表达猪流行性腹泻病毒N蛋白Vero细胞系的构建及初步鉴定

Construction and Preliminary Identification of Vero Cell Line Expressing N Protein of Porcine Epidemic Diarrhea Virus

【目的】构建表达猪流行性腹泻病毒(PEDV)N蛋白的Vero细胞系,将有助于PEDV的进一步深入研究。【方法】本研究将N基因克隆到慢病毒载体中,构建重组质粒pLenti-CMV-N,利用慢病毒载体包装系统转染293T细胞,包装表达N蛋白的慢病毒颗粒,将慢病毒颗粒转导至Vero细胞,用潮霉素B初步筛选阳性细胞,采用终点稀释法,筛选表达PEDV N蛋白的Vero细胞系,最后进行鉴定。【结果】RT-PCR结果表明构建的细胞系基因组中存在N基因序列,Western blot和IFA试验验证了N蛋白在细胞系中的表达。【结论】本研究成功构建了表达PEDV N蛋白的Vero细胞系。

【Objective】The construction of Vero cell line expressing porcine epidemic diarrhea virus(PEDV)N protein will contribute to the further study of PEDV.【Methods】N gene was cloned into lentiviral vector to construct recombinant plasmid pLenti-CMV-N,which was transfected into 293T cells by lentiviral vector packaging system in this study.The lentivirus particles expressing N protein were packaged and transferred to Vero cells.The positive cells were initially screened by hygromycin B.The Vero cell lines expressing PEDVN protein were initially screened by end point dilution method,and finally identified.【Results】The result of RT-PCR showed that there was a N gene sequence in the genome of the constructed cell line.Western blot and IFA tests confirmed the expression of N protein in the cell line.【Conclusion】A Vero cell line expressing PEDVN protein was successfully constructed by our research.