肿瘤转移抑制基因KiSS-1的克隆与真核表达载体的构建

目的克隆不含信号肽的人肿瘤转移抑制基因KiSS-1,构建KiSS-1基因的真核表达载体并鉴定。方法从正常人膀胱组织中提取总RNA,经RT-PCR得到KiSS-1基因开放阅读框cDNA序列,将其克隆到真核表达栽体pcDNA3.1( ),构建真核表达质粒pcDNA3.1( )/KiSS-1。结果经基因序列分析及PCR和酶切鉴定,证实目的基因片段已成功插入载体pcDNA3.1( )且序列正确。结论重组质粒pcDNA3.1( )/KiSS-1构建成功,为下一步KiSS-1蛋白的表达和研究该蛋白的功能奠定了良好的基础。

Cloning of Tumor Metastasis-suppressor Gene KiSS-1 and Construction of its Eukaryotic Expression Vector

Objective To clone the tumor metastasis suppressor gene Kiss-1 from normal human urinary bladder tissue which doesn't contain the signal peptide,and to construct its eukaryotic expression vector.Methods Total RNA was extracted from human urinary bladder tissue and the opening reading frame of KiSS-1 cDNA was isolated through RT-PCR as PCR template.Then,cloned it into the eukaryotic expression vector pcDNA3.1( ).The expression vector of pcDNA3.1( )/KiSS-1 was constructed successfully.Results The recombinant plasmid was identified by sequencing analysis,PCR and restriction enzyme digestion.It was testified that the target gene fragment was successfully cloned into the recombinant plasmid pcDNA3.1( )as expected.Conclusions The successful construction of the recombinant plasmid pcDNA3.1( )/KiSS-1 will be benefit to the further study in the protein expression and the protein's function.