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

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (5): 684-692.doi:

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

阴彦辉1，韦光辉1，李伟1，朱才业1，张亚妮1，杜立新2，曹文广2，李碧春1*

1. 扬州大学动物科学与技术学院江苏省动物遗传繁育与分子设计重点实验室，扬州 225009； 2. 中国农业科学院北京畜牧兽医研究所，北京 100193

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2012-05-28 发布日期:2012-05-28
通讯作者: 李碧春

Gene Clone, Subcellular Localization of Expression Products of HFABPand the Preparation of Transgenic Mice in Xuhuai Goat

YIN Yanhui1，WEI Guanghui1，LI Wei1，ZHU Caiye1，ZHANG Yani1，
DU Lixin2，CAO Wenguang2，LI Bichun1*

1. Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science & Technology, Yangzhou University, Yangzhou 225009, China; 2. Institute of Animal Science，Chinese Academy of Agricultural Sciences，Beijing 100193,China

Received:1900-01-01 Revised:1900-01-01 Online:2012-05-28 Published:2012-05-28
Contact: LI Bichun

摘要/Abstract

摘要： 旨在克隆徐淮山羊心脏型脂肪酸结合蛋白(HFABP)基因的cDNA，探讨其生物信息学功能。通过EGFP融合蛋白对该基因亚细胞水平的表达定位，探究该基因在异种生物体内表达情况,探索制备异种转基因动物的可能性。采用反转录PCR(RTPCR)方法克隆徐淮山羊HFABP基因cDNA，进行生物信息学分析，构建其融合表达载体pEGFPHFABP。脂质体(LTX)介导基因转染山羊成纤维细胞（GEF），48 h后进行荧光检测，RTPCR检测mRNA在细胞内的表达。利用睾丸注射将目的基因转入小鼠体内，在DNA和蛋白水平上检测目的基因的表达情况。结果，徐淮山羊HFABP基因完整编码区(CDS)大小为402 bp，编码133个氨基酸,GenBank登录号为AY466498.1。徐淮山羊HFABP序列与人、鸡、褐家鼠、奶牛、野猪、驴及斑马鱼相应编码区同源性为89%、76%、85%、84%、93%、91%、70%，氨基酸序列同源性为90%、79%、88%、97%、95%、94%、72%。成功构建融合表达载体pEGFP HFABP，目的基因在mRNA水平上成功表达。目的蛋白定位于细胞质中,与在线预测结果相同(无信号肽,定位于细胞质中)。通过尾静脉注射和睾丸注射,该基因可以在小鼠体内实现暂态表达和持续性表达。本研究成功克隆了徐淮山羊HFABP基因cDNA，而且HFABP基因在进化过程中是保守的。该蛋白无信号肽，在单细胞水平上可表达于细胞质中，在小鼠体内也可以成功表达。

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 homology 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.

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.

YIN Yanhui;WEI Guanghui;LI Wei;ZHU Caiye;ZHANG Yani;DU Lixin;CAO Wenguang;LI Bichun. Gene Clone, Subcellular Localization of Expression Products of HFABPand the Preparation of Transgenic Mice in Xuhuai Goat[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(5): 684-692.

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