摘要
目的克隆猪干扰素α(IFNα)基因,并在毕赤酵母中进行表达。方法用植物血凝素诱导猪外周血中的淋巴细胞,Trizol试剂提取总RNA,经RT-PCR扩增猪IFNα基因,将其克隆至pPIC9K载体中,构建重组真核表达质粒pPIC9K-IFNα,经PCR、酶切及测序鉴定正确后,电转化至毕赤酵母GS115中。筛选阳性菌株,经甲醇诱导表达后,进行SDS-PAGE及Westernblot分析。结果RT-PCR扩增获得521bp的猪IFNα基因;所构建的重组表达质粒经PCR、酶切及测序鉴定正确;表达的目的蛋白相对分子质量约为19000,可溶性蛋白约占菌体总蛋白的13%;该蛋白能与相应抗体产生特异免疫反应。结论已成功克隆并在毕赤酵母中表达了猪IFNα基因,为进一步研究其生物学性状奠定了基础。
Objective To clone porcine interferon α（IFNα）gene and express in Pichia pastoris. Methods Lymphocytes were isolated from porcine peripheral blood and induced by phytohemagglutinin（PHA）, from which total RNA was extracted with Trizol reagent for amplification of porcine IFNα gene by RT-PCR. The amplified gene was cloned into vector pPIC9K, and the constructed recombinant plasmid pPIC9K-IFNα was identified by PCR, restriction analysis and sequencing, then transformed to P. pastoris GS115 by electrotransformation. The recombinants were screened for expression under induction of methanol. The expressed product was identified by SDS-PAGE and Western blot. Results The porcine IFNα gene at a length of 521 bp was amplified by RT-PCR. PCR, restriction analysis and sequencing proved that recombinant plasmid pPIC9K-IFNα was constructed correctly. The expressed protein showed a relative molecular mass of about 19 000, and that in soluble form contained about 13% of total somatic protein. Specific immune reaction of the expressed protein with the corresponding antibody was observed. Conclusion Porcine IFNα gene was successfully cloned and expressed in P. pastoris, which laid a foundation of further study on biological characters of porcine IFNα.
出处
《中国生物制品学杂志》
CAS
CSCD
2010年第4期369-372,共4页
Chinese Journal of Biologicals
基金
辽宁省博士启动资金(20081099)
辽宁医学院博士启动资金
关键词
猪IFNα基因
克隆
真核表达
毕赤酵母
Porcine interferon α（IFNα）gene
Cloning
Eukaryotic expression
Pichia pastoris
