禽传染性支气管炎病毒S1基因在毕赤酵母中的表达  

Expression of avain infectious bronchitis virus S1 gene in Pichia pastoris

作　　者：余祖华[1] 王红宁[1] 周生[1] 黄勇[1] 柳萍[1] 

机构地区：[1]四川农业大学动物科技学院

出　　处：《中国兽医学报》2007年第6期799-802,共4页Chinese Journal of Veterinary Science

基　　金：国家"863"计划资助项目(2003AA241120)

摘　　要：根据GenBank已公布的传染性支气管炎病毒(Infectious bronchitis virus,IBV)株S1基因序列及pPIC9K表达载体序列,设计1对IBV S1基因表达片段的PCR引物,用RT-PCR方法扩增出长度为1 566 bp IBV S1基因表达片段,5′端不含信号肽序列,3′端添加了终止密码子。用限制性内切酶SnaB和Not将S1基因和载体pPIC9K酶切回收后连接,构建了重组表达载体pPIC9K-S1。用限制性内切酶Bgl将表达质粒pPIC9K-S1线性化,然后用电转化的方法导入毕赤酵母GS115,在MD平板上生长的转化子经过PCR鉴定和表型筛选后,获得了整合型阳性重组菌株GS115/pPIC9K-S1 His+Muts。将重组菌株在1%甲醇中进行诱导分泌表达,并对表达产物进行SDS-PAGE、Western blot分析。结果显示,IBV S1基因在毕赤酵母中成功获得了表达,表达蛋白的分子量约为76 000,能与IBV阳性血清特异性结合,表达的蛋白占上清中总蛋白量的12.5%。A pair of primers were designed according to isolated IBV＇s S1 gene sequences which were accepted by GenBank and the sequence of Pichia pastoris expression vector pPIC9K＇s Multiple Cloning Site （MCS）. Then the S1 gene was amplified by RT-PCR, and the nucleotide sequence length of S1 gene was 1566 bp and the deduced amino acid sequence length is 521aa,and the sequence included stop codon in 3＇ terminal but didn＇t include signal peptide in 5＇ terminal. The S1 fragment was digested by SnaBI and NotI, then ligated with the yeast secreted expressing vector pPIC9K which was also digested by SnaBI and NotI, a recombination vector was obtained （named pPIC9K-S1）. The expressing plasmid pPIC9K-S1 was linearized with Bgl Ⅱ , then it was transformed into Pichia pastoris GS115 by electroporation. After the electroporatic transformants were selected by PCR and Methanol-utilization-plus, the positive recombination yeast was obtained （named GS115/pPICgK-S1 His^＋ Mut^s）. GS115/ pPIC9K-S1 His^＋Mut^s was induced with 1% methanol for target protein expression. IBV S1 protein was analysed by SDS-PAGE and Western blotting assay. The results showed that IBV S1 gene was expressed successfully in Pichia pastoris and the product was 76 000 which could act with the positive serum of IBV. The amount of target protein shares 12.5% of the total protein which consist in the supernatant fluid of yeast culture medium.

关 键 词：禽传染性支气管炎病毒 S1基因 毕赤酵母 分泌表达 

分 类 号：S852.65[农业科学—基础兽医学] Q786[农业科学—兽医学]