基于流式细胞术分选的高效表达外源基因细胞的筛选

目的：以增强型绿色荧光蛋白（EGFP）作为报告基因,用流式细胞术筛选高表达EGFP的细胞,从而获得外源基因高效表达细胞株。方法：构建在EGFPC端编码区融合新霉素（neomycin）抗性基因的融合基因EGFP-Neomycin,将其插入pcDNA3.1（＋）载体,构建EGFP-Neomycin融合基因表达载体pcDNAEN,转染CHO-K1细胞,G418加压筛选和倒置荧光显微镜观察证实所表达的EGFP-Neomycin融合蛋白具有新霉素抗性和激发EGFP荧光双功能;将编码组织型纤溶酶原激活剂（tPA）的cDNA插入pcDNAEN中CMV启动子下游,构建表达tPA的表达载体pcDNAEN/tPA。结果：流式细胞术分析和tPA纤维蛋白溶解活性测定表明,pcDNAEN/tPA转染CHO-K1细胞的EGFP相对荧光强度（RFT）的自然对数值与tPA表达水平呈明显的直线相关关系,相关系数为0.983;比较部分未经流式细胞仪分选的pcDNAEN/tPA转染阳性细胞克隆和RFT分布在100～1000的pcDNAEN/tPA转染阳性细胞克隆的tPA表达水平,经流式细胞术分选获得的细胞克隆的tPA平均表达水平和最高表达水平分别是未经分选获得的细胞克隆的3.9倍和4.1倍。结论：构建的EGFP-Neomycin融合基因具有双功能,建立了利用流式细胞术筛选外源基因高效表达物细胞株的方法。

A FACS-Based Screening Method for High Producing Cells

Objective： The cell line expressing enhanced green fluorescence protein（EGFP） highly was screened by flow cytometry, then high producing cell line was obtained. Methods： A fusion gene, EGFP-Neomycin was constructed by linking neomycin resistance gene to the gene region coding C-terminus of EGFP and was inserted into pcDNA3.1（＋） for forming a EGFP-Neomycin expression plasmid, pcDNAEN. The resultant fusion protein of the fusion gene was validated in CHO-K1 cells to have neomycin resistance and EGFP fluorescent bi-function via G418 selection and inverted fluorescent microscopy observation. A tissue-type plasminogen activator（tPA） expression vector, pcDNAEN/tPA, was constructed by inserted the tPA cDNA into the downstream of CMV promoter of pcDNAEN plasmid. Results： The data from cytometric analysis and in vitro fibrinolytic activity assay shown that there is obvious correlation between the natural logarithm of the relactive fluorescence tensity（RFT） and the tPA expression level in the pcDNAEN transfected CHO-K1 cells with a coefficient of 0.983. The average tPA expression level and the maximum tPA expression level of the clones from the pcDNAEN transfected CHO-K1 with the RFF ranging at 100 to 1 000 was 3.9-fold and 4.1-fold as the clones from random selection, respectively. Conclusion： The fusion gene EGFP-Neomycin was bi-function, and the method of screening high producing cell was constructed with fluorescence-activated cell sorting.