日本七鳃鳗IL-17基因原核表达、蛋白纯化及多抗制备与鉴定

Prokaryotic expression, purification, and polyclonal antibody preparation of IL-17 from Lampetra japonica

目的从日本七鳃鳗(Lampetra japonica,Lj)类淋巴细胞中提取总RNA,通过RT-PCR方法扩增七鳃鳗IL-17基因,并对其氨基酸序列进行分析。方法将IL-17基因连接到原核表达载体pET-28a上,成功构建了重组表达质粒pET-28a-Lj-IL-17;经IPTG诱导表达并纯化重组Lj-IL-17蛋白。用重组Lj-IL-17蛋白免疫新西兰大白兔,制备多克隆抗体进行ELISA与Western blot检测。结果经ELISA检测抗体效价为1∶512 000,Western blot结果显示该多克隆抗体能特异性结合重组和天然IL-17蛋白。流式细胞实验证明该多抗能特异识别七鳃鳗类淋巴细胞表达的IL-17分子。结论重组Lj-IL-17蛋白表达及多克隆抗体的成功制备,为研究日本七鳃鳗适应性免疫起源与进化提供了重要的工具。

Interleukin-17 is a pro-inflammatory factor secreted by a lineage of T cells known as T helper cells 17 （Thl7）. This study designed to express IL-17 from Lampetra japonica, and purify the expression product and prepare the polyclonal antibody. Total RNA was extracted from lymphocytes-like cells in lamprey, and then IL-17 gene was obtained by RT-PCR and analyzed by bioinformaties methods. In addition, Lj-IL-17 gene was successfully constructed to pET-28a prokaryotie expression vector, and recombinant Lj-IL-17 protein was expressed with IPTG induction and purified by Ni2＋ affinity chromatography column. The recombinant Lj-IL-17 protein was used to immunize New zealand rabbit for preparing specific anti-Lj-IL-17 polyclonal antibodies. The results of ELISA indicated that the valence of anti-Lj-IL-17 antibody was 1：512 000; Western blot assay showed that the antibody was able to detect both recombinant Lj-IL-17 protein and native IL-17 protein. While flow cytometry revealed that the prepared polyelonal antibody could specifically recognize the IL-17 expressed on the lymphocytes-like cells. All this data showed that recombinant Lj-IL-17 protein was expressed and the polyclonal antibody was prepared successfully, which lay a foundation for studying origin and evolution of adaptive immune system in lamprey.