人组织激肽释放酶基因的克隆及测序分析(论文资料).pdfVIP

2001 4 22 4 # 291 # 李体远 戴 勇 杜 珙 文锦丽 暨南大学医学院第二附属医院, 广东省深圳市人民医院(518020) 克隆中国人组织激肽释放酶基因, 为开展基因治疗高血 研究奠定基础 提取人胰腺组织 总RNA, 逆转录后进行PCR 扩增将PCR 产物回收插入质粒KS, 酶切鉴定后双向测序分析 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% 克隆的中国人组织 激肽释放酶基因可用于基因治疗等深入研究工作 激肽释放酶/ 组织 基因克隆 序列分析 Molecular cloning and sequence analysis of the human pancreatic kallikrein gene Li Tiyuan, Dai Yong , Du Gong , et al. Shenzhen P eop le! s Hosp ital, Shenzhen 518020 Abstract Objective To clone the h man tiss e kallikrein gene in Chinese. Methods Total RNA was extracted from h man pancreas and h man tiss e kallikrein ( KK) gene cDNA was amplified by PCR after reverse- transcription sing Oligo( dT) primer. The original kallikrein cDNA was recovered and inserted into the Sma I site of KS plasmid. After restriction endon clease di gestion analysis, KK cDNA was seq enced by ABI 377 seq encer . Results The cloned kallikrein gene is abo t 832 bp in length. A seq ence comparison of this with the reported KK seq ence in the Genbank indicated that the cloned Chinese kallikrein gene has one n cleotide difference . The coded amino acid is Asp in the Genbank reported gene, while the coding amino acid of Chinese kallikrein gene is Asn. Conclusion The cloned kallikrein gene can be sed for f rther st dy in gene therapy and other st dy. Key words Kallikrein/ tiss e Gene cloning Seq ence analysis , RNA oligo( dT) ( kallikrein, KK) eppendorf PCR , 95 ∀ , 40s; 60 ∀ , 1 [ 1~ 3] KK - min; 72 ∀ , 1min; 10∀ 30 , 72 ∀ (KKS) , KKS 10 min PCR 1 Klenow , 25 ∀ 30 min [ 1~ 5] 1. 6 重组质粒的构建 PCR , KSXL1- Bl e