蓝舌病病毒NS4蛋白原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.07.032

摘要/Abstract

摘要： 【目的】制备蓝舌病病毒（Bluetongue virus，BTV） NS4蛋白多克隆抗体，为BTV NS4基因在病毒增殖及颉颃宿主细胞天然免疫应答中的作用研究奠定基础。【方法】构建pCzn1-NS4原核表达载体，经PCR和测序鉴定后将阳性质粒转化大肠杆菌BL21（DE3）感受态细胞，用IPTG诱导表达，使用His标签镍柱纯化NS4蛋白，并经SDS-PAGE、Western blotting鉴定。利用NS4蛋白免疫新西兰大白兔制备多克隆抗体，经SDS-PAGE和间接ELISA法测定多克隆抗体纯度及效价，以制备的NS4蛋白多克隆抗体为一抗，通过间接免疫荧光试验（IFA）检测BTV感染的BHK-21细胞内NS4蛋白的表达。【结果】成功构建了原核表达质粒pCzn1-NS4，在37 ℃、IPTG浓度为0.6 mmol/L时诱导8 h获得大量NS4重组蛋白，SDS-PAGE及Western blotting试验结果显示，重组蛋白分子质量约为18 ku，可与兔His-tag抗体发生特异性反应；制备的NS4蛋白多克隆抗体效价在1：512 000以上。经SDS-PAGE和IFA鉴定，制备的BTV NS4多克隆抗体具有良好的纯度，且在BTV感染的BHK-21细胞中能特异性识别病毒表达的NS4蛋白。【结论】本研究利用原核表达的NS4蛋白制备了特异性多克隆抗体，并初步将该多克隆抗体用于NS4蛋白免疫荧光试验研究，为BTV NS4基因的功能和应用研究提供参考依据。

关键词: 蓝舌病病毒(BTV); NS4蛋白; 原核表达; 多克隆抗体

Abstract: 【Objective】 The purpose of this study was to prepare polyclonal antibody against Bluetongue virus (BTV) NS4 protein and lay a foundation for studying the role of BTV NS4 gene in virus proliferation and antagonizing the innate immune response of host cells.【Method】 The pCzn1-NS4 prokaryotic expression vector was constructed, and the positive plasmid was transformed into Escherichia coli BL21(DE3) competent cells after identification by PCR and sequencing.The recombinant NS4 protein was induced by IPTG and purified with His-tag Ni-sepharose purification, and then identified by SDS-PAGE and Western blotting.Then, New Zealand White rabbits were immunized with the purified protein to prepare its polyclonal antibody.The titer of polyclonal antibody was determined by SDS-PAGE and indirect ELISA.The expression of NS4 protein in BHK-21 cells infected with BTV was detected by indirect immunofluorescence assay (IFA) using NS4 polyclonal antibody as the primary antibody.【Result】 The prokaryotic expression plasmid pCzn1-NS4 was successfully constructed.At 37 ℃ and the induced concentration of IPTG was 0.6 mmol/L, a large amount of recombinant NS4 protein was obtained when induced 8 h.SDS-PAGE and Western blotting results showed that the molecular weight of the recombinant protein was about 18 ku, which could detect by rabbit His-tag antibody.The titer of polyclonal antibody prepared by immunizing New Zealand white rabbits was more than 1:512 000 detected by indirect ELISA.SDS-PAGE and IFA results showed that the polyclonal antibody had good purity and could specifically recognize the NS4 protein expressed in BHK-21 cells infected by BTV.【Conclusion】 In this study, a specific polyclonal antibody was prepared using the prokaryotic expression of NS4 protein, and the polyclonal antibody was preliminarily used for the IFA, which provided a powerful tool for the function and application study of BTV NS4 gene.

Key words: Bluetongue virus (BTV); NS4 protein; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65

罗世美, 赵瑶, 马鲜平, 张金阳, 易华山. 蓝舌病病毒NS4蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(7): 2923-2930.

LUO Shimei, ZHAO Yao, MA Xianping, ZHANG Jinyang, YI Huashan. Prokaryotic Expression and Polyclonal Antibody Preparation of Bluetongue Virus NS4 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2923-2930.

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