柔嫩艾美耳球虫微线4N端蛋白序列分析与真核表达  

Sepuencing and expression of Eimeria tenella microneme protein 4N in Pichia pastoris

作　　者：王黎霞[1] 张鹏[2] 张建军[2] 安健[2] WANG Lixia;ZHANG Peng;ZHANG Jianjun;AN Jian(College of Animal Science and Veterinary,Beijing Vocational College of Agriculture,Beijing 102442,China;College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China)

机构地区：[1]北京农业职业学院牧医系,北京102442 [2]北京农学院动物科学技术学院,北京102206

出　　处：《北京农学院学报》2021年第3期62-66,共5页Journal of Beijing University of Agriculture

基　　金：北京市特色高水平骨干专业群项目-动物医学专业群项目-技术平台与社会服务建设项目(PXM2020-157102-000060-15);北京农业职业学院院级科研项目(XY-JT-03)。

摘　　要：[目的]为了获得重组柔嫩艾美耳球虫微线4N端蛋白。[方法]根据Genebank设计柔嫩艾美耳球虫微线4N端基因特异引物,RT-PCR扩增该基因,经Blast与Genebank公布的该基因比对,而后构建该蛋白的真核表达载体pPICZαA/EtMIC4N,用毕赤酵母表达系统小规模表达该蛋白,利用SDS-PAGE和Western Blot对表达的蛋白鉴定,再以摇瓶的方式进行大规模表达。[结果]RT-PCR扩增获得柔嫩艾美耳球虫微线4N端基因序列,大小为773 bp,经比较,与目的基因开放阅读框相似度99.5%,其中有4个碱基发生突变,1个gap。PCR证实成功构建真核表达载体,电转后的毕赤酵母用甲醇诱导表达,SDS-PAGE和Western Blot发现在上清中有45 kD的条带,说明成功表达该蛋白,且该表达系统可持续获得较高纯度的目的蛋白,以摇瓶的方式进行大规模表达,每升能够纯化到约10 mg的目的蛋白。[结论]毕赤酵母成功表达重组柔嫩艾美耳球虫微线4N端蛋白,为该蛋白免疫原性的研究奠定基础。[Objective] The aim of the study is to eukaryotic express the rEtMIC4 N in Pichia pastoris. [Methods] RNA was extracted from Eimeria tenella sporulated oocysts. EtMIC4 N premier were designed according to its sequence in genebank. EtMIC4 N gene was cloned by RT-PCR. The amplified sequence was compared with its sequence in genebank by blast, then the recombinant expression vector was constructed. The rEtMIC4 N was induced expressing by methanol in P. pastoris. The rEtMIC4 N was identified by SDS-PAGE and Western Blot. Mass rEtMIC4 N was expressed by shake flask fermentation. [Results] Blast analysis showed that 773 bp of 5’ end shared more than 99.5% identy to EtMIC4 fragment in genebank, 4 bp changed and 1 gap. Then, rEtMIC4 N was successfully expressed in P. pastoris and identified by SDS-PAGE and Western Blot. The results indicated that rEtMIC4 N was secretory expressed in P. pastoris expression system. The expression was up to 10 mg/L by shake-flask culture. [Conclusion] The rEtMIC4 N was successfully expressed in P. pastoris. The results is a foundation for further research on coccidiosis immunity.

关 键 词：柔嫩艾美耳球虫 毕赤酵母 微线4N端蛋白 序列分析 真核表达 

分 类 号：S852.34[农业科学—基础兽医学]