| E.tenella河北株EtMIC-2基因的克隆及毕赤酵母系统表达 |
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| 引用本文： | 张召兴,李佩国,李蕴玉,张香斋,贾青辉,张艳英,丁咚.E.tenella河北株EtMIC-2基因的克隆及毕赤酵母系统表达[J].中国兽医学报,2020(5):939-942. |
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| 作者姓名： | 张召兴 李佩国 李蕴玉 张香斋 贾青辉 张艳英 丁咚 |
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| 作者单位： | 河北科技师范学院;河北旅游职业学院;沧州职业技术学院 |
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| 基金项目： | 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)。 |
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| 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。
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| 关 键 词： | 柔嫩艾美耳球虫 EtMIC-2基因 毕赤酵母系统 Western blot |
Cloning and expression of EtMIC-2 gene of E.tenella Hebei strain in pichia pastoris |
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| ZHANG Zhao-xing,LI Pei-guo,LI Yun-yu,ZHANG Xiang-zhai,JIA Qing-hui,ZHANG Yan-ying,DING Dong.Cloning and expression of EtMIC-2 gene of E.tenella Hebei strain in pichia pastoris[J].Chinese Journal of Veterinary Science,2020(5):939-942. |
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| Authors: | ZHANG Zhao-xing LI Pei-guo LI Yun-yu ZHANG Xiang-zhai JIA Qing-hui ZHANG Yan-ying DING Dong |
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| Institution: | (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) |
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| Abstract: | 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. |
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| Keywords: | E tenella EtMIC-2 gene pichia pastoris system Western blot |
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