中文摘要：

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。

英文摘要：

This experiment was aimed to clone apolipoprotein A1（ApoA1）gene of Congjiang Xiang pig,and study the subcelluar localiztion of ApoA1 gene in eukaryocyte.The recombination plasmid pEGFP-C1-ApoA1 was constructed with RT-PCR and other methods,and detected by colony PCR,double digestion and sequencing,after successful construction of the recombination plasmid pEGFP-C1-ApoA1,the subcellular localization of ApoA1 protein were analyzed by fluorescence co-localization technique in the 36h-transfected HEK-293 Tcells.Compared with ApoA1 gene of Sus scrofa submission in GenBank,the results showed that six base mutations were found in ApoA1 gene of Congjiang Xiang pig,five of above mentioned mutations were sense mutations,causing alanine to glutamic acid,histidine to glutamine,valine to leucine and aspartic acid to glycine in 180,185,186 and 209amino acid residues,respectively.Using PSOR Ⅱ Prediction and fluorescence co-localization,it was found that the expression of ApoA1 protein was observed mainly in the extracellular matrix（77.8%）.In conclusion,ApoA1 gene of CongjiangXiang pig was cloned successfully,and the expression of ApoA1 protein was mainly concentrated in the extracellular matrix.These results would provide a knowledge for further constructing the ApoA1 gene transgenic animal models,and contribute to understanding the relation between ApoA1 gene and the human obesity-induced diseases.