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目的利用逆病毒表达载体快速构建梯度过表达人MTA1稳转单克隆细胞系。方法利用噬菌斑原位杂交筛选人肺噬菌体文库,以阳性克隆为模板,RT-PCR获得人MTA1完整CDS序列。以逆病毒表达载体pMX为基础,经过多次酶切连接,构建逆病毒表达载体pMX-MTA1-flag-IRES-EGFP。将构建好的逆病毒载体系统,转染293-gag/pol细胞,包装出含该表达载体的逆病毒颗粒。以含病毒上清多次感染HCT116细胞,获得稳定转染MTA1的HCT116多克隆细胞系。将该多克隆细胞系按合适比例稀释后接种至10cm大皿,获得大量单克隆细胞系,以GFP为筛选标志,筛选不同荧光强度的单细胞克隆进行扩增并分析MTA1与GFP表达水平的线性关系。结果测序结果显示成功克隆出人MTA1表达序列。免疫荧光、Western Blot结果证实成功构建出梯度过表达MTA1的稳转细胞系。相关性验证结果显示构建的非融合载体MTA1与GFP的表达呈明显的线性相关,相关系数r=0.932,P<0.01。结论利用逆病毒表达系统,快速成功构建并筛选出MTA1与GFP非融合梯度过表达稳转单克隆细胞系,为MTA1基因功能研究提供了良好模型。该逆转录病毒平台可用于快速建立其他基因的稳转单克隆细胞系。
OBJECTIVE: To rapidly construct a monoclonal cell line stably over-expressing human MTA1 by retroviral expression vector. Methods The human lung phage library was screened by plaque in situ hybridization and the positive clone was used as a template. The complete CDS sequence of human MTA1 was obtained by RT-PCR. Based on the retroviral expression vector pMX, the retroviral expression vector pMX-MTA1-flag-IRES-EGFP was constructed after multiple digestion. The constructed retroviral vector system was transfected into 293-gag / pol cells and the retroviral particles containing the expression vector were packaged. HCT116 cells were infected with the virus supernatant for several times to obtain HCT116 polyclonal cell lines stably transfected with MTA1. The polyclonal cell line was diluted to an appropriate ratio and inoculated into a 10 cm dish to obtain a large number of monoclonal cell lines. The single cell clone with different fluorescence intensities was screened with GFP as a screening marker to amplify and analyze the linear relationship between the expression level of MTA1 and GFP . Results Sequencing results showed that human MTA1 expression sequence was successfully cloned. Immunofluorescence, Western Blot results confirmed the successful construction of gradient-overexpressing MTA1 stable cell line. Correlation analysis showed that the constructed non-fusion vector MTA1 and GFP expression showed a significant linear correlation, the correlation coefficient r = 0.932, P <0.01. Conclusion The retroviral expression system was used to rapidly and successfully construct and screen MTC1-GFP non-fusion gradient-stable transgenic monoclonal cell lines, which provided a good model for the study of MTA1 gene function. The retroviral platform can be used to rapidly establish metastatic monoclonal cell lines for other genes.