牛病毒性腹泻病毒NS5A蛋白原核表达及多克隆抗体制备

蓝秋菊; 马晓霞; 安乐乐; 刘倩芸; 刘伊铭; 张丽娟; 刘俊林

牛病毒性腹泻病毒NS5A蛋白原核表达及多克隆抗体制备

Prokaryotic Expression of NS5A Protein and Preparation of Polyclonal Antibodies of Bovine Viral Diarrhea Virus

摘要

摘要:
目的通过原核表达系统表达牛病毒性腹泻病毒（bovine viral diarrhea virus, BVDV）非结构蛋白5A（non-structural protein 5A, NS5A），并制备其多克隆抗体，为BVDV的防控提供技术支撑。
方法利用生物信息学软件对BVDV NS5A蛋白理化性质进行分析；构建pET-28a-NS5A重组质粒并转化至感受态BL21细胞，诱导表达并通过尿素变性处理后使用Ni²⁺-NTA亲和层析法纯化NS5A蛋白；将重组蛋白与佐剂乳化，多次免疫BALB/c小鼠制备多克隆抗体；通过间接ELISA方法检测鼠源多克隆抗体效价，并用间接免疫荧光（indirect immunofluorescence assay, IFA）和Western blot进行鉴定。
结果生物信息学分析结果显示，NS5A蛋白不存在信号肽和跨膜结构域，为亲水性蛋白；成功构建原核表达质粒pET-28a-NS5A，诱导表达成功获得重组蛋白NS5A，分子质量约为58 kDa，通过尿素变性及Ni^{2 +}-NTA亲和层析纯化后得到纯度较高的重组蛋白NS5A。间接ELISA检测结果显示效价达到1:409 600；Western-blot和IFA 鉴定结果显示，该多克隆抗体具有良好的反应性及特异性。
结论本研究成功制备了特异性强的NS5A多克隆抗体，为BVDV的基础研究和临床诊断奠定了基础。

Abstract:
Objective Prokaryotic expression of bovine viral diarrhea virus (BVDV) non-structural protein 5A(NS5A) protein was obtained to prepare a polyclonal antibody against the disease.
Methods Physicochemical properties of BVDV NS5A were analyzed using bioinformatics software. The recombinant plasmid pET-28a-NS5A was constructed and transformed into competent E. coli BL21 cells. NS5A was induced for expression, followed by urea denaturation and purification via Ni²⁺-NTA affinity chromatography. The purified recombinant protein was emulsified with adjuvant to immunize BALB/c mice multiple times for generating polyclonal antibodies. Titer of the antibody was determined by indirect ELISA, and properties characterized by indirect immunofluorescence assay (IFA) and western blot.
Results The hydrophilic NS5A lacked a signal peptide and transmembrane domains. The successfully constructed prokaryotic expression plasmid pET-28a-NS5A was transformed to obtain the recombinant NS5A with an approximate molecular weight of 58 kDa. After urea denaturation and Ni²⁺-NTA affinity chromatography, highly purified recombinant NS5A was used to prepare polyclonal antibodies with a titer exceeded 1:409 600. High reactivity and specificity of the antibody was confirmed by western blot and IFA.
Conclusion The successfully prepared polyclonal antibody was highly specific against NS5A. It made further research as well as development of clinical diagnostic means of BVDV possible.

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