CHD5基因慢病毒载体的构建及在结直肠癌细胞中的表达  

Construction of CHD5 gene lentiviral vector and its expression in colorectal cancer cells

作　　者：赵蕊[1] 吕静野[1] 严启滔[1] 张宝[1] 郑文岭[2] 马文丽[1] 

机构地区：[1]南方医科大学基因工程研究所,广东广州510515 [2]华南基因组研究中心,广东广州510800

出　　处：《基础医学与临床》2013年第4期418-422,共5页Basic and Clinical Medicine

基　　金：国家青年科学基金(81000226);广东省医学科研基金(A2011360)

摘　　要：目的构建重组慢病毒载体TREAutoR3-CHD5,并检测其在结直肠癌细胞系LOVO中的表达。方法采用PCR扩增技术从CHD5 cDNA克隆中获得CHD5的全长读码框,将CHD5基因导入慢病毒载体TREAutoR3中,酶切,测序验证后,重组慢病毒载体TREAutoR3-CHD5与慢病毒包装质粒pCMV-VSV-G,pRSV-Rev,pMDLg-pRRE共转染293FT细胞,将包装重组慢病毒感染结直肠癌细胞系LOVO。通过荧光定量PCR和Western bolt验证CHD5基因在细胞中的转录和表达情况,应用MTT检测CHD5过表达对LOVO细胞增殖的影响。结果成功构建了慢病毒载体TREAutoR3-CHD5,荧光定量PCR结果显示慢病毒感染LOVO细胞能稳定转录CHD5基因,Western bolt显示感染后LOVO细胞稳定表达CHD5蛋白。感染后的LOVO细胞的增殖受到抑制。结论包装的慢病毒能够成功的感染LOVO细胞,并且CHD5能抑制结直肠癌细胞系LOVO的增殖。expression Objective To construct the recombinant lentiviral vector containing human CHD5 gene and measure the level of CHD5 in LOVO cell. Methods The CHD5 fragment was amplified by PCR and cloned into lentiviral vetor TREAutoR3. The lentiviral voter TREAutoR3 CHD5 was identified. Recombinant lentiviral was pro- duced by 293FT cells following co-transfection of TREAutoR3 CHD5 with the packaging plasmids pCMV-VSV-G, pRSV-Rev and pMDLg-pRRE. Human Colorectal cancer LOVO cell Were infected by the recombinant lventiviruse. The expression of CHD5 was confirmed by RT-PCR and Western blot, and the proliferation of LOVO cells was eval- uated by MTY assay. Results The recombinant lentiviral vecor carried the CHD5 gene was successfully construc- ted. RT-PCR and western blot analysis revealed that the CHD5 can be correct transcript and translated in human colorectal cancer LOVO cell which infected by the recombinant lentiviral lenti CHD5. After infected, the growth of LOVO cell is inhibited obviously. Conclusions It can be deliver target gene CHD5 to human colorectal cancerLOVOcell, and CHD5 can inhibited the proliferation of colorectal cancer LOVO cell.

关 键 词：CHD5 慢病毒载体 肿瘤 

分 类 号：R735.3[医药卫生—肿瘤]