Prokaryotic Expression Of Vp7 Gene From Human Group B Rotavirus And Generation Of Antibody

Rotavirus,belonging to the family Reovoridae, is the main cause of severe, dehydrating diarrhea in infants and young children, leading to significant morbidity and mortality worldwide. Human group B rotavirus became well known following major outbreaks of watery diarrhea in adults in the People's Republic of China that have occurred since 1982. To date, these outbreaks have been confined to China, and occur up to the present. Comparated with group A rotavirus, Human group B rotavirus was more severe,so it was widely researched in the world. In this study, The ORF of vp7 gene was amplified by PCR from the vp7 gene sequence of Group B rotavirus. The PCR product was cloned into the expression vector pGEX-KG. After induction with IPTG, The purified fusion protein was then used to immunize the New Zealand rabbits to obtain the polyclonal antibody against GST-VP7. Western Blot analysis using the polyclonal antibody derived from the rabbits indicated that these antibodies could react with the target protein. The preparation of the polyclonal antibody against VP7 lay a foundation for the further study of VP7 expression at protein levels and its function. The research contents are as follows:Chapter one: a brief review on recent advance on Rotavirus. It focuses on the fundamental researches and vaccine developed.Chapter two: a report on prokaryotic expression of structure Protein genes from Group B rotavirus and generation of specific antibody. A pair of primers were designed according to the vp7 gene of human group B rotavirus stain WH-2.The ORF of vp7 gene was amplified by PCR using the primer pair from the cloning plasmid pUCmT-vp7 of human Group B rotavirus stain WH-2. The GST fusion expression vector of the VP7 pGEX-KG-VP7 was constructed by cloning the PCR product of vp7 gene ORF into the pGEX-KG. and was transformed into E.coli. The transformed E.coli was induced with IPTG to express GST-VP7 fusion protein. The purified fusion protein was then used to immunize the New Zealand rabbits to produce the polyclonal antibody against VP7. The sequence of VP7 in the recombinant pGEX-KG-VP7 was confirmed by restriction endonuclease digestion and sequence analysis. SDS-PAGE analysis showed that fusion protein GST-VP7 was expressed in E.coli with a molecular weight of 53.4kDa. Western Blot analysis using the polyclonal antibody derived from the rabbits indicated that these antibodies could react with the target protein.