柔嫩艾美尔球虫14-3-3基因的原核表达与鉴定

Prokaryotic expression and identification of 14-3-3 gene of Eimeria tenella

目的 对柔嫩艾美尔球虫(E.tenella)14-3-3基因进行克隆表达,对其表达产物进行鉴定及反应原性分析,旨在探索其作为鸡球虫基因工程疫苗候选抗原的可行性。方法 以柔嫩艾美尔球虫RNA为模板,通过RT-PCR扩增14-3-3基因,连接至pET-32a(+)载体,构建重组质粒pET-32a(+)-14-3-3,转化感受态细胞BL21(DE3)后经IPTG诱导表达,利用His标签镍离子蛋白纯化柱对表达产物进行纯化,采用SDS-PAGE和Western blot鉴定重组蛋白的表达及反应原性。结果 经双酶切和测序鉴定,重组质粒pET-32a(+)-14-3-3构建正确。SDS-PAGE检测重组蛋白14-3-3在原核表达系统中主要以可溶性形式表达,融合蛋白的分子质量约为50ku,表达的重组蛋白14-3-3能被相应抗体识别。结论 成功构建重组质粒pET-32a(+)-14-3-3,表达的重组蛋白14-3-3具有反应原性,为该蛋白的生物学功能研究奠定了基础。

Objective This experiment explored the of the 14-3-3 gene of Eimeria tenella as a candidate antigen for a ally against chicken coccidia through the process of cloning and expressing the 14-3-3 gene,identifying the expression produce and analyzing the reactogenicity.Methods The recombinant plasmid pET-32 a(+)-14-3-3 was constructed through amplifying the 14-3-3 gene by RT-PCR using Eimeria tenella RNA as template and ligating it into the pET-32 a(+) vector.Recombinant plasmid was transformed into competent cells BL21(DE3),and its expression product was induced with 1 mol/L IPTG.The 14-3-3 fusion protein was expressed and purified SDS-PAGE and Western-blot were used to identify the protein and analyze its reactogenicity.Results 1% agarose gel electrophoresis and sequencing results indicated that the 14-3-3 gene was successfully amplified.Double restriction enzymes identification and sequencing verified that the recombinant plasmid pET-32 a(+)-14-3-3 was successfully constructed.12% SDS-PAGE indicated that the 14-3-3 recombinant protein was mainly expressed in supernatant and the molecular weight was about 50 ku.Western-blot indicated that the 14-3-3 recombinant protein was ed by the corresponding antibody.Conclusion All the results showed that the recombinant plasmid pET-32 a(+)-14-3-3 was constructed and the 14-3-3 fusion protein was expressed and purified,its reactogenicity was certified by Western-blot assay,which laid the foundation for the biological function study of 14-3-3 protein.