腺病毒介导的秦川牛固醇调节元件结合蛋白2基因(SREBP2)过表达载体的构建与鉴定

摘要

Sterol regulatory element binding protein 2(SERBP2) is a basic-helix-loop-helix-luecine zipper factor which regulates the metabolization process of cholesterol.We cloned the SREBP2 gene from Qinchuan cattle (Bos taurus) and constructed the overexpression adenoviral vector,and packed and amplified the virus for a high titer,as a antecedent work for the further study of cellular level function of SERBP2 gene.Total RNA was extracted from the adipose tissue of Qinchuan cattle and then reversely transcripted to cDNA.A pair of exclusive primers were designed according to the GenBank sequence information of SREBP2 gene(Accession No.NM_001205600) to amplify the complete coding sequence(CDS) area of SREBP2 gene by polymerase chain reaction (PCR).The fragments containing CDS area of SREBP2 gene were inserted into the shuttle vector to construct the pAdTrack-CMV-SREBP2 plasmid.The recombinant plasmid and the blank control pAdTrack-CMV were linearized by digesting with restriction endonuclease Pine Ⅰ and subsequently transformed into Escherichia coli B J5183 containing pAdEasy-1 to homologous recombine and obtain the recombinant adenovirus plasmid pAd-SREBP2 and pAd-CMV.And then,the confirmed recombinant adenovirus plasmid pAd-SREBP2 was digested with Pac Ⅰ and transfected into 293A cell line to package and amplify the recombinant adenovirus Ad-SREBP2 and Ad-CMV,and to collect virus of high titer.The viral titer of AdSREBP2 and Ad-CMV was 7 × 108 and 1.3 × 109 GFU/mL respectively,measured by green fluorescent protein (GFP) labelled method.Qinchuan cattle derived preadipocyte was infected by Ad-SREBP2 and Ad-CMV to verify the availability of the virues.The expression of SREBP2 increased by 102.3 times after infected with the recombinant adenovirus for 48 h,determined by quantitative Real-time PCR.The cloning of SERBP2 gene of Qinchuan cattle obtaining of recombinant adenoviru and virus of high titer are set as foundation for the studies of the gene function on cellular level.%固醇调节元件结合蛋白2(sterol regulatory element binding protein 2,SREBP2)属于碱性螺旋-环-螺旋亮氨酸拉链转录因子家族,在胆固醇的代谢过程中具有重要的调控作用.克隆秦川牛(Bos taurus)的SREBP2基因并构建相应腺病毒过表达载体,包装扩繁获得高滴度腺病毒,对于在细胞水平上研究SREBP2基因的功能和作用机制具有重要作用.本研究以秦川牛脂肪组织为实验材料,提取总RNA并反转录为cDNA,依据GenBank收录的牛的SREBP2基因(Accession NO.NM_001205600)mRNA序列设计引物,克隆出SREBP2基因编码区(coding sequence,CDS)全长.将SREBP2基因与穿梭载体连接构建pAdTrack-CMV-SREBP2表达载体,用PmeⅠ分别酶切线性化pAdTrack-CMV-SREBP2和空白对照pAdTrack-CMV载体并转化含有骨架载体pAdEasy-1的大肠杆菌(Escherichia coli)BJ5183感受态进行同源重组,得到腺病毒重组载体pAd-SREBP2和pAd-CMV,分别用Pac Ⅰ酶切后胶回收DNA大片段,并将回收产物转染293A细胞包装得到腺病毒Ad-SREBP2和Ad-CMV,增殖并提高病毒滴度,得到高滴度病毒后,用绿色荧光蛋白(green fluorescent protein,GFP)标记法测定显示Ad-SREBP2和Ad-CMV的病毒滴度分别为7×108和1.3×109 GFU/mL.将Ad-SREBP2和Ad-CMV腺病毒侵染秦川牛前体脂肪细胞检测病毒的有效性,实时定量PCR检测结果显示,侵染48h时SREBP2基因的表达量提高了102.3倍.本研究成功克隆了秦川牛的SREBP2基因,构建了病毒重组体,包装增殖获得了高滴度病毒,为在细胞水平上基因功能的研究提供了基础资料.