EB病毒融合蛋白ZtaN-p23在大肠杆菌中的表达、纯化及活性鉴定  

Expression,purification and activity identification of ZtaN-p23 fusion protein in Escherichia coli of Epstein-Barr virus

作　　者：张毅[1,2,3,4] 周淑艳[1,2,3,5] 孙业红[1,2] 涂惠英 秦晓林[1,2] 陈锐 李体远[1,2] 

机构地区：[1]暨南大学附属第二医院 [2]深圳市人民医院临床医学研究中心,广东深圳518020 [3]深圳市人民医院 [4]暨南大学药学院,广东广州510632 [5]暨南大学医学院,广东广州510632

出　　处：《细胞与分子免疫学杂志》2012年第2期120-123,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：暨南大学科研培育与创新基金研究项目(216113103);深圳市卫生局重点课题(200608)

摘　　要：目的:克隆EB病毒立即早期蛋白ZtaN和衣壳蛋白p23,构建原核表达载体,并在大肠杆菌中表达融合蛋白,为后续利用蛋白进行疾病诊断奠定基础。方法:采用逆转录聚合酶链反应(RT-PCR)技术从B95-8细胞中分别扩增出目的基因BZLF1N和BLRF2,利用融合PCR法将两个基因进行连接,构建重组质粒pGEX-4T-1-BZLF1N-BLRF2,并转化大肠杆菌BL21(DE3)。加入IPTG诱导表达融合蛋白ZtaN-p23,通过SDS-PAGE和Western blot确定蛋白表达量及活性鉴定。结果:重组质粒pGEX-4T-1-BZLF1N-BLRF2经双酶切鉴定和序列分析正确,证实已成功构建原核表达载体;SDS-PAGE显示诱导的蛋白Mr约46 000,与预期值一致;对融合蛋白表达条件进行优化后,发现其主要以包涵体的形式存在于细胞中;亲和层析结果显示纯化后的ZtaN-p23纯度>95%;Westernblot显示ZtaN-p23具有良好的生物活性。结论:成功构建原核表达载体pGEX-4T-1-BZLF1N-BLRF2,并在大肠杆菌中进行了表达纯化,融合蛋白显示出良好的活性。AIM： To construct the prokaryotic expression plasmid pGEX-4T-1-BZLF1N-BLRF2,and express it in Escherichia coli.METHODS： The EB virus BZLF1N gene and BLRF2 gene were amplified by RT-PCR respectively.Then,the two genes were linked by splicing overlap extension PCR method and inserted into the vector pGEX-4T-1,and the recombinant plasmid pGEX-4T-1-BZLF1N-BLRF2 was transformed into E.coli BL21（DE3） strain.The expression protein ZtaN-p23 was analysed by SDS-PAGE and immunoreactivity was proved by Western blotting.RESULTS： Restriction enzyme digestion and DNA sequencing showed recombinant plasmid constructed successfully.The expression product ZtaN-p23 with the molecular weight 46000 was located in the cytoplasm and insoluble.The ZtaN-p23 up to 95% purity was obtained after purified using affinity chromatography.Western blotting showed fusion protein possessed a well bioactivity and specificity.CONCLUSION： The fusion gene BZLF1N-BLRF2 is successfully constructed and effectively expressed in E.coli,which lay the foundation for further research on its biological properties and functions.

关 键 词：EB病毒 BZLF1N基因 BLRF2基因 表达 纯化 

分 类 号：R446.62[医药卫生—诊断学]