猪带绦虫六钩蚴表膜结合蛋白45WB_2基因在毕赤酵母中的表达  

Expression of Taenia solium Oncosphere 45WB_2 Gene in Pichia pastoris System

作　　者：王谨[1] 骆学农[1] 岳城[2] 胡志敏[2] 才学鹏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所农业部畜禽病毒学重点实验室,兰州730046 [2]新疆农业大学化工学院,乌鲁木齐830052

出　　处：《中国寄生虫学与寄生虫病杂志》2005年第3期171-174,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家重大基础研究发展规划项目(No.G1999011906)~~

摘　　要：目的从猪带绦虫六钩蚴总RNA中扩增45WB2基因,并在甲醇酵母分泌表达载体pPIC9K中获得高效表达。方法采用逆转录-聚合酶链反应(RT-PCR),从孵化激活的六钩蚴克隆获得459bp的基因,亚克隆至酵母分泌表达载体pPIC9K中,经测序验证读码框正确后,重组质粒电转化毕赤酵母SMD1168菌株。用PCR方法检测结果表明,目的基因整合进毕赤酵母染色体DNA中。重组菌株用1%甲醇诱导,分别取诱导72和96h的上清和沉淀进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Westernblotting)分析。结果重组菌株在毕赤酵母中的表达蛋白分子量为Mr16000,表达产物占上清总蛋白量的32.8%,且能被猪囊尾蚴病患者血清识别。结论基因在毕赤酵母中成功表达,表达产物有望作为免疫原用于猪囊尾蚴病的免疫预防。Objective To amplify and express the gene from Taenia solium oncosphere in Pichia pastoris system. Methods Total RNA was extracted from hatched and activated oncospheres. A 459bp specific fragment was amplified by RT-PCR. It was subcloned into the secreted expression vector pPIC9K, and identified by sequencing and then transformed into P. pastoris SMD1168 by electroporation. PCR analysis showed that 45WB2 was integrated into the genomic DNA of yeast. The recombinants were induced by 1% methanol and the culture supernatant was collected and tested by SDS-PAGE and Western blotting. Results 45WB2 gene was expressed successfully in P. pastoris and the product was recognized by human positive serum against cysticercosis. Conclusion The recombinant 45WB2 expressed in P. pastoris may be used to prevent pigs from the infection of Taenia solium.

关 键 词：猪带绦虫 六钩蚴表膜结合蛋白基因 毕赤酵母菌 基因表达 

分 类 号：R383.32[医药卫生—医学寄生虫学] R392.11[医药卫生—基础医学]