瑶山亚种树鼩ISG15蛋白表达及其多克隆抗体制备

【目的】克隆瑶山亚种树鼩干扰素刺激基因15(interferon stimulating gene 15,ISG15)基因,在大肠杆菌中高效表达并纯化、制备多克隆抗体,为其生物学应用及检测方法的建立奠定基础。【方法】提取瑶山亚种树鼩外周淋巴细胞总RNA,经RT-PCR扩增出ISG15基因,亚克隆到真核表达载体构建重组真核表达质粒pcDNA3.1-ISG15,并瞬时转染仓鼠肾细胞(baby hamster syrian kidney, BHK-21);同时亚克隆到原核表达载体pET-28a(+)构建重组表达质粒pET-28a-ISG15,转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达树鼩ISG15蛋白;重组蛋白经镍离子亲和层析法纯化并免疫小鼠,获得鼠抗树鼩ISG15多克隆抗体,利用Western blotting和间接免疫荧光技术(IFA)检测其反应性。【结果】成功克隆瑶山亚种树鼩ISG15基因,并构建了其真核和原核表达载体,真核表达载体在BHK-21细胞中能高效表达;原核表达载体在大肠杆菌中30℃、0.5 mmol/L IPTG诱导6 h获得重组蛋白(分子质量22 ku),...

Expression of ISG15 Protein of Tupaia belangeri yaoshanensis and Preparation of Its Polyclonal Antibody

[Objective] The interferon stimulating gene 15 (ISG15) gene of Tupaia belangeri yaoshanensis (T. b. yaoshanensis) was cloned, the ISG15 protein was highly expressed and purify in Escherichia coli, then its polyclonal antibody was prepared, which laid the foundation for its biological application and the establishment of detection methods. [Method] Total RNA was extracted from the peripheral lymphocytes of T. b. yaoshanensis, and ISG15 gene was amplified by RT-PCR. ISG15 gene was subcloned into the eukaryotic expression vector to construct the recombinant eukaryotic expression plasmid pcDNA3.1-ISG15, and transiently transfected into renal cells of Cricetidae baby hamster syrian kidney (BHK-21) cells. At the same time, it was subcloned into the prokaryotic expression vector pET-28a(+) and the recombinant expression plasmid pET-28a-ISG15 was constructed. The pET-28a-ISG15 was transformed into E. coli BL21(DE3), and the ISG15 protein of T. b. yaoshanensis was induced by IPTG. The recombinant protein was purified by nickel ion affinity chromatography and immunized with mice to obtain mouse anti-ISG15 polyclonal antibody, and its reactivity was detected by Western blotting and IFA. [Result] ISG15 gene of T. b. yaoshanensis was successfully cloned, and its eukaryotic and prokaryotic expression vectors were constructed. The eukaryotic expression vector could be highly expressed in BHK-21 cells. The prokaryotic expression vector was induced with 0.5 mmol/L IPTG at 30 ℃ for 6 h to obtain the recombinant protein with molecular weight at approximately 22 ku. The protein was expressed in the form of inclusion body and purified by nickel ion affinity chromatography. The mice were immunized with the emulsified recombinant protein to prepare the polyclonal antibody against ISG15 of T. b. yaoshanensis. Western blotting showed that the mouse-anti-T. b. yaoshanensis ISG15 polyclonal antibody could still bind to 0.01 μg of ISG15 recombinant protein at the dilution of 1:8 000. IFA tests showed that the antibody could react with the proteins overexpressed by eukaryotic cells, with good reactivity. [Conclusion] The constructed prokaryotic expression vector highly expressed ISG15 protein of T. b. yaoshanensis in E. coli, and the recombinant protein had good immunogenicity after purification and renaturation. The obtained polyclonal antibody laid a good foundation for further study on the antiviral infection immunity of T. belangeri.