摘要
为了获得具有良好免疫活性的基因工程抗原蛋白,利用毕赤酵母真核表达系统,表达猪传染性胃肠炎(河北分离株)的纤突糖蛋白。根据Gen Bank TGEV S基因设计一对引物,经PCR扩增出长2.2 kb的目的基因S,将S基因亚克隆于p PIC9k载体,转化TOP10感受态,PCR、酶切鉴定阳性克隆,SalⅠ线性化重组穿梭质粒pPIC9k-S,然后电转GS115感受态,利用G418抗性进行多拷贝整合子初步筛选,提取基因组并做PCR鉴定,得到阳性菌株,经诱导发酵后,上清发酵液经SDS-PAGE电泳检测,显示获得分子量为82 kDa左右的目的蛋白,同时用Western-Blotting检测到一条特异的目的条带;用目的蛋白免疫小鼠,ELISA检测免疫S蛋白小鼠血清抗体效价为1∶100,表达蛋白具有免疫活性。
For the good immunogenicity recombinant protein,the Pichia pastoris expression system is used for the production of transmissible gastroenteritis virus recombinant proteins. In this study,the partial spike（ S） gene（ 2. 2 kb long） of transmissible gastroenteritis virus（ TGEV） was amplified using polymerase chain reaction（ PCR）with the primers designed according to the complete genome sequence of TGEV in Gen Bank. The c DNA fragment was sub-cloned into Pichia expression vector p PIC9k（ digested with EcoR Ⅰand Not Ⅰ）. Recombinant shuttle plasmid DNA,digested with SalⅠ,named p PIC9k-S. The p PIC9k-S was integrated into the genome of the methylotrophic yeast Pichia pastoris GS115 by electroporation. The partial fragment of spike protein（ S） of approximately 82 k Da was confirmed by SDS-PAGE and Western-Blotting. The specific antibody against S recombinant protein could be detected by ELISA,the title was 1 ∶ 100,the immunogenicity was good.
出处
《华北农学报》
CSCD
北大核心
2015年第6期84-90,共7页
Acta Agriculturae Boreali-Sinica
基金
北京市科技新星计划资助项目(2007B044)
