有效引导肝细胞生成素分泌的外源信号肽的确定

Selection of the exogenous signal peptide that conducts the secretion of hepatopoietin

目的:寻找能有效引导肝细胞生成素(HPO)分泌的外源信号肽序列,构建高效分泌HPO的真核表达载体,以提高蛋白的分泌量。方法:将不同的信号肽编码序列通过PCR方法与HPO基因融合,克隆到真核表达载体中,构建HPO真核表达载体,通过脂质体介导瞬时转染COS-7细胞,采用Western blotting方法检测HPO的分泌情况。结果:采用了白细胞介素-1受体拮抗剂的信号肽、一种人工信号肽和小鼠抗体kappa链信号肽,分别构建HPO分泌型表达载体,通过Western blotting检测显示,前2种信号肽引导HPO分泌的效率很低,只有来自小鼠抗体kappa链的信号肽能够高效引导HPO从细胞中分泌出来,分泌蛋白以二聚体形式存在,大小为30kD。结论:成功构建了高分泌型HPO真核表达载体,为进行肝纤维化的基因治疗奠定了基础。

AIM： To look for a suitable signal peptide which may effectively conduct hepatopoietin （HPO） secretion, various recombinant eukaryotic expression vectors were constructed. METHODS： Different exogenous signal sequences were spliced with HPO cDNA by PCR, and the spliced genes were cloned into eukaryotic expression plasmids. The different recombinants were respectively tansfected into COS - 7 cells by Lipofectamine 2000 method and the secretion of HPO was analyzed by Western blotting. RESULTS ： Western blotting analysis indicated that the signal peptides from interleukin - 1 receptor antagonist （IL -lra） and an artifical signal peptide did not secret HPO directly and effectively, but the signal peptide from murine Ig kappa secreted HPO directly with great efficiency. The molecular weight of the secreted HPO was 30 kD, which means that the secreted HPO existed in homodimer. CONCLUSION： Secreted recombinant expression plasmid is. successful constructed. The result may pave the way for the gene therapy of hepatic fibrosis.