柔嫩艾美尔球虫EGF-like结构域基因的克隆及原核表达

Cloning and prokaryotic expression of an EGF-like domain of Eimeriatenella

目的克隆并原核表达柔嫩艾美尔球虫(Eimeria tenella)微线蛋白4(EtMIC4)的EGF-like结构域。方法收集并纯化柔嫩艾美尔球虫子孢子,用Trizol法提取总RNA,并反转录成cDNA,利用RT-PCR技术扩增EtMIC4EGFlike基因;回收PCR产物,与pMD18-T载体连接,构建重组克隆质粒pMD18-T-EGF-like,然后亚克隆至原核表达载体pET-30a中,构建重组表达质粒pET-30a-EGF-like并转化至Transetta感受态,经IPTG诱导后进行SDS-PAGE和Western blot分析。结果成功克隆了EtMIC4EGF-like(390bp)基因,双酶切鉴定重组表达质粒pET-30a-EGF-like构建正确。SDS-PAGE分析重组EGF-like蛋白的分子质量单位约为30ku,Western blot显示该蛋白能被鸡抗柔嫩艾美尔球虫血清识别。结论成功构建了pET-30a-EGF-like原核表达质粒,并证明其原核表达产物EGF-like蛋白具有反应原性,为该蛋白的功能研究奠定了基础。

Objective To clone and express the EGF-like domain of microneme protein 4 （EtMIC4） from Eimeria tend- la. Methods E. tenella sporozoites were harvested and purified. Their total RNA was extracted for amplification of the EtMIC4 gene using RT-PCR. The amplified EtMIC4 EGF-like gene was then subcloned into the prokaryotic expression vector pET-30a. The pET 30a-EGF-like recombinant plasmid that was constructed was then transferred into Escherichia coli Transetta for expression via induction with IPTG. The expressed product was identified using SDS-PAGE and West- ern blotting. ResultsRestriction analysis showed that the pET-30a-EGF-like recombinant plasmid was constructed cor- rectly. SD-PAGE showed that the recombinant protein had a molecular mass of about 30 ku, and Western blotting showed that the recombinant protein was recognized at 30 ku. Purification of EGF-like protein was done with an elution buffer containing 40 mmol/L imidazole, and the concentration of purified protein was 0.75 mg/ml. Conclusion The re- combinant EGF-like protein of E. tenella was successfully expressed in Transetta. This provides a foundation for analysis of the function of this protein.