【摘要】： 人肝細(xì)胞生長因子激活因子抑制劑-2(human hepatocyte growth factor activator inhibitor 2,hHAI-2)是一種Kunitz型絲氨酸蛋白酶抑制劑,對胰蛋白酶、胰凝乳蛋白酶、纖維蛋白溶酶和血漿組織激肽釋放酶等多種絲氨酸蛋白酶有較強(qiáng)的抑制作用;且對肝細(xì)胞生長因子激活因子(hepatocyte growth factor activator inhibitor,HGFA)具有特異性地抑制作用。因此,在抑制腫瘤的形成、腫瘤細(xì)胞的擴(kuò)散和轉(zhuǎn)移及新生血管的形成方面具有很大的應(yīng)用價(jià)值。 本研究從人乳腺癌組織總RNA中釣取hhai-2 cDNA序列,利用基因重組技術(shù)構(gòu)建hhai-2基因的克隆載體pMD18-T-hhai-2;以此為基礎(chǔ),通過PCR技術(shù)獲得了hhai-2基因的胞外區(qū)(extracellular domain, ECD)和Kunitz結(jié)構(gòu)域Ⅰ( Kunitz domainⅠ, KDⅠ),并分別構(gòu)建了表達(dá)載體pPICZα-hhai-2/ECD和pPICZα-hhai-2/KDⅠ。將這兩種表達(dá)載體分別電轉(zhuǎn)化X-33畢赤酵母,建立了穩(wěn)定表達(dá)hhai-2/ECD和hhai-2/KDⅠ基因的畢赤酵母真核表達(dá)體系。應(yīng)用特異性PCR引物進(jìn)行PCR鑒定。結(jié)果表明,hhai-2/ECD和hhai-2/KDⅠ基因已經(jīng)穩(wěn)定整合到酵母基因組中。通過測定hHAI-2/ECD和hHAI-2/KDⅠ對胰蛋白酶作用后的殘留胰蛋白酶活性檢測其活力,篩選出高表達(dá)活性的重組hHAI-2/ECD和hHAI-2/KDⅠ酵母菌,結(jié)果顯示,這兩種蛋白的表達(dá)活性具有累積效應(yīng),產(chǎn)量在一段時(shí)間內(nèi)隨表達(dá)時(shí)間延長而增加。經(jīng)SDS-PAGE檢測其培養(yǎng)液上清,分別獲得了相對分子量約為26kDa的hHAI-2/ECD蛋白和11kDa的hHAI-2/KDⅠ蛋白。本研究成功獲得了天然結(jié)構(gòu)的重組hHAI-2/ECD和hHAI-2/KDⅠ蛋白,為進(jìn)一步研究hHAI-2蛋白的生物學(xué)功能奠定了基礎(chǔ)。
[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，

本文編號：2450915