猪圆环病毒2型Cap蛋白在毕赤酵母中的分泌表达  

Secretory expression of Cap protein of porcine circovirus type 2 in Pichia pastoris

作　　者：郑光来 卢晓冉 张静远[1] 陈腾[1] 王东方[1] 严妍[1] 张守峰[1] 扈荣良[1] 

机构地区：[1]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国生物制品学杂志》2016年第7期686-689,695,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31472176)

摘　　要：目的在毕赤酵母中分泌表达猪圆环病毒Ⅱ型(porcine circovirus 2,PCV2)Cap蛋白。方法将PCV2-Cap蛋白基因(Gen Bank登录号:KC620508.1)按毕赤酵母偏好密码子优化后,插入毕赤酵母分泌型表达载体p PICZαA中,重组质粒p PICZαA-Cap经SacⅠ线性化,通过电击转化整合至毕赤酵母GS115菌株的基因组中,构建p PICZαA-CapGS115重组酵母菌。经甲醇诱导表达重组Cap蛋白,通过SDS-PAGE、Western blot和间接ELISA法进行鉴定。结果重组表达质粒p PICZαA-Cap经双酶切和测序证明构建正确;重组酵母菌p PICZαA-Cap-GS115经菌落PCR鉴定,可扩增出1 001 bp的目的基因片段;表达的重组Cap蛋白相对分子质量约49 000,可与猪圆环2型阳性血清和小鼠抗His单克隆抗体发生特异性反应。结论成功利用毕赤酵母表达系统分泌表达了PCV2 Cap蛋白,表达的重组蛋白具有良好的抗原性,为PCV2亚单位疫苗和ELISA诊断试剂盒的研制奠定了基础。Objective To express the Cap protein of porcine circovirus type 2（PCV2） in a secretory form in Pichia pastoris.Methods The PCV2-Cap gene（Gen Bank Accession No.：KC620508.1）was optimized according to the P.pastorispreferred codon,and inserted into P.pastoris secretory expression vector p PICZαA.The constructed recombinant plasmid p PICZαA-Cap was linearized by Sac Ⅰ and transformed to P.pastoris GS115 strain by electric shock to construct an engineering recombinant yeast p PICZαA-Cap-GS115 in which Cap protein was expressed under induction of methanol.The expressed product was identified by SDS-PAGE,Western blot and indirect ELISA.Results Both restriction analysis and sequencing proved that recombinant plasmid p PICZαA-Cap was constructed correctly.The target gene fragment at a length of 1 001 bp was amplified from recombinant yeast p PICZαA-Cap-GS115 by colony PCR.The expressed Cap protein,with a relative molecular mass of about 49 000,showed specific reactions with PCV2 positive serum and mouse anti-His monoclonal antibody.Conclusion PCV2 Cap protein was expressed in a secretory form by using P.pastoris expression system,which showed good antigenicity.It laid a foundation of preparation of PCV2 subunit vaccine and ELISA diagnostic kit.

关 键 词：猪圆环病毒2型 CAP蛋白 毕赤酵母 分泌表达 

分 类 号：S852.659.2[农业科学—基础兽医学] Q786[农业科学—兽医学]