牛传染性鼻气管炎病毒gI蛋白截短表达与单克隆抗体制备
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S855.3

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内蒙古农业大学高层次人才引进项目(DC1900008441)


Truncated Expression of GI Protein of Infectious Bovine Rhinotracheitis Virus and Preparation of Monoclonal Antibody
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    摘要:

    为获得牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)gI蛋白单克隆抗体,构建了gI截短基因重组原核表达质粒pET-32a-gI,并对其进行了诱导表达和纯化,通过SDS-PAGE和Western blot验证了gI蛋白在大肠杆菌内的表达情况。将纯化的gI蛋白免疫BALB/c小鼠,通过细胞融合、筛选及亚克隆,得到针对IBRV gI蛋白的单克隆细胞株gI14。利用体内诱生法制备抗IBRV gI蛋白单克隆抗体腹水,使用Western blot和间接免疫荧光(IFA)对单克隆抗体特异性进行检测和验证。Western blot结果显示:原核表达的gI融合蛋白相对分子质量为34 ku,gI蛋白单克隆抗体与gI表达蛋白反应性良好,与pET-32a(+)空载体蛋白无特异性结合;天然表达的gI蛋白相对分子质量为45 ku,gI蛋白单克隆抗体与细胞内感染的IBRV所表达的gI蛋白反应性良好。IFA结果显示,gI蛋白单克隆抗体及阳性对照组IBRV多抗均能与细胞内感染的IBRV发生特异性结合,使细胞出现特异性绿色荧光信号,而空白及阴性对照组均未见绿色荧光信号。结果表明,本研究基于高效表达的gI蛋白,成功制备了1株能稳定表达gI单克隆抗体的细胞株gI14,获得的gI蛋白单克隆抗体与原核表达的gI蛋白及IBRV全病毒均能特异性结合,这为进一步研制IBRV诊断试剂盒及探究IBRV gI蛋白抗原表位及蛋白功能奠定了基础。

    Abstract:

    In order to obtain the monoclonal antibody of gI protein of infectious bovine rhinotracheitis virus(IBRV),a recombinant prokaryotic expression plasmid pET32a-gI with truncated gI gene was constructed,induced,expressed and purified,the expression of gI protein in E.coli. was verified by SDS-PAGE and Western blot. BALB/C mice were immunized with purified gI protein,and then the monoclonal cell line gI14 targeting IBRV gI was obtained by cell fusion,screening and sub-cloning. Monoclonal antibody ascites against IBRV gI protein was prepared by the in vivo induction method to test and verify the specificity of IBRV gI monoclonal antibody fluid by Western blot and indirect immunofluorescence assay(IFA). The results showed that,by Western blot,the relative molecular weight of the prokaryotic expressed gI fusion protein was 34 ku,the monoclonal antibody against gI protein reacted well with the gI expression protein,and failed to specifically bind to pET-32a(+)empty body protein;the relative molecular weight of the naturally expressed gI protein was 45 ku,and the monoclonal antibody of gI protein reacted well with the gI protein expressed by intracellular infected IBRV;and by IFA,both the monoclonal antibody against gI protein and the positive control group's IBRV polyclonal antibody could specifically bind to the intracellular infected IBRV,resulting in specific green fluorescence signals in the cells,while no green fluorescence signals were found in the blank and negative control groups. In conclusion,based on the highly expressed gI protein,one cell strain gI14 that could stably express gI monoclonal antibody was successfully prepared in this study,the obtained gI protein monoclonal antibody could specifically bind to the prokaryotic expressed gI protein and IBRV whole virus,which laid a foundation for further development of any IBRV diagnostic kits as well as research on the antigen epitope and protein function of IBRV gI protein.

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侯丽娜,于明华,刘春羽,赵洪哲,郑秉武,温永俊,王凤雪.牛传染性鼻气管炎病毒gI蛋白截短表达与单克隆抗体制备[J].《中国动物检疫》编辑部,2022,39(8):112-119.

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