schistosomiasis japonica efficient natural molecular genetic vaccine development pace. Research methods using phage display technology to build highly efficient the Schistosoma japonicum UV-irradiation attenuated cercariae single chain antibody library, from the identification of storage capacity, diversity of the library. Schistosoma japonicum using UV-irradiation attenuated cercariae multiple infection of BALB / c mice, and sterile UV-attenuated cercariae serum in normal cercariae Infection (cercariae number of 40 ± 2) before and after infection, one week, three weeks, the transfer of a total of three times by tail vein injection to BALB / c mice, 45 days after the mice were sacrificed, worm reduction rate and egg reduction rate were calculated to ensure the efficiency of its attenuated cercariae serum. Extracted on the basis of successful to build the attenuated cercariae mouse model of mouse spleen RNA by RT-PCR amplification of a full set of VH and VL gene amplified by overlapping PCR method ScFv fragment, ScFv fragment was cloned into PCANTAB 5E transform competent TG1 bacteria carrier, electricity, save by the helper phage M13K07 obtained the Schistosoma japonicum UV-weak the cercariae single chain antibody library. Target molecule, and take advantage of building a database efficient UV-attenuated cercariae serum at different developmental stages of Schistosoma japonicum antigen immune response to identify and select immune screening candidate antigen SCA66-68 the natural molecular immunology and biochemical characteristics and preliminary study of the sequence: cut plastic by electrophoresis, electro-elution, the separation and purification methods such as ultrafiltration and lyophilization the SCA66-68KDa antigen, containing a single the SCA66-68KDa antigen PAGE gel binding trace prepared lymph injection immunize New Zealand rabbits specificity the SCA66-68KDa rabbits immunized serum, the detection and identification of its potency and specificity by ELISA and Western blot; determined by silver staining and PAS staining the SCA66-68KDa antigen chemical nature; N-terminal sequencing method SCA66-68KDa the protein sequences of the antigen. 3. Use of the separation and purification of the vaccine candidate molecules SCA66-68kDa antigen screening of high-throughput UV-radiation attenuated cercariae single portfolio strategy by the antigen directly Law and double antibody sandwich joint nitrocellulose membrane enriched colony dug set method chain antibody library, identification of specificity the SCA66-68kDa single chain antibody obtained by Western blot. Results: 1.UV-irradiated Schistosoma japonicum attenuated cercariae serum passive transfer of BALB / c mice obtained 42.5% worm reduction rate, 28.0%, significantly higher than the rate of the infection serum metastasis worm reduction. Successfully constructed on the basis of a mouse model of UV-radiation attenuated cercariae of Schistosoma japonicum highly efficient the Schistosoma japonicum UV-weak cercariae single chain antibody library, and the determination of its storage capacity to 1.9 ~ 10 ~ 8, 100% of the rate of recombination Diversity is good. Was found, the efficient cause of building a database weak cercaria schistosome serum can identify different stages of the near 66-68kDa antigen a common identification bands, and the basis of previous studies of this room, select cercariae 66-68kDa natural molecules as immune screening target molecule antigen. Successfully isolated and purified antigens the electrophoretically pure immune pure SCA66-68kDa, and successfully prepared the monospecific the SCA66-68KDa immune rabbit serum antibody titer as high as 1:12800; silver nitrate and PAS staining method to determine SCA66-68KD the chemical nature of a protein component and a non-glycoprotein; N-sequencing method SCA66-68KDa protein sequence of the native molecule, result in the N-terminal peptides confinement phenomenon proposed SCA66-68KDa natural molecular protein sequencing optimization strategy. Japan schistosomiasis vaccine candidate molecules SCA66-68kDa efficient UV-irradiation attenuated cercariae serum identified SCA66-68kDa antigen, the use of the separation and purification of UV-irradiation attenuated cercariae single chain antibody library screening. Antigen direct method and the double-antibody sandwich nitrocellulose membrane after many rounds of panning the enriched colony mining Screening caused by a the weak the cercariae single chain antibody library: the first round of the double-antibody sandwich method of false positives is too high, do not get re-screened. positive clones; antigens direct method to obtain a total of six SCA66-68kDa positive clones, will be screening the positive clones was transformed into the Ecoli HB2151 bacteria to induce the expression of soluble scFv. SDS-PAGE and Western blot analysis, the results showed that soluble scFv was correctly expressed and SCA66-68kDa with the corresponding antigen-specific binding. The results show that by direct antigen screening method to obtain a specific anti the SCA66-68kDa single chain antibody. Conclusion: for the first time successfully build efficient Schistosoma japonicum UV-induced weak cercariae single-chain antibody library, it is constructed to compensate for the limitations of the application of an attenuated vaccine to provide a reference for the development of new vaccine development strategy and the design of new vaccines, for further screening for the diagnosis and treatment of schistosomiasis-specific single-chain antibody epitopes and vaccine development to provide a high-throughput library source. 2 candidate vaccine SjSCA66-68kDa natural molecules as a primary screening target antigens, the successful separation and purification SjSCA66-68kDa natural molecular and biochemical properties and sequence their immune preliminary study for further screening the attenuated cercariae single chain antibody library provides a theoretical basis, but also to lay the foundation for the natural molecules encoded genetically engineered vaccine prepared. 3 successfully use the antigen directly combined with nitrocellulose membrane enriched colony digging screening specific anti-SCA66-68kDa single chain antibody. The SCA66-68kDa get specific ScFv the advantage embodied in the screening of the vaccine candidate molecules, build an effective anti-Japan schistosomiasis efficient natural molecular genetic engineering vaccines and expanding field applications lay the foundation for success. In addition, in many aspects of the use of single-chain antibody to carry out immune diagnostics, immunotherapy and disease pathogenic molecular mechanisms, showing a broad application prospects.

Construction of Phage Display Single-chain Fv Antibody Library Against UV-attenuated Schistosoma Japonicum Cercariae and the Screening of the Sj SCA66-68kDa ScFv

Abstract

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