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

[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.