中文摘要：

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。

英文摘要：

Objective To construct stable HCT116 sublines overexpressing MTA1 at gradient levels. Methods We cloned human MTA1 CDS by RT-PCR using the positive clones from a template screened by in situ plaque hybridization with the human lung phage library. The MTA1 CDS was cut out and ligated into the retroviral vector pMX （pMX-MTA1- flag-IRES-EGFP）. Retroviral particles were packaged by co-transfecting the constructed retroviral vector together with pM- DG plasmid into 293-gag/pol packaging cells. HCT116 cells were then infected with the supemant containing the generated retroviral particles to produce MTA1-expressing polyclonal cell pool. The polyclonal cell pool was diluted and seeded into a 10 cm-diameter dish at a very low density. About two weeks later, when the separated cells grew into clones, the MTA1- overexpressing subclones were selected with GFP as a screening marker. Results DNA sequencing verified the successful cloning of human MTA1 CDS. Immunofluorescence and Western Blot results screened a series of monoclonal sublines ex- pressing gradient MTA1. We also confirmed that levels of upstream MTA1 and downstream GFP, which were bridged with IRES sequence, showed a significant linear correlation （r = 0. 932, P 〈 0. 01 ）. Conclusion We have successfully es- tablished a method to rapidly develop a HCT116-based cancer cell line model expressing a gradient hMTA1 and this ap- proach can be extended to construction of cell lines over-expressing other genes.