单纯疱疹病毒2型潜伏相关转录本开放读码框的表达及其抗凋亡功能分析  

Expression of Herpes Simplex Virus Type 2 Latency-associated Transcript Open Reading Frames and Its Anti-apoptotic Effects

作　　者：钟菲菲 杨慧兰[1] 吕芳彪[1,2] 薛礼长[1,2] 白利利[1,2] 樊建勇[1] 

机构地区：[1]广州军区广州总医院,广州510010 [2]华南理工大学生物科学与工程学院,广州510006

出　　处：《中国生物工程杂志》2013年第10期36-43,共8页China Biotechnology

基　　金：国家自然科学基金资助项目(30972666;81071401;81171511)

摘　　要：目的:研究单纯疱疹病毒2型潜伏相关转录体(LAT)开放读码框(ORF)对凋亡诱导剂诱导Vero细胞凋亡的影响。方法:PCR扩增LAT ORF1、ORF2、ORF3片段,构建重组质粒pEGFPORF1、pEGFP-ORF2、pEGFP-ORF3,重组质粒pEGFP-ORF1、pEGFP-ORF2、pEGFP-ORF3转染Vero细胞;RT-PCR鉴定ORF1、ORF2、ORF3的表达。用凋亡诱导剂放线菌素D、顺铂、5-氟尿嘧啶(5-FU)诱导Vero细胞凋亡,通过荧光显微镜观察细胞形态学的改变,MTT检测细胞活性,流式细胞术检测细胞凋亡率。结果:双酶切和测序确认pEGFP-ORF1、pEGFP-ORF2、pEGFP-ORF3构建成功,RT-PCR表明这3个真核表达载体能在Vero细胞中高效表达。转染各种pEGFP-ORF的Vero细胞经凋亡诱导剂诱导后,细胞形态正常。MTT结果表明转染了各种重组质粒pEGFP-ORF的Vero细胞经凋亡诱导剂诱导后Vero细胞活性与未经任何处理的正常对照组相比,差异无统计学意义(P>0.05),但高于凋亡诱导剂诱导凋亡的Vero细胞组及与转染空质粒pEGFP-C2,且经凋亡诱导剂诱导的Vero细胞组,差异具有统计学意义(P<0.05)。流式细胞术结果表明,转染各种重组质粒pEGFP-ORF且经凋亡诱导剂诱导凋亡组与正常对照组凋亡率差异无统计学意义(P>0.05),而显著低于转染空质粒pEGFP-C2且经凋亡诱导剂诱导凋亡组,差异有统计学意义(P<0.05)。结论 HSV-2 LAT ORF对凋亡诱导剂诱导的Vero细胞的凋亡具有抵抗作用。Objective：To study the effects of herpes simplex virus type 2（HSV-2） latency-associated transcript（LAT） open reading frames（ORFs） on Vero cells which were induced apoptosis by agent. Methods：PCR amplified LAT ORF1, ORF2, ORF3 fragment,then construct recombinant plasmid pEGFP-ORF1,pEGFP-ORF2,pEGFP-ORF3,transfect them into Vero cells and identify its expression by RT-PCR.Using actinomycin D,5-FU and cisplatinere to induce Vero cells apoptosis,the changes of Vero cells morphology was observed by fluorescence microscopy.MTT assay was used to evaluate Vero cells viability and flow cytometry was used to calculate the cells apoptosis rate. Results：The pEGFP-ORF1,pEGFP-ORF2,pEGFP-ORF3 was constructed successfully which was confirmed by Double digestion and sequencing. Vero cells morphology were normal when transfected with pEGFP-ORFs and induced by apoptosis-inducing agent. MTT assay showed that the viabilities of Vero cells in recombinant plasmid group were no significant difference compared with the normal control group（P〉 0.05）, but remarkable higher than empty plasmid pEGFP-C2 group,the difference was significant（P〈0.05）.Flow cytometry assay showed that the cells apoptosis rate in pEGFP-ORFs group were no significant difference compared with the normal control group（P〉 0.05）,but was remarkable lower than the pEGFP-C2 group（P〈0.05）. Conclusion HSV-2 LAT ORFs gene can effectively expressed in Vero cells and can protect Vero cells from apoptosis.

关 键 词：单纯疱疹病毒 LAT ORFs 凋亡诱导剂 凋亡 VERO细胞 

分 类 号：Q819[生物学—生物工程]