摘要
将人工优化合成的山羊痘病毒P32基因和表达载体p PIC9K同时双酶切后相连,构建p PIC9K-IL-2重组表达载体。将线性化的载体p PIC9K-P32电转化毕赤酵母GS115,对重组酵母转化子经MD平板筛选和PCR分析鉴定后,经G418抗性梯度筛选,获得多拷贝重组菌株,甲醇诱导表达后进行SDS-PAGE和Western blot。SDS-PAGE电泳显示出相对分子质量约为30 k Da目的蛋白表达条带,Western blot分析表明表达的蛋白具有反应原性。本研究成功构建载体p PIC9K-P32,并且P32基因已整合到酵母基因组中,为亚单位疫苗和鉴别诊断试剂奠定了基础。
The recombinant Pichia pastoris GS115 was constructed for further research and development of the subunit vaccine and diagnosis reagent.The artificial synthetic P32 gene was subcloned into the Pichia pastoris expression vector pPIC9 K,resulting in recombinant plasmid pPIC9K-P32.The plasmid was then transformed into Pichia pastoris GS115 by electroporation.The expressed product had a molecular weight of 30 kDa band in SDS-PAGE and reacted with the positive antiserum of CPV.The multi-copy recombinant P.pastoris strains were selected out with G418 and induced with methanol.The expressed product was analyzed in SDSPAGE and Western blot.The result showed that P32 gene was integrated with chromosome of Pichia pastoris as identified in PCR.
出处
《中国动物传染病学报》
CAS
北大核心
2015年第6期31-36,共6页
Chinese Journal of Animal Infectious Diseases
基金
甘肃省科技计划资助(1104NKCA167)
