Construction of Eukaryotic Expression Vector for Pig Ghrelin Gene  

猪Ghrelin基因真核表达载体构建(英文)

作　　者：曹月胜[1] 陈俏俏 孙金海[1] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]莱阳市畜牧局,山东莱阳265200

出　　处：《Agricultural Science & Technology》2012年第6期1184-1185,1197,共3页农业科学与技术（英文版）

基　　金：Supported by Special Funds for Cultivation and Breeding of New Transgenic Organisms (2011ZX08006-003, 2009ZX08010-006B);Shandong Modern Agricultural Technology Innovation Program;the National Natural Science Foundation of China (No.30871778);Taishan Scholar Project of Shandong in China~~

摘　　要：[Objective] This study aimed to investigate the functions of transgenic growth related gene in pig growth. [Method] A pair of primers containing Nhe I and Hind Ⅲ restriction sites were designed by referring to the pig Ghrelin mRNA sequence published in Genbank. Total RNA was extracted from the small intestine tissue of 13/17 Robertson translocation heterozygous pig, and then was purified and used as the template in later RT-PCR reaction to amplify the full-length pig Ghrelin gene. The correct pig Ghrelin gene fragment was cloned into the pMD19-T simple vector for sequencing analysis. The obtained full-length cDNA of pig Ghrelin gene fragment was digested with both Nhe I and Hind Ⅲ, and then was linked into the eukaryotic expression vector pEGFP-N1 to obtain the recombinant plasmid pEGFPGhrelin. The recombinant plasmid was transected into the fibroblast cells to detect the fluorescence labeled gene expression. [Result] The nucleotide sequence extracted from 13/17 Robertson translocation heterozygous pig was the same as expected; and the eukaryotic expression vector pEGFP-Ghrelin was successfully constructed. [Conclusion] The eukaryotic expression vector constructed in this study can be further used in research on transgenic pigs, but also lays foundation for research on the regulatory mechanism of Ghrelin gene.[目的]为研究转生长相关基因对猪的作用。[方法]采用RT-PCR方法,从13/17罗伯逊易位杂合子猪小肠组织中提取总RNA,将其纯化后作为PCR扩增模板,参考GenBank公布的猪Ghrelin的mRNA序列设计合成具有Nhe I和Hind III双酶切位点引物,扩增获得Ghrelin基因cD-NA全长序列。将准确的Ghrelin基因片段克隆于 pMD19-T simple Vector后进行序列分析,获得猪Ghrelin基因cDNA全长基因片段。经Nhe I和Hind III双酶切,将 Ghrelin基因cDNA片段连接到真核表达载体pEGFP-N1,获得真核表达载体的重组质粒pEGFP-Ghrelin。重组质粒转染猪成纤维细胞,观察标记基因荧光蛋白的表达。[结果]13/17罗伯逊易位杂合子猪的Ghrelin基因与已发表的序列相同,并成功获得具备猪Ghrelin基因全长cDNA序列的真核表达载体pEGFP-Ghrelin。[结论]构建的真核表达载体可以用于转基因猪的进一步试验,同时也为研究Ghrelin的调节机制奠定基础。

关 键 词：Porcine growth hormone gene Eukaryotic expression vector TRANSGENIC 

分 类 号：S828[农业科学—畜牧学]