人KiSS-1基因克隆及其真核表达载体的构建

Cloning of human KiSS-1 gene and construction of its eukaryotic expression vector

目的:克隆转移抑制基因KiSS1的不含信号肽的表达序列,构建人KiSS1基因的真核表达载体。方法:从人正常胰腺组织中提取总RNA,经RT PCR得到KiSS1基因开放阅读框cDNA序列,并将其克隆到真核表达载体pcDNA3中,构建真核表达质粒pcDNA3/KiSS1。结果:经PCR、酶切鉴定和基因序列测定,证实重组入载体pcDNA3的片段为目的基因开放阅读框的核苷酸序列。结论:重组质粒pcDNA3/KiSS1的成功构建,为该基因相应蛋白质的表达和其抗体的制备研究提供基础。

Objective: To clone the metastasis suppressor gene KiSS-1 from human normal human pancreas tissue and construct its eukaryotic expression vector. Methods: Total RNA was extracted from human pancreas tissue. The opening reading frame of KiSS-1 cDNA was isolated by using RT-PCR, and cloned into eukaryotic expression vector pcDNA3. The expression plasmid pcDNA3/KiSS-1 was constructed. Results: The recombinant plasmid was identified with PCR, digestion of internal restriction enzyme, and sequencing. The nucleotide sequence isolated from the recombinant plasmid pcDNA3/KiSS-1 was the same as expected. Conclusion: The successful construction of the recombinant plasmid pcDNA3/KiSS-1 will be benefit to further study in the protein expression and the antibody production.