Ei sh单纯疱疹病毒1型ICP34.5在Vero细胞的 表达及对Bax/Bcl-2的影响

摘要:目的　构建单纯疱疹病毒1 型（HSV-1）真核表达载体pmR-mcherry-ICP34.5，验证ICP34.5对宿主细胞Bax、Bcl-2 mRNA相对表达量及对Bax/Bcl-2的影响。方法　以提取HSV-1病毒DNA为模版，PCR扩增HSV-1 ICP34.5序列，双酶切连接至pmR-mcherry真核表达载体上，并对重组真核表达载体进行验证，然后重组载体瞬时转染Vero细胞，mRNA水平表达用逆转录PCR方法检测，融合蛋白的表达的检测用荧光显微镜，MTT检测对Vero的细胞活性，喜树碱诱导凋亡后，实时定量PCR检测Bax、Bcl-2 mRNA在细胞中的相对表达量。结果　转染后RT-PCR 验证有目的基因的转录，荧光显微镜观察到融合蛋白在转染的Vero细胞中表达。重组质粒可以抵消空质粒对细胞的损伤作用，转染重组质粒的Vero细胞和正常细胞的中Bax mRNA表达量和Bcl-2 mRNA表达量相差不大，但Bax/Bcl-2得比值明显低于转染pmR-mcherry空质粒的Vero细胞和正常加药的Vero细胞。结论　pmR-mcherry-ICP34.5 真核表达载体能在Vero细胞中高效表达，并能减弱空质粒转染对细胞活性作用，ICP34.5能使Vero细胞中Bax/Bcl-2稳定性增强，使细胞抗凋亡。

Expression of HSV-1ICP34.5 eukaryotic expression vector and its effect on Bax/Bcl-2 in vero cells

Abstract: Objective To construct herpes simplex virus type 1 (HSV-1) eukaryotic expression plasmid pmR-mcherry-ICP34.5. To observe the effects of pmR-mcherry-ICP34.5 on the relative value of Bax and Bcl-2 mRNA in Vero cells. Methods The target sequence of ICP34.5 gene was obtained from HSV-1 genome, amplified by PCR, and then cloned into a eukaryote plasmid pmR-mcherry after restrictive endonucleases digestion. The construction of pmR-mcherry-ICP34.5 was verified by double digestion and DNA sequence analysis. Vero cells were transiently transfected with pmR-mcherry-ICP34.5 in vitro. The expression of fusion protein was observed by inverted fluorescence microscope, and its expression was identified by RT-PCR. The cells viability was evaluated by MTT assay. After camptothecin-induced apoptosis of Vero, the levels of Bax and Bcl-2 mRNA were measured by real time PCR. Results PmR-mcherry-ICP34.5 fusion protein expression was observed after transfection. RT-PCR showed that the target gene was highly expressed in Vero cells. MTT assay showed that the viabilities of Vero cells transfected with recombinant plasmidpmR-mcherry-ICP34.5 had no statistically significant difference compared with the untreated normal control group, but remarkable higher than Vero cells transfected with empty plasmid pmR-mcherry (P<0.05). Bax and Bcl-2 had no statistically significant difference in Vero cells transfected with pmR-mcherry-ICP34.5 and normal Vero, and compared with transfected with pmR-mcherry Bax/Bcl-2 and Vero cells induced culture was significantly decreased. Conclusion Recombinant plasmid can effectively expressed in Vero cells and can offset cells injury caused by empty plasmid. PmR-mcherry-ICP34.5 could stabilize Bax/Bcl-2 in Vero cells and can protect Vero cells from apoptosis.