【摘要】 本研究旨在对人激肽释放酶7 (KLK7)基因进行原核与真核表达，研究其功能，并为后续KLK7新型生物抑制剂的研制提供基础材料。采用PCR扩增KLK7基因，将其克隆至原核表达载体pGEX4T-1和真核表达载体pEGFP-C1，构建重组表达质粒pGEX4T-1-KLK7和pEGFP-C1-KLK7，分别转化Transetta (DE3)和DH5α感受态细胞，筛选阳性菌株，用IPTG诱导重组KLK7蛋白（rKLK7）表达，SDS-PAGE和Western blot分析表达情况。将pEGFP-C1-KLK7转染人角质形成细胞HaCaT，观察KLK7在细胞中的定位，检测KLK7基因过表达对角质化细胞增殖的影响。KLK7基因开放阅读框为762 bp，编码254个aa。原核系统表达的rKLK7分子量为53.5 kDa。EGFP-KLK7融合蛋白定位在细胞质。KLK7的过表达对HaCaT细胞的增殖有一定的抑制作用，但差异不显著（P>0.05）。DNA检测表明，KLK7过表达可促进HaCaT细胞的凋亡。KLK7在原核表达系统和真核细胞中均成功表达，KLK7蛋白过表达对HaCaT细胞的增殖有一定的抑制作用，而对细胞凋亡有一定的促进作用。更多还原

【Abstract】 The purpose of this study was to investigate the prokaryotic and eukaryotic expression of human kallikin-releasing enzyme 7(KLK7) gene, and to provide basic materials for the subsequent development of novel biological inhibitors of KLK7. KLK7gene was amplified by PCR and cloned into prokaryotic expression vector pGEX4T-1 and eukaryotic expression vector PEGFP-C1.Recombinant expression plasmids pGEX4T-1-KLK7 and pEGFP-C1-KLK7 were constructed and transformed into Transetta(DE3)and DH5α competent cell, respectively. The recombinant KLK7 protein(rKLK7) was expressed in Transetta(DE3)-pGEX4T-1-KLK7 strain induced by IPTG. The expression of recombinant KLK7 protein(rKLK7) was analyzed by SDS-PAGE and Western blot. pEGFP-C1-KLK7 was transfected into human keratinocyte HaCaT, and the localization of KLK7 was observed. The effect of KLK7 gene overexpression on keratinocyte proliferation was detected by CCK-8 kit.(Results) The open reading frame of KLK7gene was 762 bp, encoding 254 aa. The molecular weight of rKLK7 expressed in prokaryotic system was 53.5 kDa. The EGFPKLK7 fusion protein was localized in the cytoplasm, and the overexpression of KLK7 had a certain inhibitory effect on the proliferation of HaCaT cells, but the difference was not significant(P > 0.05). DNA analysis showed that KLK7 overexpression could promote apoptosis of HaCaT cells.(Conclusion) KLK7 was successfully expressed in both prokaryotic expression system and eukaryotic cells. Overexpression of KLK7 protein could inhibit proliferation and promote apoptosis of HaCaT cells to a certain extent.更多还原