重组含有Epstein-Barr病毒潜伏膜蛋白1(LMP1)基因的杆状病毒在昆虫细胞中的表达

用杆状病毒表达系统重组病毒，在昆虫细胞中表达了完整的含有ＥＢＶ－ＬＭＰ１基因３个外显子开放读码框架的长２．３ｋｂ的ｃＤＮＡ片段。用重组病毒感染Ｓｆ９细胞，用免疫荧光染色，结果表明：４８小时表达重组蛋白，７２小时细胞较完整，免疫荧光染色强阳性，９６小时后细胞出现破碎。我们采集７２小时的组织培养上清和细胞破碎裂解液，分别采用ＳＤＳ－ＰＡＧＥ、ＨＰＬＣ分子筛法，用免疫蛋白印迹法实验证明，表达的蛋白能被抗ＬＭＰ１的单克隆抗体所识别，测定表达蛋白的分子量为６０ｋＤ。经蛋白含量扫描图分析，采用Ｓｅｐｈａｄｅｘ－７５柱初步纯化表达的ＬＭＰ１蛋白，将后者进行裸鼠体内致瘤实验，未见肿瘤生长。

A STUDY ON THE EXPRESSION OF EPSTEIN-BARR VIRUS LATENT MEMBRANE PROTEIN IN INSECT CELLS

Using the baculovirus expression vector system,Epstein-Barr virus latent membrane protein1(EBV-LMP1)was expressed in the insect cell,which contained a 2.3kb cDNA fragment encoded by three exon ORFs.The recombinant virus was used to infect Sf9 cell and the immunofluorescent test indicated that the recombinant protein was produced after 48 hours of infection,the cells were kept intact and showed strong positivity by the immunofluorescent test after 72 hours,but the cells were broken down after 96 hours.Collecting the supernatant and cell lysate after 72 hours,the SDS-PAGE and SEC-HPLC respectively revealed that the MW of the expressed protein was 60 kD,Western-blot showed that the protein can be recognized by LMP1 monoclonal antibody.After analysing the expressed protein by Image-Master VDS and purifying initially with Sephadex-75, the purified recombinant LMP1 protein in the tumor-promoted study of nude mice did not induce tumors.