重组人肝细胞生长因子激活因子抑制剂-2在毕赤酵母中表达的研究
发布时间：2019-03-31 12:47
【摘要】： 人肝细胞生长因子激活因子抑制剂-2(human hepatocyte growth factor activator inhibitor 2,hHAI-2)是一种Kunitz型丝氨酸蛋白酶抑制剂,对胰蛋白酶、胰凝乳蛋白酶、纤维蛋白溶酶和血浆组织激肽释放酶等多种丝氨酸蛋白酶有较强的抑制作用;且对肝细胞生长因子激活因子(hepatocyte growth factor activator inhibitor,HGFA)具有特异性地抑制作用。因此,在抑制肿瘤的形成、肿瘤细胞的扩散和转移及新生血管的形成方面具有很大的应用价值。 本研究从人乳腺癌组织总RNA中钓取hhai-2 cDNA序列,利用基因重组技术构建hhai-2基因的克隆载体pMD18-T-hhai-2;以此为基础,通过PCR技术获得了hhai-2基因的胞外区(extracellular domain, ECD)和Kunitz结构域Ⅰ( Kunitz domainⅠ, KDⅠ),并分别构建了表达载体pPICZα-hhai-2/ECD和pPICZα-hhai-2/KDⅠ。将这两种表达载体分别电转化X-33毕赤酵母,建立了稳定表达hhai-2/ECD和hhai-2/KDⅠ基因的毕赤酵母真核表达体系。应用特异性PCR引物进行PCR鉴定。结果表明,hhai-2/ECD和hhai-2/KDⅠ基因已经稳定整合到酵母基因组中。通过测定hHAI-2/ECD和hHAI-2/KDⅠ对胰蛋白酶作用后的残留胰蛋白酶活性检测其活力,筛选出高表达活性的重组hHAI-2/ECD和hHAI-2/KDⅠ酵母菌,结果显示,这两种蛋白的表达活性具有累积效应,产量在一段时间内随表达时间延长而增加。经SDS-PAGE检测其培养液上清,分别获得了相对分子量约为26kDa的hHAI-2/ECD蛋白和11kDa的hHAI-2/KDⅠ蛋白。本研究成功获得了天然结构的重组hHAI-2/ECD和hHAI-2/KDⅠ蛋白,为进一步研究hHAI-2蛋白的生物学功能奠定了基础。
[Abstract]:Human hepatocyte growth factor-activating factor inhibitor-2 (human hepatocyte growth factor activator inhibitor-2, hHAI-2) is a Kunitz serine protease inhibitor, which inhibits trypsin and chymotrypsin. Many serine proteases, such as lysosome and plasma tissue kallikrein, had strong inhibitory effects. It also has specific inhibitory effect on hepatocyte growth factor-activating factor (hepatocyte growth factor activator inhibitor,HGFA). Therefore, it has great application value in inhibiting the formation of tumor, spreading and metastasis of tumor cells and the formation of neovascularization. In this study, the hhai-2 cDNA sequence was extracted from the total RNA of human breast cancer tissue, and the cloning vector pMD18-T-hhai-2; of hhai-2 gene was constructed by gene recombination technique. On the basis of this, the extracellular domain (extracellular domain, ECD) and Kunitz domain 鈪，
本文编号：2450915
[Abstract]:Human hepatocyte growth factor-activating factor inhibitor-2 (human hepatocyte growth factor activator inhibitor-2, hHAI-2) is a Kunitz serine protease inhibitor, which inhibits trypsin and chymotrypsin. Many serine proteases, such as lysosome and plasma tissue kallikrein, had strong inhibitory effects. It also has specific inhibitory effect on hepatocyte growth factor-activating factor (hepatocyte growth factor activator inhibitor,HGFA). Therefore, it has great application value in inhibiting the formation of tumor, spreading and metastasis of tumor cells and the formation of neovascularization. In this study, the hhai-2 cDNA sequence was extracted from the total RNA of human breast cancer tissue, and the cloning vector pMD18-T-hhai-2; of hhai-2 gene was constructed by gene recombination technique. On the basis of this, the extracellular domain (extracellular domain, ECD) and Kunitz domain 鈪，
本文编号：2450915
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