小鼠白介素17基因的克隆、表达及其活性鉴定

Cloning,expression and activity identification of mouse interleukin-17

目的通过基因工程技术获得有活性的重组小鼠白介素17(rmIL-17)蛋白,为研究IL-17的生物学作用及其作用机制提供基础。方法用RT-PCR技术得到小鼠IL-17编码基因,将其克隆入原核表达载体pET32a,转化至大肠埃希菌(E.coli)BL21株,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过亲和层析得到纯化的rmIL-17蛋白,免疫家兔,制备多抗,Westernblot和酶联免疫吸附实验鉴定该蛋白免疫原性;用该蛋白进行体外细胞刺激实验、体内中性粒细胞招募实验鉴定蛋白的生物学活性。结果成功构建了重组质粒pET32a-mIL17并在大肠杆菌中表达。过柱得到纯化的rmIL-17蛋白,Westernblot和酶联免疫吸附实验证实其具有良好的免疫原性;体外细胞刺激实验显示该蛋白可刺激小鼠3T3成纤维细胞产生白介素6(IL-6)且具有量效关系;体内细胞招募实验显示纯化的rmIL-17蛋白具有招募中性粒细胞的作用。结论小鼠IL-17基因在体外成功表达,且表达的重组蛋白具有明显的生物学活性。

Objective To provide the basis for investigating the biological function and study its mechanism of IL-17 through producing actively recombinant mouse interleukin-17 protein by genetic engineering. Methods Mouse IL-17 gene was amplified in vitro by RT-PCR,then,the target gene was cloned into prokaryotic plasmid Pet32a （ ＋ ） . After the recombinant plasmid was transformed into E. coli BL21,it was expressed after inducted with IPTG （ Isopropyl beta-D-thiogalactopyranoside） . Through affinity chromatography column got the purified IL-17,Rabbit were immunized with rmIL-17 and polyclonal antibody was produced. The immunogenicity was identified by Western blot and ELISA. The biological activity was examined by stimulating IL-6 secretion in vitro and by stimulating neutrophil infiltration in vivo. Results We obtained recombinant plasmid pET32a-mIL17. The purified rmIL-17 protein was produced. rmIL-17 stimulates IL-6 secretion by 3T3 mouse fibroblasts and in a dose-dependent manner in vitro. rmIL-17 induced neutrophil infiltration in vivo. Immunogenicity was identified by Western blot and ELISA. Conclusion mIL-17 can be expressed in vitro,and this expressed recombinant protein has biological activity.