乙型脑炎病毒SA14-14-2株E蛋白主要抗原片段的高效表达  

High Level Expression of Major Antigenic Segment of Japanese Encephalitis Virus Strain SA 14-14-2 E Protein

作　　者：葛菲菲[1] 邱亚峰[1] 杨耀武[2] 陈溥言[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,江苏南京210095 [2]中国预防医学科学院病毒研究所,北京100052

出　　处：《中国病毒学》2005年第2期117-120,共4页Virologica Sinica

基　　金：国家自然科学基金(30340013);广东省科技攻关项目(532014202028)

摘　　要：在本实验室已构建的原核表达载体(含乙脑疫苗株SA14 14 2株E蛋白基因主要抗原片段)的基础上用巴斯德毕赤酵母系统表达,该片段长1113bp,编码371个氨基酸残基,将其亚克隆入酵母表达载体pPICZαA,以电穿孔法转化酵母X 33,用Zeocin平板筛选重组子,经甲醇诱导表达后,SDS PAGE和免疫印迹分析表达产物。由于糖基化不同,所表达产物有两种,其相对分子质量分别为44kDa和50kDa,表达量较高,约为290mg/L,经Western印迹验证,有较好的抗原性。在ELISA试验中,我们直接以PBS透析后的酵母上清包被,能够很明显地区分出乙脑阴阳性血清,与RT PCR检测的相符率达95%,为制备JEV的诊断抗原和基因工程疫苗提供了依据。Based on a constructed prokaryotic expression plasmid which carries a ma-jor antigenic segment of E gene of JEV strain SA14-14-2 in our lab, we expressed the gene in methylotrophic yeast Pichia pastoris. The segment was choosn based on an- alysis provided by a computer software because of its higher antigenic index. The seg- ment was 1113bp, encoding 371 aa. The gene was subcloned into the vector pPICZα-A. The constructed plasmid was transformed into yeast X-33 by electrophoration. The recombinant transformants were selected by Zeocin. Induced by the addition of metha- nol every 24h, the product analyzed by SDS-PAGE was about 44kDa and 50kDa at a yield of 290 mg per litter of culture. The result of Western blotting indica- ted that the two bands both have specific antigenicity mainly owing to heterogeneous glycosylation. Coating with the supernatant, we can easily discriminate the positive serum from the negative, which should be useful for the production of diagnostic agents and genetically engineered vaccine of JEV.

关 键 词：乙脑疫苗株SA14—14—2株 E蛋白基因主要抗原片段 巴斯德毕赤酵母 高效表达 

分 类 号：R392[医药卫生—免疫学]