平滑肌肌球蛋白轻链激酶N端删除载体的构建

Construction of Recombinant Vector of N-terminus Deleted Smooth Muscle Myosin Light Chain Kinase

目的:构建重组平滑肌肌球蛋白轻链激酶(myosin light chain kinase,MLCK)N端删除载体,为研究平滑肌MLCK的分子机制提供研究模型。方法:以重组质粒pCold/155为模板,根据其待删除序列(N端1-41个氨基酸)设计上下游引物,行PCR扩增。将扩增片段以Nde I/EcoR I双酶切,产物行琼脂糖凝胶电泳回收得到目的基因。将目的基因与载体连接,转化至大肠杆菌。筛选阳性克隆,并对阳性克隆进行测序。结果:用Nde I和EcoR I双酶切重组质粒pCold/155,琼脂糖凝胶电泳显示得到约4.4kb载体和约3.4kb的MLCK片段。阳性克隆经测序证实MLCK的N端41个氨基酸序列已被成功删除。结论:成功构建了重组MLCK N端删除载体pCold/155/D41。

Objective：To construct the recombinant vector of the N-terminus deleted smooth muscle myosin light chain kinase（MLCK）,and study the molecular mechanism of MLCK furthermore.Methods： We designed forward primer1 and reverse primer2 based on the sequence of pCold I /Bovine Stomach 155K MLCK（pCold/155）,and explored the PCR technique to delete the N-terminus 1-41 amino acid of MLCK.The PCR products were digested with restriction endonucleases Nde I/EcoR I.We retrieved target gene by agarose gel electrophoresis and ligated the target gene and vector fragment by TaKaRa DNA ligation kit and then transformed the recombinant vector in E. coli. The positive colonies were picked out and sequenced. Results： We obtained the vector （4.4kb） and the N-terminus deleted MLCK fragments （3.4kb） after the recombinant plasmid pCold/155 were digested with restriction endonucleases Nde l/EcoR I. Conclusions： The recombinant vectors of the N-terminus deleted MLCK（ pCold/155/D41） are constructed successfully.