人MxA基因重组真核载体的构建及鉴定  

Construction and identification of the recombinant eucaryotic plasmid PcDNA3.1-MxA

作　　者：黄琴[1] 赵瑞秋[1] 刘爱平[1] 许红梅[1] 

机构地区：[1]重庆医科大学附属儿童医院感染消化科,重庆400014

出　　处：《重庆医科大学学报》2008年第2期152-154,249,共4页Journal of Chongqing Medical University

摘　　要：目的:应用真核载体PcDNA3.1构建含我国健康人目的基因MxA的重组真核载体PcDNA3.1-MxA。方法:在大肠杆菌JM109中扩增前期已构建的含有目的基因MxA的PAdtrack-MxA和真核载体PcDNA3.1,使用两者共同的限制性内切酶XbaⅠ、NotⅠ进行双酶切,并连接以构建重组真核载体PcDNA3.1-MxA,然后通过氨苄青霉素抗性筛选,双酶切及PCR鉴定,选取鉴定正确的克隆测序,并应用DNAssist 2.0软件对序列与前期获得的基因序列以及已知GenBank中MxA基因序列进行同源性分析。结果:经限制性内切酶、PCR鉴定重组真核载体PcDNA3.1-MxA构建成功。测序结果表明本实验所克隆片段长2012bp,包含人MxA基因序列,核苷酸序列分析表明与前期获得的目的基因序列完全相同,而与GenBank中人MxA基因序列同源性也达99.75%,仅有5个碱基不同,且所编码的氨基酸仅1个发生改变,此氨基酸位于MxA蛋白的非功能区。结论:重组真核载体PcDNA3.1-MxA构建成功,为下一步研究MxA抗乙型肝炎病毒作用奠定了基础。Objective：To construct and identify the recombinant eukaryotic plasmid PcDNA3.1-MxA encoding MxA with the eukaryotic plasmid PcDNA3.1. Methods：Recombinant plasmid PAdTrack-MxA constructed before and PcDNA3.1 were amplified in Escherchia coli JM109. Plasmids were double-digested with restrictive endonucleases Not Ⅰ and Xba Ⅰ after extracted, and then ligated to construct recombinant eukaryotic plasmid PcDNA3.1-MxA. The recombinant plasmid was selected for Ampicillin resistance and then confirmed by digestion with Not Ⅰ and Xba Ⅰ ,PCR and sequencing. The sequence was homology-analysed compared with the sequence of MxA gene in PAdTrack-MxA and the sequence of MxA gene published in Genebank（NM_002462） with sofeware DNAssist 2.0. Results：The restrictive endonuclease and PCR analysis confirmed that the recombinant eukaryotic plasmid PcDNA3.1-MxA was constructed successfully. Sequencing analysis revealed that the cloned segment was 2012bp including MxA gene sequence, and the nucleotide sequence of MxA gene was same to that in PAdTrack-MxA exactly. There were five variations in nucleotide sequence compared with published sequence in Genebank, which caused only one amino acid residue replaced from isoleucine to valine. This replaced amino acid residue was located in the non functional area of MxA protein. Conclusions：The recombinant eukaryotic plasmid PcDNA3.1-MxA is constructed successfully, which lays foundation for the further study on anti-HBV effects of MxA.

关 键 词：MXA PCDNA3.1 PcDNA3.1-MxA 

分 类 号：R512.3[医药卫生—内科学]