EB病毒潜伏膜蛋白2A基因重组逆转录病毒载体和稳定表达细胞株的构建及鉴定  

Construction and identification of recombinant retroviral vector and stable cell line expressing latent membrane protein 2A of Epstein-Barr virus

作　　者：陈云[1,2] 周锋[1] 孙倍成[2] 刘根焰[1] 王冰[1] 姚堃[1] 

机构地区：[1]南京医科大学微生物与免疫学系江苏省病原重点实验室,江苏南京210029 [2]南京医科大学第一附属医院肝脏外科卫生部活体肝移植重点实验室,江苏南京210029

出　　处：《细胞与分子免疫学杂志》2009年第11期1013-1015,1019,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30170880;30772003);江苏省"九五"攻关课题(BJ98100);江苏省重点人才资助项目(RC2007057);南京医科大学科技发展基金重点项目(NMUZ009)

摘　　要：目的:克隆EB病毒(EBV)潜伏膜蛋白2A(LMP2A)全长基因,并构建含有该目的基因的重组逆转录病毒载体pGEZ-LMP2A,转染真核细胞后获得稳定表达LMP2A的细胞株。方法:采用RT-PCR技术从B95.8细胞中获得EB病毒LMP2A全长基因,克隆到测序载体pMD18-T中,经测序后再亚克隆到逆转录病毒载体pGEZ-Term中,与两辅助病毒载体PHIT456、PHIT60通过Lipofectamine2000共转染包装细胞293T,测定产生的重组逆转录病毒滴度并感染小鼠成纤维细胞L929,经Zeocine筛选后得到稳定的细胞克隆,用RT-PCR和Western blot法检测细胞中LMP2A mRNA和蛋白表达情况。结果:重组逆转录病毒载体pGEZ-LMP2A经测序鉴定正确;转染后上清中病毒的滴度为5×108cfu/L,感染L929细胞后用Zeocine筛选出稳定的细胞克隆;RT-PCR和Westernblot检测结果表明筛选出的细胞克隆能稳定表达EBV-LMP2A。结论:表达EBV-LMP2A细胞株的构建为蛋白制备与纯化奠定了物质基础,也为后续的EBV相关性疾病疫苗的研究提供了新思路。AIM： To clone EBV-LMP2A gene, construct and identify the recombinant retroviral vector and stable cell strains expressing EBV LMP2A. METHODS： The full-length EBV LMP2A gene was generated by RT-PCR amplification from 1395.8 cells which contain complement nucleotide sequence of EBV LMP2A gene. The gene was ligated to T- vector and sequenced to construct retroviral vector consisting with LMP2A. To produce retroviral virus, packing cells, 293T cells were co-transfected with recombinant retroviral expression vector pGEZ-LMP2A and two auxiliary viral vectors pHIT456 and pHIT60 by lipofectAMINE2000. Viral titration was performed according to the instructions of the manufacturer. To establish L929 cell line stable expressing LMP2A, L929 cells were infected with recombinant retrovirus three times and selected by Zeocine. The Zeocine-resistant clones （L929/LMP2A） were screened for LMP2A expression by RT-PCR and Western blot. RESULTS： The recombinant retrovirus vector carrying LMP2A gene was constructed successfully. Transfection yield a titer of .5 ×10^8 infectious particles/L. The infected L929 cells were selected by Zeocine. Results of RT-PCR and Western blot indicated that L929 transgenetic cells could stably express EBV- LMP2A. CONCLUSION： The L929 cell line stably expressing LMP2A provides suitability for extraction of the LMP2A protein and preparations of the vaccine for the therapy of EBV-associated diseases.

关 键 词：EB病毒 LMP2A 逆转录病毒 重组 

分 类 号：R373.9[医药卫生—病原生物学] R730.3[医药卫生—基础医学]