日本血吸虫Nanos样蛋白基因的获得、表达及初步鉴定

发布时间：2018-11-15 08:41

【摘要】：目的获得日本血吸虫Nanos样蛋白基因的cDNA序列,克隆、表达和纯化原核蛋白并初步鉴定。方法应用PCR法扩增获得日本血吸虫Nanos样基因的完整开放阅读框(ORF),将该基因亚克隆到原核表达载体pET28a(+),诱导表达并纯化融合蛋白。用纯化后的蛋白免疫家兔,制备该蛋白的多克隆抗体,分别用ELISA和Western blot法对表达蛋白进行初步鉴定。结果该基因为日本血吸虫的一个新基因,其编码序列的ORF为672 bp,编码223个氨基酸,理论相对分子量为25 ku;半定量RT-PCR技术分析显示该基因在日本血吸虫的虫卵及各期虫体有不同的表达量;成功构建了该基因的重组表达质粒pET28a(+)-SjNanos,并在大肠埃希菌中获得表达,Western blot分析表明该重组蛋白具有较好的免疫原性。结论获得日本血吸虫Nanos蛋白编码基因的全长ORF,在大肠杆菌中克隆表达了日本血吸虫Nanos样基因,并制备了特异性多克隆抗体,为进一步研究其在日本血吸虫生殖生物学中的功能奠定了基础。
[Abstract]:Objective to obtain the cDNA sequence of Schistosoma japonicum Nanos like protein gene, clone, express and purify prokaryotic protein and identify it. Methods the complete open reading frame (ORF), of Nanos like gene of Schistosoma japonicum was amplified by PCR. The gene was subcloned into prokaryotic expression vector pET28a (), and the fusion protein was purified. The polyclonal antibody of the purified protein was prepared by immunizing rabbits. The expressed protein was preliminarily identified by ELISA and Western blot. Results this gene is a new gene of Schistosoma japonicum. Its ORF encoding sequence is 672 bp, and encoding 223 amino acids. The theoretical relative molecular weight is 25 ku;. Semi-quantitative RT-PCR analysis showed that the expression of the gene was different in the eggs of Schistosoma japonicum and in the body of different stages of Schistosoma japonicum. The recombinant expression plasmid pET28a ()-SjNanos, was successfully constructed and expressed in Escherichia coli., Western blot analysis showed that the recombinant protein had good immunogenicity. Conclusion the full-length ORF, encoding Nanos protein gene of Schistosoma japonicum was cloned and expressed in Escherichia coli, and the specific polyclonal antibody was prepared. It lays a foundation for further study of its function in the reproductive biology of Schistosoma japonicum.
【作者单位】：安徽医科大学病原生物学教研室;
【基金】：国家自然科学基金(编号:30771882)
【分类号】：R383.24

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