毕赤酵母表达的猪传染性胃肠炎病毒纤突糖蛋白及其免疫原性分析

doi:10.7668/hbnxb.2015.06.013

摘要/Abstract

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GenBank 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pPIC9k载体,转化TOP10感受态,PCR、酶切鉴定阳性克隆,Sal Ⅰ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.

关键词: 毕赤酵母, 猪传染性胃肠炎病毒, 纤突糖蛋白S基因, 免疫原性

Abstract: 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 GenBank.The cDNA fragment was sub-cloned into Pichia expression vector pPIC9k (digested with Eco R Ⅰand Not Ⅰ).Recombinant shuttle plasmid DNA,digested with Sal Ⅰ,named pPIC9k- S.The pPIC9k- 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 kDa 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.

Key words: Pichia pastoris, TGEV, Spike gene, Immunogenicity

中图分类号:

S432.4
Q78

张莉, 康雪燕, 章振华, 李永清, 盖丽丽, 姜世金, 张培君. 毕赤酵母表达的猪传染性胃肠炎病毒纤突糖蛋白及其免疫原性分析[J]. 华北农学报, 2015, 30(6): 84-90. doi: 10.7668/hbnxb.2015.06.013.

ZHANG Li, KANG Xue-yan, ZHANG Zhen-hua, LI Yong-qing, GAI Li-li, JIANG Shi-jin, ZHANG Pei-jun. Expression and Immunogenicity Analysis of Spike Glycoprotein of Transmissible Gastroenteritis Virus in Yeast Pichia pastoris[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(6): 84-90. doi: 10.7668/hbnxb.2015.06.013.

http://www.hbnxb.net/CN/Y2015/V30/I6/84

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