Ⅰ～Ⅳ型登革病毒E蛋白Ⅲ区在毕赤酵母中的分泌表达及鉴定  

Characterization and secreted expression of dengue virus type I- IV envelope glycoprotein domain m in Pichia pastoris

作　　者：蔡建飘[1] 钱菲[2] 王嘉颖[2] 赵莹[2] 徐晓晶[2] 金维荣[2] 车小燕[1] 

机构地区：[1]南方医科大学珠江医院医学检验中心,广州510282 [2]生物芯片上海国家工程研究中心

出　　处：《中华预防医学杂志》2010年第8期721-725,共5页Chinese Journal of Preventive Medicine

基　　金：国家杰出青年科学基金（30725031）;国家自然科学基金（30671874）

摘　　要：目的利用毕赤酵母真核系统表达Ⅰ～Ⅳ型登革病毒E蛋白Ⅲ区（DENV-1—4 ED Ⅲ），获得可分泌表达的重组蛋白ED Ⅲ。方法PCR分别扩增Ⅰ～Ⅳ型DENV EDⅢ区基因，构建至pPIC9K毕赤酵母表达质粒。酶切线性化后电转化毕赤酵母菌GS115，涂板后经G418梯度浓度筛选，获得多株抗G4184．0mg／ml的高拷贝菌株，扩大培养后，利用甲醇诱导分泌表达重组蛋白，表达上清用Ni—NTA亲和树脂纯化后以免疫印迹鉴定。结果成功构建pPIC9K—DENV-1～4 ED Ⅲ重组质粒，高拷贝菌株经甲醇诱导后分泌表达Ⅰ～Ⅳ型DENV EDⅢ重组蛋白，纯化后获得的可溶性重组蛋白经变性凝胶电泳，相对分子质量约为12×10^3，与实际相符。免疫印迹鉴定结果表明该重组蛋白能与抗His单抗和抗Ⅰ～Ⅳ型DENV小鼠血清特异结合。结论成功通过毕赤酵母高效分泌表达I～Ⅳ型DENV EDⅢ蛋白，这些重组蛋白可进一步用于疫苗、诊断试剂和E蛋白生物学功能的研究。Objective To achieve secretory and extracellular production of recombinant dengue virus serotypes Ⅰ - Ⅳ envelope glycoprotein domain m （ DENV-1 - 4 ED m ） in Pichia pastoris. Methods EDm genes of DENVⅠ- Ⅳ were amplified and cloned into vector pPIC9K, respectively. These recombinant plasmids were then linearized and transferred into Pichia pastoris strain GSll5. Clones highly produced in 4. 0 mg/ml G418 were amplified and induced by methanol to achieve the secreted recombinant proteins. Ni- NTA agarosc beads were used for purification, while SDS-PAGE and Western blotting were used for identification. Results The recombinant plasmids pPIC9K-DENV-1 -4 ED m were constructed and successfully transferred into Pichia pastoris strain GS115. The recombinant ED Ⅲ proteins were expressed in a secretory way with the molecular weight about 12 x 103 and specifically identified by anti-His monoclonal antibody and anti-DENV Ⅰ- Ⅳ mice sera. Conclusion DENV Ⅰ- Ⅳ ED m proteins are successfully achieved from Pichia pastoris expression system and could be used for development of dengue vaccines, diagnostic reagents and study of biological function of the E protein.

关 键 词：登革热病毒 毕赤酵母 质粒 基因表达调控 病毒 

分 类 号：R3[医药卫生—基础医学]