免疫抑制蝎毒活性肽Im169的重组表达和功能鉴定

目的:筛选鉴定新型蝎毒免疫活性肽。方法:从海南斑等蝎(Isometrus maculates)毒腺组织c DNA文库中筛选和分离蝎毒肽基因,通过GST融合蛋白表达纯化、肠激酶酶切和高压液相色谱(HPLC)分离的方法,获得色谱纯重组蝎毒活性肽,酶联免疫吸附(ELISA)实验检测细胞因子,鉴定免疫调节活性。结果:筛选得到一个新的蝎毒活性肽基因Im169,其编码的成熟肽由29个氨基酸组成,其中含有6个半胱氨酸。Im169多肽对T淋巴细胞IL-2、TNF-α和IFN-γ等细胞因子的分泌具有明显的抑制作用,且作用呈浓度依赖性(P<0.05)。序列分析显示:Im169多肽和蝎钾通道毒素的相似性最高,提示其是一个潜在的钾通道毒素多肽。功能机制分析显示:Im169多肽通过抑制T细胞表面的钾通道,从而发挥免疫抑制功能。结论:发现了一个新的蝎毒免疫调节肽,丰富了对蝎毒活性肽的功能认识,为自身免疫疾病的药物开发提供了新的多肽模板。

Recombinant Expression and Functional Characterization of a New Immunosuppression Scorpion Toxin Peptide Im169

Objective To screen and test a new immunosuppression toxin peptides from scorpion. Methods Screening new scorpion toxin peptide genes from the venom gland c DNA library of the scorpion isometrus maculates. Recombinant peptides were obtained through the cleavage of GST fusion protein with enterokinase. ELISA experiments tested the immune suppression effect of recombinant peptides. Results A new scorpion toxin peptide gene named Im169 was cloned from the venom gland c DNA library of the scorpion isometrus maculates. The mature peptide of Im169 has 29 residues,including six cysteines. Im169 inhibited IL-2,TNF-α and IFN-γ secretions of T cells with a concentration-dependent manner,and statistical analyses showed that the inhibition effect of 100 nmol / L Im169 peptides was markedly different from the negative control（ P〈0. 05）,which indicated that Im169 is a new immunosuppressive peptide from scorpion. Sequence analyses showed that Im169 might be a potassium channel inhibitor. Functional mechanism analyses showed that Im169 might inhibit T cell cytokine secretions through inhibiting potassium channels. Conclusion Our findings enriched the functional role of scorpion toxin peptides,and provided a new peptide scaffold to design new immunosuppressant targeting T cell-associated autoimmune diseases.