高致病性禽腺病毒血清4型Hexon蛋白的原核表达及多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2023.05.032

摘要/Abstract

摘要： 【目的】原核表达禽腺病毒血清4型(Fowl adenovirus serotype 4,FAdV-4)Hexon蛋白,并制备兔抗Hexon多克隆抗体,为FAdV-4 Hexon功能研究提供材料。【方法】采用基于PCR的长DNA序列准确合成(PCR-based accurate synthesis,PSA)方法,设计全长拼接引物,在引物两端各设计保护性碱基合成FAdV-4 Hexon基因;利用同源重组技术将其连接至pCzn1-Hexon表达载体,获得pCzn1-Hexon重组质粒进行原核表达,采用纯化重组蛋白免疫新西兰白兔制备兔抗Hexon蛋白的多克隆抗体。以间接ELISA方法测定多克隆抗体效价;通过Western blotting和间接免疫荧光试验(IFA)鉴定多克隆抗体的特异性。【结果】重组原核表达质粒pCzn1-Hexon经双酶切及测序鉴定证明构建正确;获得的重组蛋白以包涵体的形式表达,分子质量约为41 ku;用纯化的重组蛋白免疫新西兰白兔后,通过间接ELISA方法检测抗体效价高达1∶512 000。以制备的多克隆抗体为一抗,通过Western blotting和IFA检测到鸡肝癌细胞(LMH)中的Hexon蛋白表达,表明制备的多克隆抗体能与Hexon蛋白发生特异性反应。【结论】FAdV-4 Hexon蛋白在大肠杆菌中实现了高效表达,制备并纯化的抗Hexon多克隆抗体具有较高的反应性和特异性,该研究为进一步探究Hexon蛋白的生物学功能及其在FAdV-4感染和致病过程中的作用奠定了基础。

关键词: 禽腺病毒4型; Hexon蛋白; 原核表达; 多克隆抗体

Abstract: 【Objective】 The purpose of this study was to express the Hexon protein of Fowl adenovirus serotype 4 (FAdV-4) by prokaryotes expressed system and prepare its polyclonal antibody,so as to provide materials for the study of FAdV-4 Hexon function.【Method】 The method accurate synthesis of long DNA sequences based on PCR (PAS) was used to design the full-length spline primer,and protective bases were designed on both ends of the primer to synthesize FAdV-4 Hexon gene.It was connected to the pCzn1-Hexon expression vector by homologous recombination technology,and the pCzn1-Hexon recombinant plasmid was obtained for prokaryotic expression.The purified recombinant protein was used to immunize New Zealand White rabbits to prepare rabbit polyclonal antibody against Hexon protein.The titer of polyclonal antibody was determined by indirect ELISA,and the specificity of polyclonal antibody was identified by Western blotting and indirect immunofluorescence assay (IFA).【Result】 The recombinant prokaryotic expression plasmid pCzn1-Hexon was confirmed to be constructed correctly by double restriction enzyme digestion and sequencing.The obtained recombinant protein was expressed in the form of inclusion body,and its molecular weight was about 41 ku.After immunizing New Zealand White rabbits with purified recombinant protein,the antibody titer was up to 1∶512 000 by indirect ELISA.Using the prepared polyclonal antibody as the first antibody,the expression of Hexon protein in chicken LMH cells was detected by Western blotting and IFA,indicating that the prepared polyclonal antibody could react specifically with Hexon protein.【Conclusion】 The recombinant Hexon protein had been highly expressed in Escherichia coli,and the prepared and purified anti-Hexon polyclonal antibody showed high reactivity and specificity,which laid a foundation for in-depth analysis of the biological function of Hexon protein and its role in FAdV-4 infection and pathogenesis.

Key words: Fowl adenovirus serotype 4; Hexon protein; prokaryothic expression; polyclonal antibody

中图分类号:

S852.659.1

王雪平, 张孝俊, 李晓月, 王国栋, 张福良, 宋玉伟, 张明亮, 马磊. 高致病性禽腺病毒血清4型Hexon蛋白的原核表达及多克隆抗体的制备[J]. 中国畜牧兽医, 2023, 50(5): 2053-2060.

WANG Xueping, ZHANG Xiaojun, LI Xiaoyue, WANG Guodong, ZHANG Fuliang, SONG Yuwei, ZHANG Mingliang, MA Lei. Prokaryotic Expression of Hexon Protein of Highly Pathogenic Fowl Adenovirus Serotype 4 and Preparation of Polyclonal Antibodies[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2053-2060.

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