单纯疱疹病毒2型潜伏相关转录体RL1的表达及其抗凋亡作用

生物技术通报 ›› 2014, Vol. 0 ›› Issue (3): 187-192.

• 研究报告 • 上一篇

单纯疱疹病毒2型潜伏相关转录体RL1的表达及其抗凋亡作用

高睿迪¹, 杨慧兰¹, 钟菲菲², 吕芳彪²

(1.南方医科大学，广州 510515；2.广州军区广州总医院皮肤科，广州 510010)

收稿日期:2013-12-10 出版日期:2014-03-29 发布日期:2014-03-31
作者简介:高睿迪，女，研究生，研究方向:皮肤性病学；E-mail:854170648@qq.com
基金资助:
国家自然科学基金项目(81171511)

The Expression and Anti-apoptotic Function of Herpes Simplex Virus Type 2 Latency Associated Transcript-RL1

Gao Ruidi¹, Yang Huilan¹, Zhong Feifei², Lü Fangbiao²

(1. Southern Medical University，Guangzhou 510515；2. Genenral Hospital of Guangzhou Military command of PLA，Guangzhou 510010)

Received:2013-12-10 Published:2014-03-29 Online:2014-03-31

摘要/Abstract

摘要： 单纯疱疹病毒2型潜伏相关转录体(LAT)-RL1对放线菌素D诱导的凋亡作用的研究。构建重组pEGFP-RL1质粒，转染Vero细胞，RT-PCR及荧光鉴定重组质粒的表达。放线菌素D诱导Vero细胞凋亡，通过Hochest33342荧光染色观察细胞形态变化，流式细胞术检测细胞凋亡率，JC-1荧光观察膜电位变化，Caspase 3 凋亡蛋白检测。RT-PCR和荧光观察表明该真核表达载体能在Vero细胞中高表达。Hochest33342染色转染了pEGFP-RL1的Vero细胞经放线菌素D凋亡诱导后，细胞形态正常。流式结果表明转染重组质粒pEGFP-RL1且经诱导凋亡组与正常对照组凋亡率无差异，而显著低于诱导凋亡组和转染空质粒pEGFP-C2诱导凋亡组。转染重组质粒pEGPF-RL1的细胞JC-1染色后，红色细胞的比例要远大于绿色细胞的比例，而转染空质粒pEGFP-C2被染成绿色细胞的数量较多。Caspase-3结果表明转染了空质粒 pEGFP-C2 的Vero细胞经诱导凋亡后，活性显著高于转染了 pEGFP-RL1 经放线菌素D诱导凋亡后的Vero细胞和正常Vero细胞。HSV-2 LAT RL1 具有抗放线菌素D诱导的Vero细胞的凋亡作用。

关键词: 单纯疱疹病毒, LAT, RL1, 放线菌素D, 凋亡, Vero细胞

Abstract: It was to study the expression of herpes simplex virus type 2 latency-associated transcript(LAT)RL1 and its anti-apoptosis function induced by actinomycin D in vero cells. The recombinant plasmid pEGFP-ORF1 was constructed and transfected into Vero cells, expression was determined by green fluorescent protein and RT-PCR .The changes of Vero cells morphology induced by Actinomycin D were observed by Hochest33342 fluorescence staining, cells apoptosis rate was detected by flow cytometry . Membrane potential JC-1 fluorescence was observed, and Caspase 3 detected of apoptosis protein activity. Results showed that the green fluorescent protein has a high expressed efficiency in Vero cells, and target gene was detected by RT-PCR. Hochest33342 staining reavealed that Vero cells transfected with pEGFP-RL1 and induced apoptosis by actinomycin D had no changes in morphology. Flow cytometry assay showed that the cells apoptosis rate were no significant difference between pEGFP-RL1group and the normal group, but the cells apoptosis rate of pEGFP-RL1 was remarkable lower than the pEGFP-C2 group. Transfected with the recombinant plasmid pEGPF-RL1 cells by JC-1 staining, the proportion is much larger than red cells green cells transfected with empty vector pEGFP-C2. Caspase-3 results indicate that induction of apoptosis by transfected with empty vector pEGFP-C2 Vero cells, and the activity was significantly higher than those transfected with pEGFP-RL1 by actinomycin D-induced apoptosis of Vero cells and normal cells. HSV-2 LAT RL1 gene can effectively expressed in Vero cells and can protect Vero cells from apoptosis induced by actinomycin D.

Key words: Herpes simplex virus, LAT, RL1, Actinomycin D, Apoptosis,Vero cells

高睿迪, 杨慧兰, 钟菲菲, 吕芳彪. 单纯疱疹病毒2型潜伏相关转录体RL1的表达及其抗凋亡作用[J]. 生物技术通报, 2014, 0(3): 187-192.

Gao Ruidi, Yang Huilan, Zhong Feifei, Lü Fangbiao. The Expression and Anti-apoptotic Function of Herpes Simplex Virus Type 2 Latency Associated Transcript-RL1[J]. Biotechnology Bulletin, 2014, 0(3): 187-192.

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单纯疱疹病毒2型潜伏相关转录体RL1的表达及其抗凋亡作用

The Expression and Anti-apoptotic Function of Herpes Simplex Virus Type 2 Latency Associated Transcript-RL1

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