H9N2亚型禽流感病毒NP蛋白原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2022.10.029

Abstract

Abstract: 【Objective】 This study was aimed to express NP protein of an epidemic H9N2 subtype Avian influenza virus (AIV) strain isolated from South China using prokaryotic expression system,and prepare its polyclonal antibody.【Method】 The NP gene of a H9N2 strain was cloned into pET-32a(+) vector to construct the prokaryotic expression plasmid of NP protein.The recombinant plasmid was transformed into both the Escherichia coli(E.coli) BL21(DE3) and Rosetta (DE3) strains for expression.The recombinant protein was induced by IPTG.Coomassie bright blue staining and Western blotting were used to analyze and compare the expression of NP protein in the two kinds of expressing bacteria.Furthermore,the concentration of IPTG,induction time,and induction temperature were optimized to improve the protein production.By affinity chromatography,the recombinant protein was purified using a Ni-NTA column,and the concentration was determined using BCA assay.The polyclonal antibody was obtained from New Zealand,which had been inoculated with the purified protein.Western blotting and indirect immunofluorescence assay were used to identify the polyclonal antibody,the antibody titer was determined using indirect ELISA.【Result】 The prokaryotic expression plasmid pET-32a-H9N2-NP was constructed and the combinant NP protein was expressed successfully.Coomassie bright blue staining and Western blotting results revealed that the recombinant protein was expressed at a much higher level in E.coli Rosetta(DE3) than that in E.coli BL21(DE3),and its size was about 73 ku.The optimal induction conditions showed that the expression of recombinant NP protein reached the maximum at 37 ℃ and IPTG concentration of 1 mmol/L for 7 h.The results of Western blotting and indirect immunofluorescence assay demonstrated that the prepared polyclonal antibody could bind specifically to H9N2 subtype AIV at a high titer.By indirect ELISA detection,the titer was about 1∶409 600.【Conclusion】 The polyclonal antibody against NP protein prepared from rabbits had a higher titer,indicating that NP protein had good immunogenicity.The results laid the foundation for the preparation of monoclonal antibodies against NP protein and establishing the ELISA detection method.

Key words: H9N2 subtype; Avian influenza virus(AIV); NP protein; prokaryotic expression; polyclonal antibody

CLC Number:

S855.3

YANG Jing, ZHANG Xinyu, LIANG Zhipeng, CHENG Qing, WANG Congying, CHI Shihong, YUAN Sheng, GUO Jinyue, HUANG Shujian, WEN Feng. Prokaryotic Expression of NP Protein of H9N2 Subtype Avian Influenza Virus and Preparation of Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3963-3971.

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Prokaryotic Expression of NP Protein of H9N2 Subtype Avian Influenza Virus and Preparation of Polyclonal Antibody

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