伪狂犬病毒的gE蛋白胞外区真核表达以及单克隆抗体制备

doi:10.13560/j.cnki.biotech.bull.1985.2019-0310

摘要/Abstract

摘要： 旨在构建gE基因胞外区真核表达载体,在HEK293F细胞中表达并纯化得到稳定的可溶性蛋白,并通过杂交瘤筛选获得表达gE蛋白的特异性单克隆抗体。生物信息学方法分析gE基因序列,构建gE胞外区真核表达载体pcDNA3.4-gE-6×His和pcDNA3.4-gE-mFc,通过瞬时转染的方法在HEK293F细胞中进行表达并进一步纯化。通过Western blot和SDS-PAGE鉴定和比较gE-6×His和gE-mFc融合蛋白表达情况。利用伪狂犬灭活全病毒免疫小鼠,电融合获得杂交瘤融合细胞,通过gE-mFc蛋白和IFA筛选出稳定且特异性结合gE蛋白的阳性杂交瘤细胞株。结果表明,成功构建pcDNA3.4-gE-6×His和pcDNA3.4-gE-mFc表达载体,表达纯化得到gE-6×His和gE-mFc可溶性蛋白。比较发现gE-6×His蛋白表达量低,稳定性差。而gE-mFc蛋白表达量高,稳定性好,一次性纯化后纯度可达85%。进一步利用gE-mFc筛选获得9株稳定性和特异性高的阳性杂交瘤细胞株。首次利用哺乳动物细胞表达系统表达并获得稳定的可溶性gE蛋白,并利用其筛选获得gE特异性单克隆杂交瘤细胞株。

关键词: 伪狂犬病毒, gE, 真核表达, 单克隆抗体

Abstract: This work aims to construct eukaryotic expression vector of the extracellular domain of gE gene for generating stable soluble gE protein in HEK293F cells,and to obtain specific monoclonal antibody of gE by hybridoma screening. Bioinformatics was used to analyze the gE gene sequence,and the expression vectors pcDNA3.4-gE-6×His and pcDNA3.4-gE-mFc were constructed and then transfected into HEK293F cells via transient transfer method for the expression and further purification. Western blot and SDS-PAGE were used to identify and compare the fusion protein gE-6×Hia and gE-mFc. Pseudorabies inactivated whole virus was used to immunize the mice,hybridoma fusion cells were obtained by electrofusion,positive hybridoma cell lines stably and specifically binding to gE protein were screened by gE-mFc protein and IFA. As results,the pcDNA3.4-gE-6×His and pcDNA3.4-gE-mFc expression vectors were successfully constructed and the gE-6×His and gE-mouse Fc soluble proteins were obtained after expression and purification. The results of SDS-PAGE and Western blot showed that gE-mFc had a higher expression level and better stability than gE-6×His,and the purity reached 85% after only one purification. Further,9 stably and specifically hybridoma positive cell lines were screened using gE-mFc protein. In sum,this is the first time that stable and soluble gE protein is expressed by mammalian cell expression system,and by which gE specific monoclonal hybridoma cell lines are screened,laying a fine material foundation for the development of pseudorabies diagnostic reagents.

Key words: pseudorabies virus, gE, eukaryotic expression, monoclonal antibody

郎巧利, 吴梦, 黄楠, 何琦琳, 葛良鹏, 杨希. 伪狂犬病毒的gE蛋白胞外区真核表达以及单克隆抗体制备[J]. 生物技术通报, 2019, 35(11): 96-103.

LANG Qiao-li, WU Meng, HUANG Nan, HE Qi-lin, GE Liang-peng, YANG Xi. Eukaryotic Expression of Extracellular Domain of Pseudorabies Virus gE Protein and Preparation of Monoclonal Antibodies[J]. Biotechnology Bulletin, 2019, 35(11): 96-103.

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