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  

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

作　　者：闪海霞[1] 范崇桂[1] 张怀宏[1] 霍丽亚[1] 翟玉峰[1] 

机构地区：[1]河南省南阳市中心医院感染性疾病科,河南南阳473000

出　　处：《现代预防医学》2015年第21期3969-3972,共4页Modern Preventive Medicine

摘　　要：目的构建单纯疱疹病毒1型(HSV-1)真核表达载体pm R-mcherry-ICP34.5,验证ICP34.5对宿主细胞Bax、Bcl-2 m RNA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pm R-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,m RNA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 m RNA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 m RNA表达量和Bcl-2 m RNA表达量相差不大,但Bax/Bcl-2得比值明显低于转染pm R-mcherry空质粒的Vero细胞和正常加药的Vero细胞。结论 pm R-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。Objective To construct herpes simplex virus type 1（HSV-1） eukaryotic expression plasmid pm R-mcherry-ICP34.5. To observe the effects of pm R-mcherry-ICP34.5 on the relative value of Bax and Bcl-2 m RNA 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 pm R-mcherry after restrictive endonucleases digestion. The construction of pm R-mcherry-ICP34.5 was verified by double digestion and DNA sequence analysis. Vero cells were transiently transfected with pm R-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 m RNA were measured by real time PCR. Results Pm R-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 plasmidpm R-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 pm R-mcherry（P〈0.05）. Bax and Bcl-2 had no statistically significant difference in Vero cells transfected with pm R-mcherry-ICP34.5 and normal Vero, and compared with transfected with pm R-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. Pm R-mcherry-ICP34.5 could stabilize Bax/Bcl-2 in Vero cells and can protect Vero cells from apoptosis.

关 键 词：单纯疱疹病毒1型 ICP34.5 VERO BAX BCL-2 

分 类 号：R117[医药卫生—公共卫生与预防医学]