2型单纯疱疹病毒毒力蛋白ICP34.5的真核表达及其对Ve-ro细胞活性的影响

目的:在非洲绿猴肾细胞(Vero细胞)中表达2型单纯疱疹病毒(HSV-2)毒力蛋白感染细胞多肽34.5(ICP34.5),并检测其对Vero细胞活性的影响。方法:PCR扩增HSV-2的ICP34.5基因,连接至pEGFP-C2载体,并对重组真核表达载体pEGFP-ICP34.5进行双酶切测序验证;将重组子瞬时转染Vero细胞,RT-PCR检测其在mRNA水平的表达,荧光倒置显微镜观察融合蛋白的表达,MTT法检测细胞活性。结果:经双酶切和测序验证表明pEGFP-ICP34.5构建成功,转染细胞后经RT-PCR验证有目的基因的转录,荧光显微镜下观察到融合蛋白在转染的Vero细胞中表达,MTT法检测结果证实重组质粒可以抵消空质粒对细胞的损伤作用。结论:构建了pEGFP-ICP34.5真核表达载体,其能在Vero细胞中高效表达,并能抵消空质粒对细胞的损伤作用。

Eukaryotic Expression of HSV-2 ICP34.5 and its Effect on Vero Cells Viability

Objective： To construct eukaryotic expression plasmid pEGFP-ICP34.5 of infected cell protein 34.5 （ICP34.5） of herpes simplex virus type 2（HSV-2）, evaluate its expression in Veto cells, and detect its effect on Vero cells viability. Methods： The target sequence of ICP34.5 gene was obtained from HSV-2 genome, amplified by PCR, and then cloned into a eukaryote plasmid pEGFP-C2 after restrictive endonucleases digestion. The con struction of pEGFP-ICP34.5 was verified by double digestion and DNA sequence analysis. Vero cells were tran siently transfected with pEGFP-ICP34.5 in vitro. The expression of fusion protein was observed by inverted fluores cence microscope and its mRNA expression was identified by RT-PCR. The Veto cells viability was evaluated by MTT assay. Results： Double digestion and sequencing confirmed that the pEGFP-ICP34.5 was constructed success- fully. ICP34.5 fusion protein expression was observed after transfection. RT-PCR showed that the target gene was highly expressed in Veto cells. MTT assay showed that the viabilities of Vero cells transfected with recombinant plasmid pEGFP-ICP34.5 has no statistically significant difference compared with the untreated normal control group, but remarkable higher than Veto cells transfeeted with empty plasmid pEGFP-C2. Conclusion： Recombi- nant plasmid pEGFP-ICP34.5 was constructed successfully and can effectively expressed in Veto ceils, and can offset cells injury caused by empty plasmid.