通用型动物转基因载体的构建及验证  

Construction and verification of an universal transgenic vector for animals

作　　者：丰秀静[1,2] 田石[2] 蒋进[2] 孟春花[1] 茆达干[2] 曹少先[1] 

机构地区：[1]江苏省农业科学院畜牧研究所,江苏南京210014 [2]南京农业大学动物科技学院,江苏南京210095

出　　处：《江苏农业学报》2012年第5期1083-1087,共5页Jiangsu Journal of Agricultural Sciences

基　　金：转基因生物新品种培育科技重大专项(2009ZX08008-009B)

摘　　要：为了构建通用型动物转基因载体,以pBluescriptⅡKS+为基础,根据标记基因所含酶切位点,设计改造多克隆位点,插入从pTARGET载体中扩增的SV40启动子及新霉素磷酸转移酶基因(Neo),其下游插入共表达序列、增强型绿色荧光蛋白质基因(EGFP)及SV40 poly A,并在SV40启动子上游和SV40 poly A下游各添加一个同向的LoxP位点,构建成通用载体pNIGFP,标记基因上游拥有Xho I、Sal I、SacⅡ多克隆位点,下游拥有Nhe I、Cla I、Sac I位点。酶切鉴定和测序表明pNIGFP构建正确。pNIGFP脂质体法转染山羊胎儿成纤维细胞,荧光显微镜下观察到EGFP高表达。遗传霉素(G418)筛选表明,Neo基因表达正常,山羊胎儿成纤维细胞的最适筛选浓度为600"g/ml。pNIGFP可利用G418进行抗性筛选,还可通过EGFP表达对外源基因的整合进行实时跟踪,另外,该载体具有LoxP位点,外源基因整合后可用Cre重组酶去除标记基因,具有高效、方便、安全的特点。In order to construct a universal transgenic vector for animals,the SV40 promoter and neomycin gene（Neo） were amplified from the pTARGET vector and cloned into the modified multiple clone sites（MCS） of pBluescriptⅡ KS＋ vector.Then,co-expression sequence,enhanced green fluorescence protein（EGFP） and SV40 polyA were cloned into MCS downstream.At the upstream of SV40 promoter and the downstream of SV40 poly A,the same direction LoxP（locus of crossing-over（x） in P1） site was inserted,respectively,and formed a universal vector pNIGFP.There were Xho I,Sal I and Sac II enzyme sites at the forward of the marker gene and Nhe I,Cla I and Sac I enzyme sites at the behind.The restriction enzymatic digestion and sequencing showed that vector pNIGFP was successfully constructed.pNIGFP was transfected into dairy goat fetal fibroblast cells（gFFCs） by LipofectaminTM LTX and PLUSTM Reagent.High-level expression of EGFP was observed under fluorescence microscope after 24 h.Successful expression of Neo was evaluated with the G418 selection in the gFFCs transfected pNIGFP,and the optimal selection dosage was 600 μg/ml.The pNIGFP vector contained a Neo marker gene to facilitate the enrichment of G418 resistant transgenic cells as well as an EGFP marker gene to facilitate the real-time tracking of exogenous gene integration.Two LoxP sites beside the markers could be used to knock out the dual markers by Cre/LoxP system.In conclusion,an efficient,convenient and safe general transgenic vector was constructed.

关 键 词：转基因通用载体 共表达序列 新霉素磷酸转移酶基因 增强型绿色荧光蛋白质基因 LOXP 

分 类 号：Q782[生物学—分子生物学]