分泌表达柔嫩艾美耳球虫EtMIC2重组枯草芽孢杆菌的构建与抗体检测

Establishment and Antibody Detection of Recombinant Bacillus Subtilis Secretory Expression Eimeria Tenella EtMIC2 Protein

鸡球虫病是一种严重危害养鸡业生产的流行性肠道寄生原虫病,可造成严重的经济损失。目前,应用抗球虫药是防治球虫病的主要手段,但随着抗药性和环境污染等问题的出现,抗球虫疫苗的研究成为防控球虫病的热点。柔嫩艾美耳球虫是鸡球虫病的主要病原,因此,研究旨在通过芽孢杆菌表达系统,构建能够分泌表达柔嫩艾美耳球虫微线体蛋白2(Eimeria tenella isolate microneme protein 2,EtMIC2)重组枯草芽孢杆菌菌株,为研发新型鸡球虫疫苗奠定基础。首先,采用PCR及质谱分析方法鉴定食用性及饲用性菌株,然后从柔嫩艾美耳球虫基因组中扩增EtMIC2基因,通过无缝克隆技术将其克隆至pBE2-R质粒中,构建重组质粒pBE2-R-EtMIC2,并采用冰浴转化将重组质粒分别整合至两株枯草芽孢杆菌中,最后采用PCR、SDS-PAGE、Western blot进行鉴定。将重组菌株饲喂雏鸡,免疫7 d后初步检测抗体效价。结果表明:EtMIC2基因克隆成功,且成功整合于饲用性枯草芽孢杆菌基因组,目的蛋白正确表达,并分泌至胞外,将重组菌株饲喂给雏鸡后,血液中检测出EtMIC2抗体。研究成功构建了分泌表达柔嫩艾美耳球虫EtMIC2蛋白的重组枯草芽孢杆菌,且重组菌株能刺激动物产生抗体,可用于后续的抗球虫免疫保护性试验。

Coccidiosis is an epidemic intestinal parasitic protozoa that seriously harms the production of intensive chicken farming and can cause serious economic losses.At present,the application of anticoccidial drugs is the most common method to prevent coccidiosis,but with the problems of drug resistance and environmental pollution appearing,the research of anticoccidial vaccines has become a hot spot for the prevention of coccidiosis.Eimeria tenella is the main pathogen of coccidiosis.Therefore,it aimed to construct recombinant Bacillus subtilis secretory expression EtMIC2(E.tenella isolate microneme protein 2,EtMIC2)and lay the foundation for developing a new coccidiosis vaccine.First,edible and feeding strains were identified by PCR and mass spectrometry analysis.Then,the EtMIC2 gene was amplified by PCR from the genomic DNA of E.tenella.Then the EtMIC2 gene was cloned into pBE2-R vector to construct recombinant plasmid pBE2-R-EtMIC2.The recombinant plasmid was integrated into B.subtilis chromosome by ice bath.The recombinant B.subtilis secretory expression EtMIC2 protein was identified by PCR,SDS-PAGE and Western blot.The recombinant strain was fed to chickens,and antibody titer was detected after 7 days of immunization.The results indicated that the EtMIC2 gene was cloned and integrated into fodder B.subtilis chromosome successfully and EtMIC2 was expressed correctly and secreted extracellularly.After feeding the recombinant strain to chickens,EtMIC2 antibodies were detected in the blood samples.In this study,a recombinant B.subtilis secretory expression E.tenella EtMIC2 protein was successfully constructed,which could be used for subsequent anti-coccidiosis immune protective test.