畜牧兽医学报2012，Vol.43Issue(3)：489-495,7.
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
Construction of the Recombinant Adenovirus of Malonyl/Acetyl Transferase of Xinong Saanen Goat
摘要
Abstract
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.关键词
奶山羊/乙酰/丙二酸单酰基转移酶/重组腺病毒Key words
dairy goat/ Malonyl/Acetyl transferase(MAT)/ recombinant adenovirus分类
农业科技引用本文复制引用
朱江江,罗军,王紫骞,许会芬,孙雨婷,石恒波,郝娟,赵丽媛..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[J].畜牧兽医学报,2012,43(3):489-495,7.基金项目
国家自然科学基因项目(31072013) （31072013）
转基因生物新品种培育项目(2009ZX08009-162B) （2009ZX08009-162B）
公益性行业(农业)科研专项(201103038) （农业）
