摘要目的 比较不同tPA信号肽突变体对目的蛋白表达和分泌水平的影响,优化并筛选通用性外源信号肽序列.方法 通过定点突变技术将人类组织型纤溶酶原激活物(tPA)的信号肽第22位氨基酸由脯氨酸(P)突变为丙氨酸(tPA22P/A)或甘氨酸(tPA22P/G),并将突变前后的3种信号肽序列分别插入HIV-1 p24基因的N末端,构建不同的p24蛋白表达载体,这些重组表达载体体外瞬时转染人胚肾HEK293T细胞,转染72 h后,通过SDS-PAGE与western blot检测并比较各组培养上清和细胞中p24蛋白的表达水平.结果 成功构建了3个p24蛋白的真核表达载体P24T.tPA、P24T.tPA22P/A和P24T.tPA22P/G,经序列测定证实基因序列和方向与预期相符;重组载体转染293T细胞72 h后均检测到分泌性p24的表达.与P24T.tPA组相比,P24T.tPA22P/A转染组培养上清中和细胞内p24蛋白表达水平分别提高62%和29%,P24T.tPA22P/G转染组分别提高8%和6%.结论 tPA信号肽22位由脯氨酸突变为丙氨酸或甘氨酸,这就提高了目的蛋白的表达和分泌水平,为优化外源蛋白在哺乳细胞中的表达和分泌提供了实验基础.

abstractsObjective To optimize the tissue plasminogen activator (tPA) signal peptide (tPA-SP) as a universal heterologous signal peptide for the secretory expression of target proteins. Methods Two tPA-SP mutants, tPA22P/A and tPA22P/G, were constructed by substituting the amino acid proline at 22 position of tPA-SP to alanine (A) or glycine (G), respectively. Then, three HIV-1 p24 expression vectors, P24T. tPA,P24T. tPA22P/A and P24T. tPA22P/G were constructed by introducing tPA-SP, tPA22P/A or tPA22P/G into the N terminus of p24 gene. HEK293T cells were transfected by these recombinant vectors. 72h post-transfection, the p24 protein in the supernatant and cell lysate of each transfectant was examined by SDS-PAGE and western blot. Results Three p24 eukaryotic expression vectors P24T. tPA, P24T. tPA22P/A and P24T.tPA22P/G were successfully constructed. The three constructs gave secretory expression of HIV-1 p24 protein.The expression levels of p24 protein driven by P24T. tPA22P/A and P24T. tPA22P/G increased by 62% and 8% in the transferred supernatants, respectively, comparing with that driven by P24T. tPA; and the expressionlevels increased by 29% and 6% in the transferred cell lysates, respectively. Conclusions The amino acid substitution of tPA-SP 22P with alanine or glycine enhanced the expression and secretory levels of the target protein. This work provides an experimental foundation for optimizing the expression and secretion of heterologous proteins in mammalian cells.