从江香猪ApoA1基因的克隆及亚细胞定位研究
发布时间:2018-10-26 20:17
【摘要】:试验旨在克隆从江香猪载脂蛋白A1(apolipoprotein A1,ApoA1)基因,研究ApoA1基因在真核细胞中的亚细胞定位情况。通过提取从江香猪总RNA,采用RT-PCR、目的基因的连接、转化等方法构建携带有绿色荧光蛋白的pEGFP-C1-ApoA1重组质粒,并经菌落PCR、双酶切及测序鉴定正确后,转染HEK-293T细胞,36h后观察荧光,分析ApoA1蛋白在真核细胞中的亚细胞定位情况。结果表明,从江香猪ApoA1基因与GenBank上公布的野猪序列相比,有6处发生了碱基突变,其中5处为有义突变,分别导致180位氨基酸由丙氨酸变为谷氨酸、185位氨基酸由组氨酸变为谷氨酰胺、186位氨基酸由缬氨酸变为亮氨酰胺、209位氨基酸由天冬氨酸变为甘氨酸;PSORTⅡPrediction和荧光共定位试验结果均表明,ApoA1蛋白的表达主要集中在细胞外基质,约占总表达量的77.8%。本试验成功克隆了从江香猪ApoA1基因CDS区,且ApoA1蛋白的表达主要集中在细胞外基质中,为进一步构建ApoA1基因转基因动物模型、开展ApoA1基因与人类因肥胖引起的相关疾病关系的研究奠定基础。
[Abstract]:The aim of this study was to clone the apolipoprotein A1 (apolipoprotein A1) gene of Congjiang Xiang pig and to study the subcellular localization of ApoA1 gene in eukaryotic cells. The recombinant plasmid carrying green fluorescent protein (GFP) was constructed by extracting total RNA, from Congjiang Xiang pig by ligation and transformation of RT-PCR, target gene. The recombinant plasmid was confirmed by double digestion of colony PCR, and sequencing. After transfection of HEK-293T cells, fluorescence was observed after 36 h, and the localization of ApoA1 protein in eukaryotic cells was analyzed. The results showed that there were 6 base mutations in the ApoA1 gene of Congjiang Xiang pig compared with the wild boar sequence published on GenBank, 5 of which were semantic mutations, which led to the amino acid change from alanine to glutamate, respectively. 185 amino acids changed from histidine to glutamine, 186 amino acids changed from valine to leucine, and 209 amino acids changed from aspartic acid to glycine. The results of PSORT 鈪,
本文编号:2296833
[Abstract]:The aim of this study was to clone the apolipoprotein A1 (apolipoprotein A1) gene of Congjiang Xiang pig and to study the subcellular localization of ApoA1 gene in eukaryotic cells. The recombinant plasmid carrying green fluorescent protein (GFP) was constructed by extracting total RNA, from Congjiang Xiang pig by ligation and transformation of RT-PCR, target gene. The recombinant plasmid was confirmed by double digestion of colony PCR, and sequencing. After transfection of HEK-293T cells, fluorescence was observed after 36 h, and the localization of ApoA1 protein in eukaryotic cells was analyzed. The results showed that there were 6 base mutations in the ApoA1 gene of Congjiang Xiang pig compared with the wild boar sequence published on GenBank, 5 of which were semantic mutations, which led to the amino acid change from alanine to glutamate, respectively. 185 amino acids changed from histidine to glutamine, 186 amino acids changed from valine to leucine, and 209 amino acids changed from aspartic acid to glycine. The results of PSORT 鈪,
本文编号:2296833
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