摘要
目的开发激肽释放酶基因工程产品,为开展基因治疗高血压奠定基础。方法提取人胰腺组织总RNA,逆转录后PCR扩增激肽释放酶cDNA。回收、补平后插入质粒KS,构建出中间载体KSKK,酶切鉴定后双向测序分析激肽释放酶基因序列。从KSKK中切出激肽释放酶原及激肽释放酶基因,插入真核表达载体PET-28b(+),经酶切鉴定后,进行核苷酸序列分析和融合蛋白表达。结果本实验克隆的激肽释放酶基因与GenBank报告的激肽释放酶基因相比,有一个碱基不同,同源性为99.8%。将IPTG诱导表达进行SDS-PAGE电泳,与蛋白标准品比较在31800处可见明显的高表达带。免疫印迹实验表明重组蛋白具有KK的抗原性。结论已成功克隆并表达了人组织激肽释放酶基因,为进一步开发基因工程产品及进行基因治疗高血压研究奠定了基础。
[Objective] To develop recombinant human pancreatic kallikrein and lay a foundation of hypertension. [Methods] Total RNA was extracted from human pancreas and then human tissue kallikrein gene eDNA was amplified by reverse-transcription PCR and cloned to the KS plasmid.Then amplify the mature kallikrein gene from pBluescripe Ⅱ KSKK（＋）and insert into pET28b（＋）. The clones were identified by enzyme digestion and sequenced, and fusion protein was expression under the instruction of IPTG. [Results] SDS-PAGE profile showed a clear protein band with a relative molecular weight of 31 800. Western blot proved that the expressed protein showed specific antigenieity to human serum kallikrein. [Conclusion] Human pancreatic kallikrein gene was successfully cloned and expressed.It laid a foundation of further study in gene therapy for treating hypertensive diseases and other renal diseases or be used in other researehs.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2005年第16期2405-2409,共5页
China Journal of Modern Medicine
基金
the Nature Science Foundation of Shenzhen city Science and Technology Bureau(No.200204005)
