AHA1蛋白与A型核纤层蛋白在细胞中的共定位  

AHA1 protein colocalizes with A-type lamins in cells

作　　者：蒋智文[1,2] 陈维春[1,2,3] 郑慧玲[1,2] 周静[1,3] 刘新光[1,2,3] 

机构地区：[1]广东医学院衰老研究所,广东东莞523808 [2]广东省医学分子诊断重点实验室,广东东莞523808 [3]广东医学院生物化学与分子生物学研究所,广东湛江524023

出　　处：《国际检验医学杂志》2013年第11期1345-1347,1350,共4页International Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(30672205;30871440;30971620;81170327);广东省自然科学基金资助项目(8452402301001450);广东省高等学校自然科学研究重点项目(06Z015);广东医学院科技创新基金资助项目(STIF201102);广东医学院青年基金资助项目(Q2010009)

摘　　要：目的构建AHA1与荧光蛋白GFP融合的真核表达载体,并在HEK293细胞中进行表达,然后检测AHA1重组蛋白在细胞中的表达,以及与A型核纤层蛋白在细胞中的共定位。方法提取HEK293细胞中总RNA,随之逆转录成cDNA,然后设计引物扩增AHA1的编码序列后构建到pEGFP-N1载体中,转染HEK293细胞后,通过免疫印迹检测重组AHA1蛋白在细胞中的表达,采用激光共聚焦显微镜观察重组AHA1蛋白与A型核纤层蛋白在细胞中的共定位。结果经PCR和DNA限制性内切酶鉴定,最后通过DNA测序并进行序列相似性分析比对,确定pEGFP-N1-AHA1重组质粒构建成功;在HEK293细胞中转染pEGFP-N1-AHA1重组质粒后,检测发现,重组AHA1蛋白在细胞中已经成功表达,并且部分AHA1蛋白与核纤层蛋白A共定位于细胞的核膜上,但是AHA1蛋白与核纤层蛋白C在细胞中不能共定位。结论成功构建了AHA1与GFP荧光蛋白的重组质粒,并且AHA1蛋白与核纤层蛋白A在细胞中能够共定位,而不与核纤层蛋白C共定位。Objective To study the function of AHA1 in cells,we constructed a recombinant plasmid pEGFP-N1-AHA1 and observed AHA1 fusion protein expression and co-localization with A-type lamin proteins in the HEK293 cells after transfection.Methods HEK293 cells′ total RNA was extracted and reversed transcribed to cDNA,and applied AHA1 gene primer constructed pEGFP-N1-AHA1 recombinant plasmid.Finaly,AHA1 fusing with GFP fluorescence protein expression detected by Western Blotting,and AHA1 protein co-localized with A-type lamin proteins were observed by confocal laser microscope.Results pEGFP-N1-AHA1 recombinant plasmid was identified by PCR and DNA digestion,and confirmed by DNA sequencing and BLAST compared.AHA1 fusion protein had been over-expressed in HEK293 cells,and compared to lamin C,some of AHA1 fusion protein co-localized with laminA on the nuclear membrane.Conclusion The pEGFP-N1-AHA1 recombinant plasmid has been successfully constructed,over-expressed AHA1 fusion in HEK293 cells.AHA1 fusion protein co-localized with laminA on the nuclear membrane has been found.

关 键 词：AHA1蛋白 A型核纤层蛋白 载体 免疫印迹 

分 类 号：R363[医药卫生—病理学]