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Clone and Expression of Porcine CD163 Gene and Preparation of Its Specific Antibodies

Author: GuoJuanJuan
Tutor: ZhangDeLi；TianZhiJun
School: Northwest University of Science and Technology
Course: Preventive Veterinary Medicine
Keywords: CD163 Cloning and expression Polyclonal antibody Monoclonal antibodies
CLC: S852.4
Type: Master's thesis
Year: 2011
Downloads: 27
Quote: 0
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Abstract


Porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome Virus, PRRSV) is a major pathogen of a serious hazard to the pig industry. PRRSV mainly alveolar macrophages (porcine alveolar macrophages, PAM) as target cells through interaction with the host cell receptor, causing the body too strong innate immune response and the adaptive immune response. CD163 is present in the monocyte - macrophage cell surface-specific receptors, belonging to type I transmembrane protein. PRRSV non-permissive cell lines in the transient and stable expression of the CD163 molecule were to support the type I and type II PRRSV infection and produce progeny virus particles, suggesting that the CD163 molecule play a role in the release of virus particles husking process. PRRSV infection process, the interaction between the receptor CD163 SRCR5 domain and glycosylated membrane protein of PRRSV GP2 and GP4, and promote viral uncoating, virus released into the cytoplasm start transcription and translation to form new virus particles from the acidic vesicles particles, and delete the cytoplasmic tail of CD163 molecule can promote the proliferation of PRRSV replication. In order to understand the function in the the PRRSV infection process in CD163 of the CD163 SRCRs domain mechanism of interaction with other receptors, this study prokaryotic expression and purification the of the cytoplasm outside the area SRCR4-SRCR5 of the CD163 protein, immunization of New Zealand white rabbits and BALB / c mice mice were prepared rabbit anti-pig CD163 polyclonal antibody and monoclonal antibodies and antibody function identification. The experimental results are as follows: 1. Construct expressing porcine the CD163 gene cytoplasm outside SRCR4-SRCR5 prokaryotic expression vector of pGEX-6P-CD163 IPTG induction in E. coli BL21, confirmed by SDS-PAGE and Western blot analysis obtained the corresponding size of the fusion protein GST-CD163 purified by plastic cutting the GST-CD163 fusion protein was purified to get the high purity of the target protein. The purified protein was used to immunize New Zealand white rabbits, preparation of rabbit anti-pig CD163 polyclonal antibody and polyclonal antibody blocking experiments were identified by flow cytometry and antibody. 3 purified fusion protein-immunized BALB / c mice, to take immune mouse spleen cells with myeloma cells were fused with 50% PEG (4000). Cells using indirect ELISA and Western Blot Detection Fusion survival. Screened by limiting dilution of an anti-GST-positive hybridoma cell lines, named 3E2F2; the anti CD163 positive clonal cell lines still further screening. In the present study, we obtain CD163 polyclonal rabbit anti-swine antibody and an anti-GST monoclonal antibody-producing hybridomas secreted CD163 monoclonal antibody hybridoma still further screening. If you can get a monoclonal cells secreting CD163 antibody, and lay the foundation for further research on the specific roles in the the PRRSV infection process in CD163 receptor.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Immunology
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