重组质粒AHA1的构建和表达及对HeLa细胞周期的影响  

Detected cell cycle and proliferation after the recombinant protein AHA1 was expressed in HeLa cells

作　　者：蒋智文[1,2] 郑慧玲[1,2] 陈维春[1,2,3] 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8期913-915,918,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的真核表达质粒,并在HeLa细胞中进行表达,然后检测过表达的AHA1重组蛋白对HeLa细胞周期的影响。方法提取HeLa细胞中总RNA,逆转录成cDNA后,设计引物扩增AHA1基因的编码序列后构建到pCMV-HA真核表达载体中,转染HeLa细胞后使用HA标签抗体通过免疫印迹检测AHA1融合蛋白在细胞中的表达,最后分别使用流式细胞仪和CCK-8方法检测HeLa细胞的周期和增殖的变化。结果 pCMV-HA-AHA1重组质粒经PCR和DNA限制性内切酶酶切鉴定,最后通过DNA测序并经序列相似性比对,确定AHA1重组质粒构建成功;在HeLa细胞中转染pCMV-HA-AHA1重组质粒后,免疫印迹结果显示AHA1融合蛋白在细胞确已经实现过表达,经流式细胞仪和CCK-8方法检测发现,过表达的AHA1蛋白对HeLa细胞的周期和增殖影响不大。结论成功构建了pCMV-HA-AHA1重组质粒,并且在HeLa细胞成功过表达,且过表达的AHA1蛋白对HeLa细胞的周期和增殖影响不大。Objeetive To construct eukaryotic expression plasmid of AHA1 and express the recombinant protein in HeLa cells, and then detect HeLa cell cycle and proliferation. Methods HeLa cells＇ total RNA was extracted and reversed transcribed to cD-NA,and then constructed pCMV-HA-AHA1 recombinant plasmid. After recombinant vector was transfected in HeLa cells,con-firmed AHA1 fusion protein over-expressed by Western Blotting with HA antibody, and applied flow cytometry and CCK-8 assay detected cell cycle and cell proliferation. Results AHA1 recombinant vector was identified by PCR and DNA digestion,and was confirmed by DNA sequencing and BLAST compared. HeLa cell cycle and cell proliferation were almost unaffected by over-ex-pressed AHA1 fusion protein. Conclusion AHA1 recombinant plasmid was constructed and over-expressed in the HeLa cells,but over-expressed AHA1 has little effect on the cell cycle and cell proliferation.

关 键 词：HELA细胞 免疫印迹法 细胞周期 

分 类 号：R363[医药卫生—病理学]