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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 and express the Rhomboid gene of Lanzhou strain of Eimeria stiedai (E.stiedai). Methods Primers were designed by analyzing the conserved regions of genes and the complete cDNA sequence of Rhomboid gene of Eimeria tenella was amplified by RT-PCR and 3’RACE. The sequence of Rhomboid gene was analyzed. The obtained Rhomboid The gene was cloned into the prokaryotic expression vector pET-28a and transformed into E.coli Rosetta (DE3) for expression under the induction of IPTG. The expressed product was analyzed by SDS-PAGE and Western blot. Results The full-length open reading frame of Rhomboid gene was 774 bp in length and encoded a protein of 257 amino acids. The predicted relative molecular mass and isoelectric point of Rhomboid gene were 28 120 and 8.64, respectively. The Rhomboid protein amino acids The sequence was 84.44%. The constructed recombinant prokaryotic expression plasmid pET-Rhomboid was confirmed by double enzyme digestion and sequencing. The relative molecular mass of the expressed recombinant protein was about 29 000, which could be compared with the rabbit anti-E.stiedai positive serum Specific reaction occurred. Conclusion The Rhomboid gene was successfully cloned and expressed, which laid the foundation for its biological functions and the study of its potential as a candidate gene for Ehrlichia spp. Vaccine.