Culture of human sapovirus GⅠ.1 in vitro and preparation of polyclonal antibody against capsid protein VP1 / 中国生物制品学杂志

ABSTRACT

@#Objective To culture human sapovirus(HuSaV) GⅠ.1 in vitro and prepare polyclonal antibody against the capsid protein VP1.Methods HuSaV GⅠ.1 positive stool specimens preserved in diarrhea department of National Institute for Viral Disease Control and Prevention were inoculated with HuTu-80 cells supplemented with different bile acid salts[glycine chenodeoxycholic acid(GCDCA) and glycine cholic acid(GCA)],and the infection,proliferation and passage of the virus were determined by PCR and RT-qPCR.The VP1 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1.The constructed recombinant expression plasmid pGEX-6P-1-VP1 was transformed into E.coli BL21(DE3) and induced by IPTG.Two female New Zealand white rabbits were immunized with the purified recombinant VP1 protein for 4 times.The blood samples were collected 18,28,38 and 48 d after immunization,and the serum titers were detected by ELISA.Results HuTu-80 cells were effectively infected by HuSaV GⅠ.1 in the presence of bile acid salt GCA,and the proliferated virus were stably and continuously transmitted for three generations in HuTu-80 cells.The expressed recom-binant GST-VP1 protein showed a relative molecular mass of about 86 000,and about 60 000 after purification(GST tag excision).The titer of polyclonal antibody against HuSaV VP1 protein was over 1:12 800.Conclusion HuSaV was successfully isolated and cultured in vitro using HuTu-80 cells supplemented with bile acid salt,and polyclonal antibody with high titer against HuSaV VP1 protein was prepared,which laid a foundation of in-depth research of HuSaV identification,infection and pathogenesis.