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摘要：【目的】克隆廣西巴马小型猪载脂蛋白A1（ApoA1）基因并进行生物信息学分析，为后续开展ApoA1基因研究提供基础数据，同时为制作广西巴马小型猪动脉粥样硬化疾病模型打下基础。【方法】以提取的广西巴马小型猪肝脏组织总RNA为模板，运用RT-PCR克隆广西巴马小型猪ApoA1基因序列，以实时荧光定量PCR（qPCR）检测ApoA1基因在广西巴马小型猪心脏、肝脏、脾脏、肺脏和肾脏组织中的表达情况，并利用MegAlign、ProtParam、SOPMA、NetPhos、SignalP和SWISS-MODEL等在线分析软件对广西巴马小型猪ApoA1基因进行生物信息学分析及构建系统发育进化树。【结果】广西巴马小型猪ApoA1基因编码区（CDS）序列长795 bp，与普通猪ApoA1基因序列的同源性为99.9%，仅第630处有1个碱基发生同义突变。ApoA1基因在广西巴马小型猪心脏、肝脏、脾脏、肺脏和肾脏组织中均有表达，且以肝脏中的相对表达量最高、脾脏中的相对表达量最低。广西巴马小型猪ApoA1由264个氨基酸残基构成，其分子式为C1343H2136N376O409S5，蛋白分子量为30254.31 Da，理论等电点（pI）为5.47，属于亲水性蛋白，存在跨膜结构，不存在信号肽，有18处磷酸化位点（丝氨酸磷酸化位点有10处，苏氨酸磷酸化位点有6处，络氨酸磷酸化位点有2处）。广西巴马小型猪ApoA1的二级结构中，α-螺旋占79.92%，β-折叠占2.65%，无规则卷曲占11.12%，延伸链占6.31%，属于全α类蛋白。广西巴马小型猪ApoA1氨基酸序列与普通猪的同源性最高，达99.6%;由基于ApoA1氨基酸序列同源性构建的系统发育进化树也可看出，广西巴马小型猪与普通猪的亲缘关系最近。【结论】广西巴马小型猪ApoA1基因保守性强，以在肝脏中的表达量最高，是广西巴马小型猪抗动脉粥样硬化的重要因子，通过敲除该基因可制作广西巴马小型猪动脉粥样硬化疾病模型。
关键词： 广西巴马小型猪;ApoA1基因;克隆;生物信息学分析;动物模型
中图分类号： S828.89 文献标志码： A 文章编号：2095-1191（2019）06-1339-08
Abstract：【Objective】The aim of this study was to clone the apoliprotein A1（ApoA1） gene of Guangxi Bama mini-pig， and analyze the biological information of ApoA1 gene， which would eich the basic data for the follow-up study of ApoA1 gene and lay a foundation for the production of the atherosclerosis disease model of Guangxi Bama mini-pig.【Method】Total RNA extracted from the heart and liver of Guangxi Bama mini-pig was as template， and the sequence of ApoA1 gene was cloned by RT-PCR. The expression of ApoA1 gene in heart， liver， spleen， lung and kidney of Guangxi Bama mini-pig was detected by real-time fluorescence quantitative（qPCR）. Online analysis softwares including MegAlign， ProtParam， SOPMA， NetPhos， SignalP and SWISS-MODEL were used to analyze the bioinformatics of ApoA1 gene and construct phylogenetic tree. 【Result】The length of coding sequence（CDS） region of ApoA1 gene of Guangxi Bama mini-pig was 795 bp and the homology was 99.9% compared with the ApoA1 gene sequence of pigs with a synonymy mutation on one base at the 630 position. The ApoA1 gene was expressed in heart， liver， spleen， lung and kidney of Guangxi Bama mini-pig with the highest expression in liver and the lowest expression in spleen.The molecular formula of ApoA1 gene encoding 264 amino acid residues in Guangxi Bama mini-pig was C1343H2136N376O409S5， the molecular weight was 30254.31 Da， and the theoretical pI was 5.47， belonging to hydrophilic protein. There was transmembrane structure， no signal peptide. There were 18 phosphorylation sites， including 10 sites of serine， 6 sites of threonine and 2 sites of tyrosine. The secondary structure of coding protein belonged to all-alpha protein， which consisted of α-helix， β-fold， random coil and extended strand， accounting for 79.92%， 2.65%， 11.12% and 6.31%， respectively. It was found that the homology of ApoA1 amino acid sequence between Guangxi Bama mini-pig and ordinary pig was the highest（99.6%）. According to phylogenetic tree based on ApoA1 amino acid sequence homology， Guangxi Bama mini-pig had the closest genetic relationship with pig. 【Conclusion】The sequence of ApoA1 gene in Guangxi Bama mini-pig is strongly conservative and expresses the highest in liver. It is an important factor of anti-atherosclerosis in Guangxi Bama mini-pig. The model of atherosclerotic disease in Guangxi Bama mini-pig can be produced by knocking out the ApoA1 gene.