E.tenella河北株EtMIC-2基因的克隆及毕赤酵母系统表达  

Cloning and expression of EtMIC-2 gene of E.tenella Hebei strain in pichia pastoris

作　　者：张召兴[1,2] 李佩国 李蕴玉[1] 张香斋[1] 贾青辉[1] 张艳英[1] 丁咚[3] ZHANG Zhao-xing;LI Pei-guo;LI Yun-yu;ZHANG Xiang-zhai;JIA Qing-hui;ZHANG Yan-ying;DING Dong(Heibei Province Key Laboratory of Veterinary Preventive Medicine,Hebei Normal University of Science and Technology,Qinhuangdao,Hebei 066604,China;Hebei Tourism Vocational College,Chengde,Hebei 067000,China;Cangzhou Technical College,Cangzhou,Hebei 061001,China)

机构地区：[1]河北科技师范学院,河北省预防兽医学重点实验室,河北秦皇岛066604 [2]河北旅游职业学院,河北承德067000 [3]沧州职业技术学院,河北沧州061001

出　　处：《中国兽医学报》2020年第5期939-942,共4页Chinese Journal of Veterinary Science

基　　金：河北省现代农业产业技术体系蛋肉鸡产业创新团队资助项目(HBCT2018150206);河北省自然科学基金资助项目(C2014407043);秦皇岛市科技局资助项目(201803B014)。

摘　　要：为表达鸡柔嫩艾美耳球虫(E.tenella)河北株EtMIC-2蛋白,本试验采用RT-PCR方法克隆出E.tenella河北株EtMIC-2基因,连接到毕赤酵母分泌表达载体pPIC9中,构建重组表达载体pPIC9-EtMIC-2。将其线化后转化到巴斯德毕赤酵母GS115中,甲醇诱导表达,饱和硫酸铵4℃沉淀浓缩后,用His选择镍-亲和层析柱纯化EtMIC-2蛋白。采用SDS-PAGE和Western blot方法验证其蛋白的表达。结果表明,构建的重组表达载体pPIC9-EtMIC-2在毕赤酵母菌中表达出相对分子质量约为45 000的目的蛋白,表达的EtMIC-2蛋白能被E.tenella阳性血清识别,具有良好的免疫反应原性。为进一步研究EtMIC-2蛋白功能及构建DNA疫苗奠定基础。To express the EtMIC-2 protein of E.tenella Hebei strain,the EtMIC-2 gene of E.tenella Hebei strain was cloned by RT-PCR and linked to the secretory expression vector pPIC9 of pichia pastoris to construct the recombinant expression vector pPIC9-EtMIC-2.The recombinant expression vector was linearized and transformed into pichia pastoris GS115.Methanol was used to induce the expression.After precipitation and concentration of saturated ammonium sulfate at 4℃,EtMIC-2 protein was purified by His selective nickel affinity chromatography column.The EtMIC-2 protein was analyzed by SDS-PAGE and Western blot.The results showed that the molecular mass about 45 000 target protein was expressed in pichia pastoris of the recombinant expression vector pPIC9-EtMIC-2.The expressed EtMIC-2 protein could be recognized by E.tenella positive serum and had good immunogenicity.It lays a foundation for further study of EtMIC-2 protein function and construction of DNA vaccine.

关 键 词：柔嫩艾美耳球虫 EtMIC-2基因 毕赤酵母系统 Western blot 

分 类 号：S852.723[农业科学—基础兽医学]