核苷二磷酸激酶B稳定表达细胞系的建立及其对新城疫病毒F48E9株复制的影响

摘要

为研究核苷二磷酸激酶B(nucleoside diphosphate kinase B,NDPK B)蛋白表达对新城疫病毒(Newcastle disease virus,NDV)F48E9株复制与增殖的影响,本试验将真核表达重组质粒pEGFP-NDPK B转染Vero细胞,经G418压力筛选出了阳性细胞单克隆,并通过RT-PCR、Western blotting和倒置荧光显微镜观察鉴定了NDPK B mRNA和蛋白质的表达,在细胞系基础上,通过HA-HI、TCID50及实时荧光定量PCR检测NDPK B蛋白对NDV F48E9株复制和增殖的影响.结果表明,试验成功地构建了高表达NDPK B蛋白的Vero/NDPK B细胞系,并在此基础上,通过检测证明了NDPK B能抑制NDV F48E9株在Vero细胞中的复制与增殖,提示以NDPK B为基础有可能设计开发抗NDV的新药物或抗病毒增效剂,对NDV进行预防或治疗.%In order to explore the influence of nucleoside diphosphate kinase B(NDPK B)on replication and proliferation of F48E9 strain of Newcastle disease virus(NDV),the eukaryotic expression plasmid pEGFP-NDPK B was transfected into Vero cells,then we got the cell subclones by G418 screening.We identified the expression of NDPK B protein by RT-PCR,Western blotting and inverted flurescence microscope.On the basis of Vero/NDPK B cell line,we explored the influence of NDPK B on replication and proliferation of F48E9 strain of NDV by HA-HI,TCID50 and Real-time PCR.The results showed that we had successfully constructed the cell line named Vero/NDPK B,which could stably express NDPK B protein,and found that NDPK B could inhibit the replication and proliferation of F48E9 strain of NDV in Vero cells.It suggested that on the basis of NDPK B,we could design and develop new drugs or antiviral synergist to prevent or treat NDV.