分泌表达牛病毒性腹泻病毒E0蛋白MDBK细胞的稳定性分析

Stability Analysis of MDBK Cell Line Secretory Expressing Bovine viral diarrhea virus E0 Protein

目的:构建分泌表达牛病毒性腹泻病毒(BVDV)E0蛋白的MDBK细胞系,并探究其传代和冻存稳定性。方法:用实验室制备的BVDV E0基因重组慢病毒感染MDBK细胞,荧光观察并收集MDBK细胞培养上清进行Western印迹分析,通过qPCR方法检测重组细胞系中重组基因拷贝数变化。结果:IgK信号肽组和预测信号肽组均分泌表达E0蛋白;通过Oligo6.0软件设计BVDV E0基因SYBR GreenⅠ染料法荧光定量PCR检测引物,经过条件优化,未出现非特异性扩增,检测下线为20拷贝/μL;通过建立的荧光定量PCR和流式分析对2组细胞系进行传代稳定性分析,结果2组细胞系在第12代之后荧光率显著下降,15代之后E0基因拷贝数显著下降,2组细胞冻存前后荧光率未发现显著变化。结论:构建了稳定分泌表达BVDV E0蛋白的MDBK细胞系,建立了BVDV E0基因定量检测方法,为研究E0蛋白的生物学功能、探索BVDV的防控措施以及开发基因工程诊断抗原和基因工程亚单位疫苗奠定了基础。

Objective:To evaluate the passage and freezing stability of MDBK cell line secretory expression Bovine viral diarrhea virus(BVDV)E0protein.Methods:Recombinant lentivirus,which packed in our library,were used to infect MDBK cell.Western blot analysis of MDBK cell culture supernatant was used to detect secretion of E0protein in IgK signal peptide group and predicted signal peptide group.Oligo6.0software was used to design a pair of primer for SYBR GreenⅠquantitative RT-PCR.Results:After reaction condition optimization,the detection limit of this quantitative PCR method was20copies/μL.Quantitative RT-PCR and flow cytometric method were used to analyze the passage and freezing stability.Passage stability analysis showed that fluorescence rate and E0gene copies numbers were decreased significantly after twelfth and fifteenth generation,respectively.And there was no significant change of fluorescence rate before and after freezing.Conclusion:Two kinds of MDBK cell lines secretory expressing BVDV E0protein were successfully constructed and quantitative PCR method for detection of BVDV E0gene was established,which not only laid the foundation for the study of biological function of E0protein,provided a new way to prevention and control this disease,but also provided a chance to investigate the possibility of been used as genetic engineering diagnostic antigen and gene engineering subunit vaccine of BVDV E0protein.