SB转座子介导的hLF乳腺表达载体的构建及鉴定  

Construction and identification of mammary gland expression vector of human lactoferrin mediated by Sleeping Beauty transposon

作　　者：宋洋[1] 王子玉[1] 张艳丽[1] 钟部帅[1] 张国敏[1] 孟立[1] 黄荣[1] 王昌龙[1] 闫益波[1] 王锋[1] 

机构地区：[1]南京农业大学动物胚胎工程技术中心,江苏南京210095

出　　处：《江苏农业学报》2011年第4期795-801,共7页Jiangsu Journal of Agricultural Sciences

基　　金：高产优质转基因奶羊新品种培育项目(2008ZXD8008004)

摘　　要：构建了以睡美人(Sleeping beauty,SB)转座子为载体元件、β-乳球蛋白质(β-lactoglobulin,BLG)为调控区的人乳铁蛋白质(Human lactoferrin,hLF)乳腺表达载体及增强型绿色荧光蛋白质(EGFP)表达载体,便于验证SB转座子的转座效率,为hLF乳腺表达载体成功转染乳腺上皮细胞(GMC)奠定基础。采用PCR法从绵羊基因组中扩增5'、3'BLG,经TA克隆,在中间载体pcDNA3.1(-)的作用下,与hLF连接,得到pcDNA3.1(-)-BLG-hLF,然后与pT2连接,构建pT2-BLG-hLF;此外,还构建了pT2-BLG-EGFP。采用测序、酶切、PCR 3种方法鉴定阳性克隆,并用脂质体法将pT2-BLG-EGFP转染至GMC细胞中进行观察。分子鉴定结果:5'BLG、3'BLG克隆成功,pT2-BLG-hLF及pT2-BLG-EGFP构建正确;细胞鉴定结果:SB转座子介导的EGFP能在GMC细胞中高效表达。成功克隆了5'BLG、3'BLG调控区,并成功构建了乳腺表达载体pT2-BLG-hLF。A mammary gland expression vector of human lactoferrin（hLF） and an enhanced green fluorescent protein（EGFP） expression vector were constructed by using Sleeping Beauty（SB） transposon as vector components and β-lactoglobulin（BLG） as regulatory region,so that the transposition efficiency of SB transposon could be affirmed.In addition,it would lay a foundation for the further research on the transfection of mammary gland expression vector of hLF mediated by SB transposon into mammary gland epithelial cells（GMC）.The 5′BLG and 3′BLG were amplified by PCR from genomic DNA of sheep,which were connected with pMD19-T vector respectively by TA cloning.After the TA cloning,connecting hLF with the help of pcDNA3.1（-） as an intermediate vector to get pcDNA3.1（-）-BLG-hLF,which was connected with pT2 to construct the finally correct vector pT2-BLG-hLF.The positive clone was testified by restriction enzyme digestion,PCR and agarose gel electrophoresis.Then pT2-BLG-EGFP were transfected into mammary gland epithelial cells by LipofectamineTM2000 for EGFP detection.The result indicated by the enzyme digestion and PCR demonstrated that the 5′BLG and 3′BLG were cloned precisely,and the construction of pT2-BLG-hLF and pT2-BLG-EGFP were under anticipation.The result indicated by cells transection demonstrated that EGFP mediated by SB transposon was able to express in mammary gland epithelial cells.It was concluded that the 5′BLG and 3′ BLG regulatory region cloning and mammary gland expression vector pT2-BLG-hLF were constructed successfully.

关 键 词：“睡美人”(SB)转座子 Β-乳球蛋白 乳腺表达载体 乳腺上皮细胞 

分 类 号：Q785[生物学—分子生物学]