一种新的遍在蛋白融合基因Uba256的克隆及表达  

Cloning and Expression of a Novel Ubiquitin Fusion Gene Uba256

作　　者：李朝飞[1] 李充璧[1] 汤慕瑾[1] 尹崇[1] 龚映雪[1] 庞义[1] 

机构地区：[1]中山大学生物防治国家重点实验室,昆虫学研究所,广州510275

出　　处：《生物化学与生物物理学报》2003年第4期360-365,共6页

基　　金：国家自然科学基金资助项目 (No .3 0 170 0 40 )~~

摘　　要：斜纹夜蛾核多角体病毒 (Spodopteralituramulticapsidnucleopolyhedrovirus ,SpltMNPV)的Uba2 5 6基因是已知昆虫病毒基因组中惟一编码遍在蛋白与GP37融合蛋白的基因。通过PCR方法扩增得到该基因及其N端的遍在蛋白编码区与C端的GP37编码区。将这 3个基因片段分别克隆至质粒pBV2 2 0与pQE30 ,构建得到重组质粒pB VUBCP、pQEUB与pQECP。pBVUBCP转化大肠杆菌DH5α ,经热激诱导表达了一条 38kD的蛋白带 ,证明Uba2 5 6表达的蛋白在大肠杆菌中没有发生剪切。pQEUB、pQECP转化大肠杆菌M15 ,经IPTG诱导 ,Western印迹分析结果表明遍在蛋白与GP37蛋白获得高效表达。以Ni2 + NTA偶联抗体检测证明所表达的蛋白质均为融合蛋白质。纯化的融合蛋白质免疫新西兰大白兔可诱导产生特异抗体。ELISA和Western印迹检测结果显示 ,SpltMNPV遍在蛋白抗体及GP37抗体均与表达的融合蛋白质呈阳性反应 ,表明所表达的融合蛋白质仍保持原有蛋白质的免疫原性。以获得的抗体对感染SpltMNPV 72h的Sl zsu 1细胞进行检测 ,发现仅有一条 34kD的蛋白质带与GP37抗体发生特异反应 ;而有 4条分子量分别为 14、2 4、33、5 1kD的蛋白质带与遍在蛋白抗体发生特异反应 ,表明Uba2 5 6基因表达的蛋白质在宿主细胞内发生了剪切。Spodoptera litura nucleopolyhedrovirus (SpltMNPV) Uba256 gene is the only Ub-gp37 fusion gene in the genome of insect viruses. With the specific primers designed for Uba256 gene that was reported recently, the coding regions of Uba256, N-terminal ubiquitin and C-terminal GP37 that lacking the signal sequence, were amplified from SpltMNPV genomic DNA by PCR. The Uba256 coding region was expressed using the expression vector pBV220, and a band of 38 kD was detected with Western blot analysis, indicating that ubiquitin-GP37 fusion protein did not undergo post-transcriptional processing in E. coli . The ubiquitin and GP37 coding region were highly expressed, respectively, using pQE30 expression vector. Antibody to the purified ubiquitin reacted not only to the recombinant ubiquitin but also to bovine ubiquitin, and an antibody to the purified GP37 also reacted to the recombinant GP37. The results indicated that the purified ubiquitin and GP37 retained their antigenicity. Western blot analysis results of SpltMNPV-infected Sl-zsu-1 cells revealed that the intact Uba256 was processed in this insect cell line to yield free ubiquitin and GP37 protein.

关 键 词：斜纹夜蛾核多角体病毒 遍在蛋白 GP37融合蛋白 Uba256基因 基因克隆 基因表达 免疫原性 

分 类 号：Q785[生物学—分子生物学]