新型H7N9禽流感病毒NA蛋白胞外区片段的生物信息学分析及其多克隆抗体制备

Bioinformatics Analysis of the Extracellular Region of NA Protein in Novel H7N9 Avian Influenza Virus and Preparation of Polyclonal Antibodies

旨为以原核表达系统表达、纯化新型H7N9禽流感病毒(安徽株)NA蛋白胞外区片段并制备该蛋白的多克隆抗体。对新型H7N9禽流感病毒神经氨酸酶(Neuraminidase,NA)蛋白胞外区片段进行生物信息学分析,基于大肠杆菌密码子偏爱性进行密码子优化并合成该蛋白编码基因。将构建的重组质粒pET28b-tN9(Truncated N9)转化至E.coli BL21(DE3)、E.coli Rosetta和E.coli Arctic Express(DE3),进行诱导表达,并进行SDS-PAGE鉴定。选取E.coli BL21(DE3)重组菌进行放大培养、IPTG诱导并对诱导产物进行镍柱纯化、SDS-PAGE分析和质谱鉴定。将纯化的重组蛋白免疫新西兰大白兔,制备多克隆抗体,以Western blotting和间接ELISA法检测抗体特异性及效价。成功表达并纯化目标蛋白,Western blotting显示制备的多克隆抗体能特异性识别重组蛋白和新型H7N9禽流感病毒(上海株),间接ELISA方法检测制备的多克隆抗体,效价为1∶256000。利用原核表达系统高效表达并纯化了tN9蛋白,制备的多克隆抗体效价高,特异性强,旨为深入探索NA蛋白结构及功能、H7N9禽流感病毒致病机制和建立快速检测方法奠定基础。

This work aims to express and purify the extracellular region of NA(neuraminidase)protein in novel H7N9 avian influenza virus(Anhui Strain)in prokaryotic expression system and to prepare its polyclonal antibodies.Firstly,bioinformatics analysis of the extracellular region of NA protein in novel H7N9 avian influenza virus was conducted.The codon optimization was performed according to the codon preferences in the Escherichia coli expression system,and the gene encoding this protein was synthetized.Subsequently the recombinant plasmid pET28b-tN9(truncated N9)carrying chemically synthesized extracellular region of NA gene was transformed into E.coli BL21(DE3),E.coli Rosetta and E.coli Arctic Express(DE3)and their expressions were induced with IPTG,and the SDS-PAGE identification was conducted.The recombinant E.coli BL21(DE3)was cultured for more mass,induced by IPTG,the induced products were purified by Ni column,analyzed using SDS-PAGE,and identified by mass spectrometry.The purified protein was used to immunize rabbit to prepare polyclonal antibodies.Western blotting and an indirect ELISA assay were performed to determine specificity and titer of the polyclonal antibody.As results,the recombinant protein was successfully expressed and purified.Western blotting analysis showed that the prepared polyclonal antibody specifically recognized the recombinant protein and H7N9 avian influenza viru(s Shanghai Strain).The titer of the antibody was about 1:256000 detected by indirect ELISA.In conclusion,the specific polyclonal antibodies with high titer were successfully prepared by immunizing the rabbit using the purified tN9 protein in in prokaryotic expression system,laying a foundation for further studying the structure and function of the NA protein,pathogenesis and building rapid detection methods of H7N9 avian influenza virus.