柔嫩艾美耳球虫EtMIC2蛋白重组枯草芽孢杆菌的构建与鉴定  

Construction and identification of recombinant Bacillus subtilis expressing Eimeria tenella EtMIC2 protein

作　　者：刘嘉韵 陈学秋[1] 阳毅敏 吴飞 潘灵韬 赵明秀 王钊 杜爱芳[1] LIU Jiayun;CHEN Xueqiu;YANG Yimin;WU Fei;PAN Lingtao;ZHAO Mingxiu;WANG Zhao;DU Aifang(Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine,College of Animal Science,Zhejiang University,Hangzhou 310012,China)

机构地区：[1]浙江大学动物科学学院浙江省动物预防医学重点实验室,浙江杭州310012

出　　处：《中国兽医学报》2022年第8期1602-1606,共5页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2017YFD0501200)。

摘　　要：旨在通过芽孢表面展示技术构建柔嫩艾美耳球虫EtMIC2蛋白的重组枯草芽孢杆菌,为防控鸡球虫感染提供新思路。首先从柔嫩艾美耳球虫和枯草芽孢杆菌基因组中分别扩增EtMIC2基因和芽孢衣壳蛋白编码基因CotB,通过重叠PCR技术将两者融合后克隆至pDG364中,构建整合型重组质粒pDG364-CotB-EtMIC2,并通过电转化将融合基因CotB-EtMIC2整合于枯草芽孢杆菌amyE基因位点,最后利用PCR、Western blot和免疫荧光等技术进行鉴定。结果显示,成功克隆目的基因EtMIC2,与锚定元件CotB融合成功,且定点整合于枯草芽孢杆菌基因组,融合蛋白正确表达并可在芽孢表面检测到特异性荧光信号。本研究成功构建能够表面展示EtMIC2蛋白的重组枯草芽孢杆菌,可用于后续评估其抗球虫免疫保护效果。To construct recombinant Bacillus subtilis spores expressing the EtMIC2 antigen of Eimeria tenella(E.tenella)and develop a new way for prevention and control of E.tenella infections.The EtMIC2 gene and spore coat protein encoding gene CotB were amplified by PCR from the genomic DNA of E.tenella and B.subtilis,respectively.The CotB-EtMIC2 fusion gene was cloned by overlap PCR and cloned into pDG364 vector to construct recombinant integrated plasmid pDG364-CotB-EtMIC2.The fusion gene was integrated into B.subtilis chromosome at the amyE locus by electroporation.The recombinant B.subtilis expressing EtMIC2 protein was sequentially identified by PCR,Western blot and immunofluorescence.The results indicated that the CotB-EtMIC2 fusion gene correctly inserted at amyE locus on B.subtilis chromosome and EtMIC2 was successfully expressed and displayed on spore surface.In this study,a recombinant B.subtilis expressing EtMIC2 protein was successfully constructed,which could be used for further evaluation of the immune protection against coccidiosis.

关 键 词：柔嫩艾美耳球虫 EtMIC2 枯草芽孢杆菌 芽孢表面展示 

分 类 号：S852.71[农业科学—基础兽医学]