Expression, Purification and Antigenicity of P Domain and Its Variant of Monkey GII.17 Norovirus Genome

doi:10.3969/j.issn.1674-5817.2016.05.002

Abstract

Abstract: Objective To express P domain and its variant of monkey GII.17 norovirus genome, purify protein and analyze antigenicity. Methods The gene of P domain was synthesized, following designation based on the nucleotide sequence that was obtained from monkey GII.17 norovirus genome and bias of E. coli. PCR with these specific primers was performed to amplify P domain and make its variant, followed by cloning into prokaryotic expression vector via in-Fusion ligation. Protein expression was induced by IPTG, purified through GST affinity column and analyzed by Coomassie staining, immunoblot and ELISA assay. Results Sequencing results showed that P domain and its variant were successfully cloned into the expression vector. Analysis of SDS-PAGE revealed that P domain was observed in both the supernatant and pellet, and its variant was mostly expressed in the supernatant. The expressed and purified proteins were further confirmed by immunoblot analysis via the anti-GST antibody. The results of immunoblot analysis and ELISA assay indicated the good antigenicity of the proteins. The ELISA titers of antibodies induced by these proteins were 1:32000. Conclusion The P domain and its variant of monkey GII.17 norovirus genome were successfully expressed. The antibodies were obtained after immunization of mice with these two proteins. The findings of this study pave the way for the development of viral detection kit, viral identification and assay of virus-receptor binding.

Key words: Monkey, Norovirus genome, P domain, Protein expression, Antigenicity

CLC Number:

Q95-33

LIU Bo, LI Chao, TAO Yu-fen, LI Xin-tong, LIU Jian-sheng, HE Zhan-long, LIU Hong-qi. Expression, Purification and Antigenicity of P Domain and Its Variant of Monkey GII.17 Norovirus Genome[J]. Laboratory Animal and Comparative Medicine, 2016, 36(5): 334-339.

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