准噶尔小胸鳖甲抗冻蛋白MpAFP698在真核细胞中的表达与鉴定  

Expression and Characterization of Microdera punctipennis dzhungarica Antifreeze Protein MpAFP698 in Eukaryotic Cell

作　　者：姜敏[1] 于继云[2] 李晨晨[1] 马纪[1] 

机构地区：[1]新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室,新疆乌鲁木齐830046 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《生物技术通讯》2011年第2期154-157,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30760056);新疆生物资源基因工程重点实验室开放课题(XJDX0201-200804)

摘　　要：目的:构建真核表达质粒pEGFP-N1-mpafp698,转染人胚肾293T细胞,并表达准噶尔小胸鳖甲抗冻蛋白MpAFP698。方法:PCR扩增出mpafp698序列,将其克隆入本室保存的真核表达载体pEGFP-N1中,转染293T细胞;利用RT-PCR、流式细胞仪、免疫荧光、Western印迹检测蛋白表达。结果:构建了真核表达载体pEGFP-N1-mpafp698,在转染后24 h可检测到绿色荧光的表达,而对照组则没有检测到荧光蛋白;Western印迹结果显示表达蛋白的相对分子质量约37 000。结论:为准噶尔小胸鳖甲抗冻蛋白的细胞表达及功能研究奠定了基础。Objective：To construction eukaryotic expression vector pEGFP-N1-mpafp698 and express Microdera punctipennis dzhungarica antifreeze protein MpAFP698 in eukaryotic cell 293T by transfection.Methods：The segments of mpafp698 was obtained by the method of PCR and was inserted into a eukaryotic expression plasmid pEGFP-N1.The recombinant expression plasmid was tansfected into eukaryotic cell 293T,and the expression was detected by RT-PCR,FACS,Western blot and IMF.Results：The recombinant plasmid pEGFP-N1-mpafp698 has been constructed and expressed successfully in the 293T cells.Green fluorescent protein could be detected in the transfected 293T cell after 24 hours,then no fluorescent protein detected in control group.The result of Western blot showed His-MpAFP698 fusion protein with the molecular weight of about 37 kD.Conclusion：This work laid a foundation for the cell level expression and the function research of antifreeze protein from Microdera punctipennis dzungarica.

关 键 词：准噶尔小胸鳖甲 抗冻蛋白 细胞表达 

分 类 号：Q78[生物学—分子生物学]