EB病毒LMP1-CTAR3对NP69细胞增殖和蛋白质表达的影响  

Influence of Epstein-Barr Virus Encoded Latent Membrane Protein 1-CTAR_3 for Proteinic Expression and Cellular Proliferation of NP69 Cells

作　　者：张志伟[1,2] 张琼[1] 余艳辉[1] 欧阳咏梅[1] 贺智敏[1] 

机构地区：[1]中南大学湘雅医学院肿瘤研究所,长沙410078 [2]南华大学医学院肿瘤研究所,衡阳421001

出　　处：《生物化学与生物物理进展》2009年第5期580-586,共7页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(30470668);湖南省自然科学基金(07JJ6164);湖南省卫生厅科研基金(B2006-100);湖南省重点科学建设经费资助项目~~

摘　　要：为了探讨EB病毒潜伏性膜蛋白1(LMP1)第三个功能活性区域(CTAR3)在鼻咽上皮细胞NP69中的转化作用机制,采用逆病毒感染的方法,将浓缩的逆病毒RV-LMP1和RV-LMP1△232～351分别感染鼻咽上皮细胞NP69,建立NP69-LMP1与NP69-LMP1△232～351稳定表达细胞系.通过绘制生长曲线、平皿克隆形成试验和软琼脂集落形成试验比较野生型和突变型LMP1对NP69细胞增殖的影响,运用蛋白质组学方法鉴定NP69-LMP1与NP69-LMP1△232～351细胞间的差异表达蛋白,选用实时荧光定量RT-PCR与Western blot对其中部分蛋白质点差异表达进行验证.结果发现:a.突变型LMP1△232～351促NP69细胞增殖的能力较野生型LMP1明显降低(n=3,P<0.05);b.鉴定了LMP1-CTAR3在NP69细胞中参与调节的16个蛋白质(表达上调的蛋白质8个,下调的8个).c.实时荧光定量RT-PCR和Westernblot证实了部分上述蛋白质的差异表达.以上结果说明,LMP1-CTAR3是其发挥促细胞增殖的重要活性部位,可能通过参与调节G蛋白和异柠檬酸脱氢酶等蛋白质的表达而起作用.To investigate transduction mechanism on carboxyl terminal activating region 3 of Epstein - Barr virus encoded latent membrane protein 1（LMP1） in nasopharyngeal epithelial cells NP69, the NP69 cell lines were respectively infected, applied the approach of retroviruses infection, by RV-LMP1 and RV-LMP1^Δ252～351 retroviruses. Therefore, the NP69-LMP1 and NP69-LMP1^Δ252～351 cell lines of stable expression LMP1 were established. Sequentially, cellular proliferation of the NP69-LMP1 and NP69-LMP1^Δ252～351 cell lines was compared to draw growth curve, experiment plate clone formation and test soft agar colony forming. Meanwhile, adopted proteomic methods, the differential expression proteins were identified between NP69-LMP1and NP69-LMP1^Δ252～351 cell lines, and the expression levels of partial identified proteins were verified by Western blotting and fluorescent real-time quantitative RT-PCR. The results showed： 1） the ability of LMP1^Δ252～351 promoting NP69 cell proliferation was obviously decreased to compare with wild type LMP1（n=3,P 〈 0.05）. 2） 16 proteins, 8 up- and 8 down-regulated ones, of LMP1-CTAR3 mediated regulation were identified from infected NP69 cell lines. 3） The differential expression of partial identified proteins was similar with 2-DE separated ones and confirmed by Western blotting and fluorescent real-time quantitative RT-PCR. These results suggested that LMP1-CTAR3 is the important activating domain of inducing cellular proliferation and the domain probably plays the role of mediating to regulate the expression of G-protein, isocitric enzyme, and so on.

关 键 词：EB病毒 潜伏性膜蛋白1 羧基端功能活性区域3 差异表达蛋白 

分 类 号：R318.0[医药卫生—生物医学工程]