摘要
潜伏膜蛋白1(Latent membrane protein 1,LMP1)是由γ疱疹病毒亚科的EB病毒基因编码的能够使正常细胞发生恶性转化的跨膜信号蛋白,C-端为LMP1的主要功能区。通过PCR从B95-8细胞和鼻咽癌活检组织中获得lmp1和lmp1 C-端30bp缺失(lmp1 C-terminal deleted,lmp1-ctd)基因;利用体外基因重组技术,将lmp1和lmp1-ctd分别插入AAV质粒载体中;应用Lipofectamine介导转染HEK-293包装细胞,获得含lmp1和lmp1-ctd的rAAV;再以rAAV感染猴肾上皮细胞。利用RT-PCR检测lmp1和lmp1-ctd在猴肾上皮细胞中的转录;MTT法、流式细胞仪技术检测细胞生长和DNA合成,探讨lmp1和lmp1-ctd对其在细胞中活性的影响。结果显示:lmp1在转化细胞中转录,导致细胞生长速度加快,其中lmp1-ctd转化细胞的生长速度和处于增殖期比例高于lmp1转化细胞。提示lmp1-ctd较lmp1具有更强的促细胞增殖作用。
Latent membrane protein 1 （LMP1） encoded by Epstein-Barr virus gene is a kind of transmembrane signal protein causing cell malignancy. LMP1 C terminus is essential for its transforming activities. Both Lmp 1 and lmp 1 C-terminal 30-base pair deleted （lmp 1-ctd） genes were respectively extracted from B95-8 cell and nasopharyngeal carcinoma biopsy by polymerase chain reaction （PCR）. These genes were separately inserted into adenovirus-associated virus （AAV） plasmid vector with DNA recombinant technique. The recombinant AAVs （rAAVs） each including lmp1 and lmp1-ctd were assembled in HEK-293 package cell line under the help of lipofectamine transfection reagents. The epithelial cells of monkey kidney were infected by rAAVs. The transcription of lmp1 and lmp1-ctd in monkey kidney cells was determined by RT-PCR. The growth and DNA synthesis of infected epithelial cells of monkey kidney were examined with MTT method and flow cytometry technology. The effect of lmp1-ctd on cell proliferation was discussed. Our data suggested mRNAs of lmp1 and lmp1-ctd genes could be detected in the infected epithelial cells of monkey kidney. The growth of infected monkey kidney epithelial cells was strongly promoted by lmp1 and lmp1-ctd. More cells infected by rAAV including lmp 1-ctd transited quiescent phase to proliferation phase than cells including lmp1 did. These results indicated lmp 1-ctd has stronger activity to promote cell propagation.
出处
《病毒学报》
CAS
CSCD
北大核心
2006年第6期440-444,共5页
Chinese Journal of Virology
基金
国家自然科学基金(30270520)
国家科技攻关项目(2003BA712A01)
关键词
γ疱疹病毒亚科
EB病毒潜伏膜蛋白1
C-端缺失
细胞增殖
Gammaherpesvirinae
Epstein-Barr virus
latent membrane protein 1
C-terminal deleted
cell proliferation
作者简介
杜海军（1970-），男，河北唐山人，硕士，助理研究员，主要从事肿瘤病毒致癌机制和鼻咽癌免疫治疗研究。
通讯作者：周玲，研究员 E-mail：zhouling@ht．roc．cn．net
