人组织激肽释放酶基因真核表达载体的构建与鉴定

Construction and identification of human tissue kallikrein gene eukaryotic expressing vector

目的 构建并鉴定人组织激肽释放酶基 (h -TKK)基因真核表达质粒。方法 设计含pnI和BamHⅠ酶切位点的人组织激肽释放酶基因引物 ,采用PCR法从原核pBluescripeⅡKSKK ( +)中扩增KKcDNA片段 ,亚克隆至pAd track -CMV真核表达载体 ,转化感受态大肠杆菌DH10B ,酶切鉴定阳性重组子 ,并进行序列测定。结果 经PCR能扩增出 73 8bp的片段 ,与预期片段大小相符 ,构建的重组体经酶切鉴定能切出插入片段 ,测序结果与预期序列完全一致。结论 成功构建了人组织激肽释放酶基因真核表达重组体pAdtrack -CMVKK。

Objective To construct human tissue kallikrein gene eukaryotic expressing vector. Methods Human tissue kallikrein gene was amplified by PCR from pBluescript Ⅱ KSKK(+) and was subcloned into eukaryotic expressing plasmid to form pAdtrack-CMV-KK. After restriction endonuclease disgestion analysis, pAdtrack-CMV-KK was sequenced by ABI 377 sequencer. Results A 738 base pair single DNA fragment was found in the products by 10 g/L agarose electrophoresis, the same size expected for the human tissue kallikrein gene. The insertion fragment could be seen after restricton digestion as expected. Conclusion pAdtrack-CMV-KK eukarytic recombinant has been successfully constructed.