人KiSS-1基因克隆及其慢病毒载体的构建**★

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。

Constructing lentivirus vector carrying human KiSS-1 gene cloning

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-1, 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-1 has been constructed successfully.