CHD5基因RNAi慢病毒载体的构建及其对结直肠癌细胞生物学功能的影响

Construction of Recombinant Lentiviral Vector of shRNA for CHD5 Gene and the Study of Its Functions in Human Colon Carcinoma Lovo Cells

目的:研究人类肿瘤相关基因CHD5基因miR-shRNA慢病毒对结直肠癌Lovo细胞的影响。方法:利用软件设计对CHD5基因干扰有效的序列,合成靶序列,退火形成双链DNA,酶切后与载体相连接。将重组慢病毒载体pPRIME-TET-GFP-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人类结直肠癌Lovo细胞。通过荧光定量PCR和Western blot验证CHD5基因在细胞中的表达情况,应用MTT检测CHD5低表达对Lovo细胞增殖的影响。结果:成功构建pPRIME-TET-GFP-CHD5重组质粒,经酶切及序列测定正确,包装的病毒滴度为3.1×106TU/ml。用制备的病毒上清感染Lovo细胞后,荧光定量PCR和Western blot分析结果显示该慢病毒可分别在转录和蛋白质水平上抑制Lovo细胞CHD5基因的表达,并使得Lovo细胞增殖失控。结论:成功构建CHD5慢病毒表达载体,表达的慢病毒可有效的感染Lovo细胞,提高Lovo细胞的增殖能力。

To study the effects of lentiviral vector of microRNA targeting CHD5 gene on human colon carcinoma Lovo cell.Methods： Specific small interference RNAs were designed on line software and screened with the optimize principles,synthesized and cloned into the pPRIME vetor.Recombinant lentiviral was producted by 293FT cells following co-transfection of pPRIME-TET-GFP-CHD5 with the packaging plasmids pCMV-VSV-G,pRSV-Rev and pMDLg-pRRE.Human colon carcinoma Lovo cell were infected by the recombinant lentivirus.The expression of CHD5 was confirmed by RT-PCR and Western blotting,and the proliferation of Lovo cells was evaluated by MTT assay.Results： The recombinant lentiviral vecor carried the CHD5 gene was successfully constructed by restriction enzyme digestion and sequencing,respectively.The packaged lentiviral titer was 3.1×106 TU/ml.RT-PCR and Western blot analysis revealed that pPRIME-TET-GFP-CHD5 infection inhibited CHD5 mRNA and protein expressions in Lovo cells.The proliferation of Lovo cells was increased.Conclusion： The recombinant lentiviral vector pPRIME-TET-GFP-CHD5 was constructed successfully.It can be deliver target gene to human colon carcinoma Lovo cell,and inhibit CHD5 can promoted the proliferation of colon carcinoma Lovo cell.