西农萨能羊乙酰/丙二酸单酰基转移酶基因重组腺病毒载体的构建

摘要

The aim of this study was to observe the role of the Malonyl/Acetyl transferase (MAT) region of goat fatty acid synthase (FASN) in fatty acids metabolism of goat mammary, and to construct the recombinant adenovirus vector with goat MAT for observing gene overex-pression in primary goat mammary epithelial cells. The primers were designed according to the MAT sequence in GenBank, and the gene was cloned and sequenced by RT-PCR. The goat gene fragments containing both MAT region and HIS sequence were inserted into shuttle vector pAdTrack/CMV to construct recommbinant shuttle vector pAdTrack/CMV-MAT-HIS. After identifying by digestion and sequencing, pAdTrack/CMV-MAT-HIS plasmid was linearized by Pme I , and then it was transformed into E. Coli BJ5183 competent cells containing backbone vector pAdEasy I to obtain recombinant vector pAdEasy-MAT-HIS by homologous recombination. pAdEasy-MAT-HIS plasmid was linearized by Pac I , and transfected into HEK 293 cells for virus packing, amplification and titer testing by LaSRT. The MAT sequence cloned had a G-A mutation at 601 bp by comparing with the corresponding sequence in GenBank, which caused an Ala-Thr mutation at the 201st amino acid residue. The results of PCR detection and Western blot showed that recombinant overexpression vector containing MAT was successfully constructed. The titer of virus obtained was 2 X 109PFU · mL-1. The recombinant adenoviral vector pAdEasy-MAT-HIS was constructed successfully in this experiment.%本研究旨在通过构建西农萨能羊脂肪酸合酶(FASN)基因乙酰/丙二酸单酰基转移酶(MAT)区域的重组腺病毒载体,为研究其在奶山羊乳腺上皮细胞中过表达以及功能和作用机制做准备.根据GenBank收录的西农萨能羊MAT序列设计引物,PCR扩增并克隆测序.将目的基因连接到穿梭载体pAdTrack/CMV上并线性化后,转化含有腺病毒骨架载体pAdEasy Ⅰ的Ecoli Bj5183感受态细胞进行同源重组,得到重组腺病毒质粒pAdMAT-HIS,并用Pac Ⅰ酶切鉴定,将经过Pac Ⅰ线性化的pAd-MAT-HIS转染HEK 293细胞进行病毒包装和扩增,用LaSRT法测定病毒滴度.将本次克隆的MAT序列与GenBank收录的山羊序列对比发现,在601 bp处碱基由G转变为A,导致氨基酸序列由丙氨酸(Ala)201转变为苏氨酸(Thr) 201.酶切鉴定、绿色荧光蛋白观察、PCR及Western blot检测均证明,重组腺病毒质粒构建成功,病毒滴度为2×109 PFU·mL-1.本研究成功构建了重组腺病毒pAdEasy-MAT-HIS.