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Preparation Of PRRSV GP2 Protein Monoclonal Antibody And Establishment Of Marc145-GP2 Cell Line

Posted on:2022-04-14Degree:MasterType:Thesis
Country:ChinaCandidate:D G W WuFull Text:PDF
GTID:2480306527489644Subject:Prevention of Veterinary Medicine
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Porcine Reproductive and Respiratory Syndrome(PRRS)is a highly contagious infectious disease caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV).PRRS is widespread worldwide.Although various measures have been taken to control PRRSV infection,the virus continues to cause economic problems in the pig industry around the world.In this study,the PRRSV ORF2 sequence was shortened and synthesized,and the prokaryotic expression vector p ET-30a-ORF2 was obtained,and the expression was induced.The recombinant GP2 protein was successfully expressed by SDS-PAGE gel electrophoresis.The purified recombinant GP2 protein was immunized to mice,and the positive hybridoma cell lines were screened.The antibody was prepared by in vivo induction method and purified,and the monoclonal antibody of recombinant GP2 protein was successfully obtained.It lays the foundation for the study of the structure and function of PRRSV GP2 protein,and also provides technical reserves for future scientific research on PRRS.At the same time,this study used the PRRSV full-length infectious clone p JXwn06 as a template to amplify the ORF2 gene sequence by PCR and clone it into a lentiviral vector to obtain the recombinant plasmid p LV-EF1a-EGFP-2A-GP2.The recombinant lentiviral particles were transfected by lentiviral packaging system and transduced into Marc145 cells.Screening was carried out with a medium containing puromycin,and the cells after the initial screening were cloned by the limiting dilution method,and the cell line was finally obtained.The stable expression of GP2 protein in the cell line was verified by PCR and Western blot experiments.In this study,the Marc145-GP2 cell line stably expressing PRRSV GP2 protein was successfully established,which laid the foundation for studying the immunological properties and functions of GP2 protein.
Keywords/Search Tags:Porcine reproductive and respiratory syndrome virus(PRRSV), ORF2 gene, Prokaryotic expression, GP2 protein, Monoclonal antibody, Cell line
PDF Full Text Request
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