H3N2流感病毒非结构蛋白-1真核表达载体的构建与表达及其对293T细胞增殖的影响

Cloning and eukaryotic expression of non-structural protein-1 of a human influenza virus subtype H3N2 and the effect of non-structural protein-1 on proliferation of 293T cell

目的:构建甲型H3N2流感病毒非结构蛋白-1(nonstructal-1,NS1)真核表达载体并表达其编码蛋白;探讨NS1对细胞增殖的影响。方法 :从江苏省甲型H3N2流感病毒毒株提取病毒RNA,采用RT-PCR技术扩增NS1全长基因,将其克隆至pMD18-T Simple Vector中构建pMD18-T/NS1质粒,双酶切pMD18-T/NS1与pXJ40-HA后,构建真核表达载体pXJ40-HA-NS1,经酶切及测序鉴定后将质粒转染到293T细胞中,通过免疫印迹法鉴定NS1蛋白的表达。3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide,MTT]法检测NS1转染细胞后对细胞增殖的影响。结果:经双酶切、测序鉴定证实NS1基因的真核表达载体构建成功;免疫印迹法证实NS1蛋白的表达。MTT法证实转染NS1质粒后对细胞增殖有抑制作用。结论:成功克隆了NS1全长基因,构建了其真核表达载体,并初步验证了NS1蛋白过表达后能抑制细胞的增殖,该表达载体的构建为后期建立稳定表达NS1的细胞模型和NS1蛋白对细胞增殖和凋亡作用机制的进一步研究提供了基础。

Objective：To construct the full-length nonstructal-1（NS1） gene of influenza A（H3N2） into a eukaryotic expression vector and express NS1 gene in mammalian cells,as well as to investigate the effects of NS1 on proliferation of 293T cells.Methods：The NS1 gene of influenza A（H3N2） was amplified by RT-PCR and cloned into pMD18-T simple vector to construct a plasmid,named pMD18-T/NS1.the pMD18-T/NS1 and the pXJ40-HA were double-digested,to yield the recombinant eukaryotic expression vector pXJ40-HA-NS1.The expression of the NS1 gene in transfected 293T cells was identified by Western blot.The effects of NS1 gene on cell proliferation were measured with 3-（4,5）-dimethylthiahiazo（-z-y1）-3,5-di-phenytetrazoliumromide（MTT） method.Results： The recombinant eukaryotic expression vector pXJ40-HA-NS1 was successfully constructed.The expression of NS1 protein was detected by Western blot.NS1 over-expression inhibited the proliferation of 293T cells.Conclusion： The full-length NS1 gene has been obtained and its recombinant eukaryotic expression vector has been successfully constructed.And we found that the proliferation was dramatically inhibited in 293T cells over-expressed with NS1 protein.The construction of eukaryotic expression plasmid of NS1 gene made it possible to set up the cell model expressing NS1 protein and further study the effects of NS1 protein on cell proliferation and apoptosis.