肝癌相关基因HTA的重组表达及蛋白功能研究  

Recombinant expression of hepatoma associated gene and its protein function

作　　者：宋婕[1] 刘艳红[1] 李亚林[1] 李官成[1] 

机构地区：[1]中南大学肿瘤研究所,癌变与侵袭原理教育部重点实验室,卫生部癌变原理重点实验室,长沙410078

出　　处：《中南大学学报（医学版）》2011年第7期655-661,共7页Journal of Central South University ：Medical Science

基　　金：湖南省研究生科研创新项目(CX2011B050)~~

摘　　要：目的:将肝癌相关基因HTA基因(hepatoma associated gene)进行重组表达,并对HTA蛋白功能进行初步研究。方法:从肝癌细胞系HepG2细胞中扩增得到HTA338-616cDNA,再将其克隆到原核表达载体pET21a(+)-MBP内,诱导表达MBP-HTA融合蛋白及MBP蛋白,His-tag磁珠纯化试剂盒纯化2种蛋白,并用Western印迹及ELISA方法进行活性鉴定。分别用MBP,MBP-HTA蛋白刺激并培养HepG2细胞,采用MTT法、平板克隆形成实验观察细胞增殖情况,流式细胞术检测刺激前后细胞周期分布的改变。结果:成功地构建了表达分子质量约为52 kD的MBP-HTA融合蛋白的原核表达质粒pET21a(+)-MBP-HTA。与MBP刺激后的HepG2细胞及阴性对照组相比,MBP-HTA刺激后的HepG2细胞增殖明显;流式细胞术显示MBP-HTA刺激后的HepG2细胞G1期细胞减少,S期细胞增多,细胞增殖活性增强。结论:HTA蛋白可以明显地促进HepG2细胞增殖,这可能与其促进细胞从G1期过渡到S期有关。Objective To recombinant express hepatoma associated gene（HTA） and pre-test the function of HTA to determine the role of HTA in the development of liver cancer.Methods HTA338-616 was amplified from HepG2 cells and cloned into the prokaryotic expression vector pET21a（＋）-MBP.The proteins MBP and MBP-HTA were induced,purified by His-tag magnetic bead purification kit and identified by Western blot and ELISA.HepG2 cells were stimulated with MBP or MBP-HTA proteins.MTT assay and colony formation assay were employed to examine the proliferation of these cells and the changes of cell cycle distribution were determined by flow cytometry.Results The prokaryotic expression plasmid pET21a（＋）-MBP-HTA was successfully constructed.We got a 52 kD purified purpose protein.The proliferation of HepG2 cells stimulated with MBP-HTA was significantly higher than those stimulated with MBP and negative controls.HepG2 cells stimulated with MBP-HTA showed significant decrease fraction in G1 phase and increase fraction in S phase,and the cell proliferation was enhanced.Conclusion HTA protein can significantly promote the proliferation of HepG2 cells,which may be related to the promotion of G1 phase to S phase.

关 键 词：肝细胞癌 肝癌相关基因 原核表达 细胞增殖 

分 类 号：R735.7[医药卫生—肿瘤]