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Prokaryotic Expression of vp7 Gene from Human Group B Rotavirus and Generation of Antibody

Author: HuYingHui
Tutor: YangJiHong
School: Central China Normal University
Course: Microbiology
Keywords: Group B rotavirus VP7 gene Clone GST fusion protein expression Antibody preparation
CLC: R392
Type: Master's thesis
Year: 2007
Downloads: 70
Quote: 0
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Abstract


Rotavirus (Rotavirus, RV) for solitary virus family Reoviridae, Rotavirus has been recognized as a cause of the most common cause of acute gastroenteritis of infants and young children worldwide. In adults, rotavirus will also cause acute diarrhea, including adult diarrhea rotavirus (ADRV) Hongtao academicians in China first discovered in 1983, a human group B rotavirus in many parts of our country, it has caused scale based adult cholera-like diarrhea outbreak. Human group B rotavirus infection, transmission intensity, cause clinical symptoms are very serious, far beyond the group A rotavirus has aroused extensive attention of scholars and research. Of this study in vitro amplification of human group B rotavirus WH-2 strains vp7 gene ORF District constructed recombinant pGEX-KG-VP7 induced by IPTG, SDS-PAGE analysis, expression pGEX-KG-VP7 fusion protein and preparation of a polyclonal antiserum, Western-blot and preliminarily purified protein identification results in the predicted corresponding molecular weight at a specific reaction with, which confirmed that the fusion protein has a good immunological activity. Protein expression and antibody prepared not only for the study of the structure and function of the material basis and can also be used to build people GBRV ELISA method, laid the epidemiological studies and clinical diagnosis of the disease caused by GBRV prevention, diagnosis and treatment foundation has important practical value. Specific studies as follows: the first comprehensive overview of the morphological structure of the group B rotavirus, Advances in the study of the physical and chemical properties, genome structure and encoded protein, cultured in vitro, epidemiological, laboratory testing and clinical diagnosis and treatment . Chapter II VP7 structural protein gene was cloned into the vector pGEX-KG expression, purification and polyclonal antibody preparation. From containing human group B rotavirus WH-2 strains of full-length gene pUCmT-vp7 vp7 recombinant plasmid on human group B rotavirus WH-2 the vp7 genes lines ORF district amplified, and the amplified products were inserted fusion expression vector constructed recombinant pGEX-KG-VP7 plasmid pGEX-KG. The recombinant plasmid was transformed into E. coli BL21 by IPTG induction, by SDS-PAGE analysis showed that expression of the fusion protein of relative molecular weight of 53.4kDa, pGEX-KG-VP7. PGEX-KG-VP7 fusion protein as an antigen to immunize New Zealand rabbits, and polyclonal antibody. 1:500 dilution antiserum prepared by the same induced expression of the expression vector pGEX-KG expression product absorption by Western Blot analysis, after the fusion protein to obtain a specific color signal and 53.4kDa the pGEX-KG-VP7, which confirmed that the The fusion protein have good immunological activity.

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