畜牧兽医学报2012，Vol.43Issue(5)：684-692,9.
徐淮山羊H-FABP基因克隆、表达产物亚细胞定位的研究及转基因小鼠的制备
Gene Clone, Subcellular Localization of Expression Products of H-FABP and the Preparation of Transgenic Mice in Xuhuai Goat
摘要
Abstract
The purpose of this study was to clone heart-type fatty acid binding protein (H-FABP) gene cDNA of Xuhuai goat, and to explore its bioinformatics function and the possibility of preparation of transgenic animals among heterogeneous species. The subcelluar location H-FABP was detected by EGFP fusion protein and its expression was observed in vitro. Reverse transcription PCR (RT-PCR) technology was used to clone the H-FABP gene cDNA of Xuhuai goat, its biological information characteristics was analyzed by online software, then the expression vector pEGFP-H-FABP was constructed. The transfection of goat fibroblasts (GEF) was performed by Liposomes (LTX), and fluorescence was observed under inverted microscope after 48 h. The RT-PCR was conducted to detect mRNA expression of H-FABP in GEF. The pEGFP-H-FABP was injected into mouse testicular and its expression was detected at the level of DNA and protein. The complete CDS size of H-FABP was 402 bp, encoding 133 amino acids with GenBank accession number (AY466498.1). The H-FABP cDNA coding sequence was compared with the corresponding regions of human, chicken, brown rat, cow, wild boar, donkey and zebra fish, the similarity was 89% , 76%, 85% , 84% , 93% , 91% , 70% , respectively, amino acid sequence homol-ogy was 90%, 79%, 88%, 97%, 95%, 94%, 72%, respectively. The signal peptide was not found in H-FABP protein. The RT-PCR results showed the H-FABP mRNA expressed successfully in vitro. pEGFP-H-FABP was successfully constructed, and H-FABP mRNA was expressed. The H-FABP protein was localized in the cytoplasm which was in line with the result of online prediction. The gene can aslo be expressed in mice transiently and persistencely after intravenous and testicular injection. The H-FABP gene cDNA of Xuhuai goat was cloned successfully, and it was conservative during the evolutionary process, there was no signal peptide in protein. The H-FABP protein was located in the cytoplasm, and also could be expressed in mice successfully.关键词
徐淮山羊/心脏型脂肪酸结合蛋白/真核表达/荧光蛋白/转基因鼠Key words
Xuhuai goat/ heart-type fatty acid binding protein/ eukaryotic expression/ fluorescent protein/ transgenic mice分类
农业科技引用本文复制引用
阴彦辉,韦光辉,李伟,朱才业,张亚妮,杜立新,曹文广,李碧春..徐淮山羊H-FABP基因克隆、表达产物亚细胞定位的研究及转基因小鼠的制备[J].畜牧兽医学报,2012,43(5):684-692,9.基金项目
国家转基因重大专项(2008ZX08008-003 （）
2009ZX08008-003B) （）
