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Establishment of Inderect ELISA for Detection of Antibodies Angainst STLV-1 and Preparation of Hybri

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Tutor: ChenChaoYin;ZhouXiaoLi
School: Kunming University of Science and Technology
Course: Biochemical Engineering
Keywords: STLV-1,gag protein,Baculovirus expression vector,Indiect ELISA,McAb
CLC: R373
Type: Master's thesis
Year:  2011
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Abstract:
According to STLV-1 virus gag gene nucleotide sequences have been documented in Genbank database(NO:AY590142),we synthesized gag gene. The gag gene fragment was cloned in the pFastBacHTB donor plasmid of Bac-to-Bac baculovirus expression system,the positive recombinant plasmid was named pFastBacHTB-gag.The recombinant plasmid was constructed and identified by PCR and enzyme digestion and sequenced. Then the plasmid pFastBacHTB-gag was transformed into DH10Bac complement cells. It was identified by antibiotics and PCR, it is shows we had constructed baculovirus expression vector to gag gene. Recombinant virus was generated by transfecting with recombinant DNA into sf9 insect cells.After transfection for 96 hours, cellvirus was harvested.Then we confirmed the recombinant gag protein via the SDS-PAGE and Western blotting,which showed that gag protein was expressed in bacmid-gag virus infected insecet cells.The gag protein had a good biological activity and was approximate 55kDa.The indirect ELISA was established to detect antibody of STLV-1 using purified gag protein.The optional working circumstances for the indirect ELISA assay(antigen concentration 0.28¦Ìg£»serum dilution:1:100)was confirmed.The working concentration of HRP-labeled goat anti-human IgG was 1:5000.The positive criterion of this ELISA assay is OD450nm=0.327,OD450nm=0.285 is negative,and it is suspicious between 0.327 and 0.285.The tests showed no cross-reactivity with antibodies to SRV and Simian B Virus. After detecting 200 serum samples collected from Yunnan provence,the coincidence rate was 98.5% between indirect ELISA and commercialization ELISA kit,indicating the recombinant gag protein could be used for primary diagnoses of Simian infection with STLV-1.The BALB/c mice were immunized with recombinant gag protein, Cell fusion was carried out by PEG 3 days after boosting immunization.we used indirect ELISA to screen hybridoma cells, and we also performed limited dilution method to subclone negligent positive hybridoma cells constantly expressing monoclonal antibodies of anti-STLV-1 gag protein. We obtain 1 positive hybridoma clones,which designated as3B6.
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