人KiSS-1基因克隆及其慢病毒载体的构建

Constructing lentivirus vector carrying human KiSS-1 gene cloning

背景:慢性病毒载体技术是目前转基因中最有效和最成功的方法,技术操作简便。目的:克隆转移抑制基因KiSS-1基因不含信号肽的表达序列,构建人KiSS-1基因的慢病毒表达载体。设计、时间及地点:开放性实验于2006-09/2007-12在福建师范大学发育学院实验室完成。材料:载体pNL-IRES2-EGFP由福建师范大学发育学院实验室保存。方法:从人正常胎盘组织中提取总RNA,经反转录-聚合酶链反应得到KiSS-1基因开放阅读框cDNA序列,并将其克隆到慢病毒载体pNL-IRES2-EGFP中,构建其表达质粒pNL-IRES2-EGFP-KiSS-1。主要观察指标:KiSS-1目的基因片段的克隆,重组表达质粒pNL-IRES2-EGFP-KiSS-1的酶切鉴定及测序。结果:经酶切鉴定和基因序列测定,证实重组入载体pNL-IRES2-EGFP的片段为目的基因开放阅读框的核苷酸序列。结论:成功构建了重组质粒pNL-IRES2-EGFP-KiSS-1。

BACKGROUND： Construction of lentivirus vector has the advantages of simplicity, it is regard as the most effective and successful method in transgene therapy. OBJECTIVE： To clone the metastasis suppressor gene KISS-1 from human normal placenta tissue and construct its lentivirus vector DESIGN, TIME AND SETTING： The open experiment was performed at the Laboratory of Fujian Normal University from September 2006 to December 2007. MATERIAL： The pNL-IRES2-EGFP vector was conservated by the Laboratory of Fujian Normal University. METHODS： Total RNA was extracted from human placenta tissue. The opening reading frame cDNA of KISS-1 was isolated by using RT-PCR, and cloned into its lentiviral vector pNL-IRES2-EGFP to construct expression plasmid pNL-IRES2-EGFP-KiSS-1. MAIN OUTCOME MEASURES： Clone objective gene fragment of KiSS-l, restriction enzyme digestion and gene sequencing of the recombinant plasmid pNL-IRES2-EGFP-KiSS-1 were observed in the study. RESULTS： The nucleotide sequence isolated from the recombinant plasmid pNL-IRES2-EGFP-KiSS-1 was confirmed the same as expected by restriction enzyme digestion and gene sequencing. CONCLUSION： The recombinant plasmid pNL-IRES2-EGFP-KiSS-I has been constructed successfully.