斯氏艾美耳球虫兰州株Rhomboid基因的克隆及原核表达

Cloning and prokaryotic expression of Rhomboid gene of Fimeria stiedai Lanzhou strain

目的克隆并原核表达斯氏艾美耳球虫(Eimeria stiedai,E.stiedai)兰州株Rhomboid基因。方法通过分析基因保守区设计引物,采用RT-PCR方法与3′RACE技术相结合,扩增获得兔斯氏艾美耳球虫Rhomboid基因cDNA全序列,并对其进行序列分析;将获得的Rhomboid基因克隆至原核表达载体pET-28a中,转化E.coli Rosetta(DE3),IPTG诱导表达,并对表达产物进行SDS-PAGE及Western blot分析。结果 Rhomboid基因开放阅读框全长774 bp,编码257个氨基酸残基,预测蛋白质相对分子质量和等电点分别为28 120和8.64,与鸡柔嫩艾美耳球虫(E.tellan)Rhomboid蛋白氨基酸序列的同源性为84.44%;构建的重组原核表达质粒pET-Rhomboid经双酶切和测序证实构建正确;表达的重组蛋白相对分子质量约为29 000,并可与兔抗E.stiedai阳性血清发生特异性反应。结论成功克隆并表达了Rhomboid基因,为其生物学功能及以其作为斯氏艾美耳球虫疫苗候选基因的研究奠定了基础。

Objective To clone the Rhomboid gene of Eimeria stiedai Lanzhou strain and express in prokaryotic cells.Methods Primers were designed based on the analysis of conserved region,by using which the full-length cDNA of Rhomboid gene of E.stiedai was amplified by RT-PCR and sequenced,then cloned into prokaryotic expression vector pET-28a.The constructed recombinant plasmid pET-Rhomboid was transformed to E.coli Rosetta（DE3） and induced with IPTG,and the expressed product was identified by SDS-PAGE and Western blot.Results The open reading frame of Rhomboid gene,at a full-length of 774 bp,encoded 257 amino acid residues,of which the predicted relative molecular mass and isoelectric point were 28 120 and 8.64 respectively.The homology of amino acid sequence of expressed protein was 84.44% to the Rhomboid protein of E.tellan.Restriction analysis and sequencing proved that recombinant plasmid pET-Rhomboid was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 29 000,showed specific reaction with rabbit antisera against E.stiedai.Conclusion Rhomboid gene was successfully cloned and expressed,which laid a foundation of study on the biological function of Rhomboid gene as a candidate gene of E.stiedai vaccine.