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The Initial Research on the Ribosome Display Technology of Selecting the SEB Antibody

Author: MengXiaMeng
Tutor: DuanHongQuan；GaoZhiXian；NingBaoAn
School: Tianjin Medical University
Course: Medicinal Chemistry
Keywords: Ribosome display In vitro screening SEB Prokaryotic expression Recombinant
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 112
Quote: 0
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Abstract


Objective To establish a ribosome display library using ribosome display technology established screening SEB antibody research. The first part of this test method for constructing a prokaryotic expression of the the SEB protein of recombinant plasmid. Selection has become the choice for expression in E. coli protein Invitrogen pET system through the the SEB gene fragment with N-terminal has six histidine PET 32a carrier is connected to the expression product of the N-terminal fusion of six histidine-tagged chelated metal ion affinity chromatography, using a HiTrap Chelating Ni-column purified target protein. Providing the the ribosome display filter desired antigen. The second part of the test ribosome display library constructed from the murine single-chain variable region gene and ribosome display technology for the preliminary study of the screening SEB antibody. By TRIzol method to extract the total RNA of Balb / c and C57 mice, the use of PCR amplified mouse heavy chain variable region (VH) and light chain variable region (VL), amplified and connected to the ribosome display all the components needed to build, including the T7 promoter, stem-loop, a ribosome binding sites, a single chain antibody gene library interval sequence of the ribosomal genes show formwork. Subsequently, the library was in vitro transcription and in vitro translation, the initial screening recombinant SEB antigen. Was successfully constructed to express the the SEB protein of recombinant plasmid named SEB321. To 3mmol/LIPTG induction, 30 ° C shaking for 4 hours, can be expressed SEB protein, further use of the HiTrap Chelating Ni-column affinity-purified, were identified by Western blot. Ribosome display library derived from mouse single-chain variable region gene was successfully constructed, and the determination of the ultimate storage capacity of 7.33 × 10 13 . . Subsequently, the library of the in vitro transcription and translation in vitro, indicating that it can be effectively carried out in vitro transcription and translation. Ribosome display technology to restructure SEB protein antigen, a preliminary screening, positive results. Conclusion storage capacity of 7.33 × 10 13 from non-immunized mice spleen ribosome display library of single-chain antibody variable region gene segments. Explore the conditions of building a database and carrier PET 32a prokaryotic expression vector was successfully expressed the recombinant SEB protein, recombinant SEB protein antigen, the initial screening, screening SEB antibody positive. Demonstrated a single-chain antibody variable region gene fragments from non-immunized mice spleen ribosome display libraries can be screened quickly and efficiently antibody-based research group using ribosome display lay the foundation for other antibody screening, but also for ribose the body display technology research to provide a reference.

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