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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。
In this study, in order to clone the coding sequence of thyroid hormone receptor β1 (TRβ1) in Guangxi Bama miniature pig and to construct the eukaryotic expression vector of the gene, we use the Bama miniature pig liver tissue of Guangxi as a material to amplify TRβ1 The gene encoding sequence was ligated with the pMD19-T-Simple vector and double-digested with the correct recombinant plasmid. The pEGFP-N1 vector was ligated to construct the eukaryotic expression vector pEGFP-N1-TRβ1. After double enzyme digestion and sequencing, the recombinant plasmid pEGFP-N1-TRβ1 was transfected into 293T cells. After cultured for 48 h, the expression of fluorescent protein was observed under a fluorescence microscope. The results showed that the cDNA sequence of TRβ1 gene was successfully cloned in Guangxi Bama miniature pig with a length of 1 368 bp encoding 455 amino acids with 99.6% homology with the reference sequence and 5 mutations at All synonymous mutations. After transfected 293T cells with pEGFP-N1-TRβ1, the expression of green fluorescent protein was observed under a fluorescence microscope.