应用重组PCR技术构建人单核细胞趋化蛋白-1CDNA的突变体-HΜMCP-1(7ND)  

Construction of human monocyte-chemoattractant protein-1 mutant-7 ND by recombinant PCR

作　　者：仲琳[1] 张运[1] 张梅[1] 季哓平[1] 陈文强[1] 李大庆[1] 张岩[2] 张冰[2] 杨军[3] 刘少荣[3] 

机构地区：[1]山东大学齐鲁医院心内科,山东济南250012 [2]山东大学医学院分子生物学实验室,山东济南250012 [3]烟台毓璜顶医院,山东烟台264000

出　　处：《中国病理生理杂志》2005年第12期2452-2456,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(NO.60271015);卫生部临床学科重点项目(NO.20012943)

摘　　要：目的:探讨用重组PCR技术对人单核细胞趋化蛋白-1(HΜMCP-1)基因CDNA进行缺失突变,构建 N末端缺失7个氨基酸的编码序列HΜMCP-1突变体-HΜMCP-1(7ND)CDNA,以期实现7ND基因治疗抑制MCP-1 活性。方法:根据缺失前后的两段基因片段A和B分别设计两对引物即内引物与外引物,第一轮PCR反应通过每段各自的内外引物,分别获得加有互补末端的A+和B+DNA片段。然后进行第二轮PCR反应,以第一轮PCR产物为模板,加入两外引物,获得大量重组体AB基因片段,将PCR产物与T载体连接,进行酶切鉴定并测序证实成功进行了 HΜMCP-1的基因改造。为便于表达HΜMCP-1突变体,通过ECOR Ⅰ/HINDⅢ酶切,将目的基因克隆入PCDNA3．1真核表达载体中。结果:经酶切鉴定并测序,表明已成功地构建了HΜMCP-1CDNA突变体-7ND的真核细胞表达载体。结论:已成功进行了HΜMCP-1基因CDNA的缺失突变,获得了7ND CDNA的克隆,为进一步研究HΜMCP-1功能奠定了基础。AIM： To construct 7ND - the deletion mutant of human monoeyte ehemoattraetant protein - 1 eDNA by recombinant PCR. METHODS： Using pBlueseript- hμMCP- 1 as template and two synthetic oligonueleotides containing restriction sites suitable for eloing as primers, the deletion mutant was introduced by recombinant PCR. Linking the 2 chains by reeombinat PCR and cloning into T vector, the sequenee was verified as 7ND eDNA with a length of 342 bp and was inserted into peDNA3.1 eukarytie expressing plasmid. RESULTS： A recombinant plasmid peDNA3.1 - 7ND for cloning human monoeyte ehemoattrctant protein 1 eDNA mutant was successfully constructed. The results of sequencing proved that 7ND was the mutant of human monoeyte ehemoattraetant protein 1, which lacked the N - terminal amino acids 2 through 8. CONCLUSION： A clone of human monoeyte ehemoattraetant protein - 1 mutant was obtained by recombinant PCR. This research has paved the way for further study on biological functions ot 7ND.

关 键 词：单核细胞化学吸引蛋白质1 DNA 克隆 序列 

分 类 号：R363[医药卫生—病理学]