广西巴马小型猪TRβ1基因真核表达载体的构建及鉴定

发布时间：2018-11-12 06:47

【摘要】：本试验为了克隆广西巴马小型猪甲状腺激素受体β1(TRβ1)基因编码序列并构建该基因的真核表达载体,取广西巴马小型猪肝脏组织作为材料,采用RT-PCR技术扩增出TRβ1基因编码序列,与pMD19-T-Simple载体连接,测序正确的重组质粒双酶切后,连接pEGFP-N1载体,构建pEGFP-N1-TRβ1真核表达载体。经双酶切和测序鉴定后,重组质粒pEGFP-N1-TRβ1转染293T细胞,培养48 h后,在荧光显微镜下观察细胞荧光蛋白的表达情况。结果表明,本试验成功克隆了广西巴马小型猪的TRβ1基因cDNA序列,序列长度为1 368 bp,编码455个氨基酸,与参考序列的同源性为99.6%,有5处位点突变,且全为同义突变。阳性重组质粒pEGFP-N1-TRβ1转染293T细胞48 h后,在荧光显微镜下观测出绿色荧光表达。
[Abstract]:In order to clone the encoding sequence of thyroid hormone receptor 尾 1 (TR 尾 1) gene from Guangxi Bama miniature pig and construct its eukaryotic expression vector, the liver tissue of Guangxi Bama miniature pig was used as the material. The coding sequence of TR 尾 1 gene was amplified by RT-PCR technique and ligated with pMD19-T-Simple vector. The recombinant plasmid was digested by double enzyme and ligated with pEGFP-N1 vector to construct pEGFP-N1-TR 尾 1 eukaryotic expression vector. After double enzyme digestion and sequencing, the recombinant plasmid pEGFP-N1-TR 尾 1 was transfected into 293T cells. After 48 h culture, the expression of fluorescent protein was observed under fluorescence microscope. The results showed that the TR 尾 1 gene cDNA sequence of Guangxi Bama mini-pig was cloned successfully. The length of the sequence was 1 368 bp, encoding 455 amino acids, and the homology was 99.6 with reference sequence. There were 5 locus mutations and all of them were synonymous mutations. The positive recombinant plasmid pEGFP-N1-TR 尾 1 was transfected into 293T cells for 48 h, and the green fluorescent expression was observed under fluorescence microscope.
【作者单位】：广西大学动物科学技术学院动物遗传育种实验室;
【基金】：国家现代农业技术体系广西生猪创新团队(nycytxgxcxtd-03-15) 广西科技基础条件平台建设项目(15-235-04)共同资助
【分类号】：Q78;R73

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