诺如病毒P-partical-GnIH抗原制备及抗原性初步检测

doi:10.16431/j.cnki.1671-7236.2019.09.025

Abstract

Abstract: In order to explore the technique of regulating the reproductive performance of domestic geese,this study designed a synthetic goose gonadotropin-inhibitory hormone (GnIH) sequence fragment,which was digested and ligated to Norovirus (Nov) P gene recombinant pEGX-4T-1 vector,transformed into E.coli DH5α competent cells,extracted positive bacterial recombinant plasmid,and verified by sequencing and enzyme digestion.The correctly sequenced plasmid was transformed into E.coli BL21 competent cells,and recombinant protein expression was induced by IPTG.The expression of recombinant protein was detected by SDS-PAGE and Western blotting.The prokaryotic expression was further carried out under different IPTG concentration and induction time conditions,and the optimal induction conditions were determined.Under the optimal conditions,the induced bacterial liquid was collected by centrifugation,and the protein was analyzed by ultrasonic disruption and centrifugation.The recombinant protein was purified using xTractor Buffer and Glutathione Superflow resin,and the recombinant protein was detected by Western blotting using self-made anti-GnIH goose serum and anti-goose secondary antibody.The results showed that the recombinant plasmid Nov P-partical-GnIH was identified by double restriction enzyme digestion to obtain the target gene band of about 96 bp.The sequencing results confirmed that the recombinant plasmid was successfully constructed.The molecular weight of the recombinant protein induced by expression was about 64 ku,which was consistent with the expected size,and the highest expression was obtained at 37℃,1.0 mmol/L IPTG and induction for 2 h.The recombinant protein was detected by SDS-PAGE after sonication.Mainly expressed in the precipitate.The recombinant protein reacted with both GST monoclonal antibody and anti-GnIH positive serum,indicating that the recombinant protein had good reactogenicity and immunogenicity.In this study,a large number of highly purified recombinant proteins were successfully obtained by prokaryotic expression and purification of Nov P-partical-GnIH protein,which would help to further establish the reproductive regulation of domestic geese and lay the foundation for studing on improving reproductive performance and regulation reproduction mechanism.

Key words: Nov P-partical; gonadotropin-inhibitory hormone(GnIH); protein immunogenicity; vaccine

CLC Number:

S853.74

HUANG Xuebing, YANG Peijie, DENG Rusen, YANG Bo, LIU Wenjun, HUANG Yunmao. Preparation of P-partical-GnIH Antigen of Norovirus and Preliminary Detection of Its Antigenicity[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2699-2706.

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Preparation of P-partical-GnIH Antigen of Norovirus and Preliminary Detection of Its Antigenicity

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