文献类型：期刊文献

中文题名：EGFP-PDGFRβ融合基因表达载体的构建及其转染COS-7细胞条件的优化

英文题名：Construction of Expression Vector for EGFP-PDGFRβ Fusion Gene and Optimization of Condition for Transfection of COS-7 Cells with The Vector

作者：任培君[1];易小萍[1];张元兴[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2008

卷号：21

期号：11

起止页码：933

中文期刊名：中国生物制品学杂志

外文期刊名：Chinese Journal of Biologicals

收录：CSTPCD;;Scopus;CSCD:【CSCD2011_2012】；

语种：中文

中文关键词：EGFP基因;PDGFRB基因;融合表达;COS-7细胞;转染

外文关键词：EGFP gene; PDGFRβ gene; Fusion expression; COS-7 cells; Transfection

摘要：目的构建EGFP-PDGFRβ融合基因表达载体,并优化PEI介导基因转染COS-7细胞的条件。方法PCR扩增EGFP和PDGFRβ基因,依次连接至pcDNA3.1(+)质粒中,构建融合基因表达载体H-E-P-pcDNA3.1(+),经PEI介导转染COS-7细胞,并对转染条件进行优化。结果融合基因表达载体经酶切及测序证明构建正确。经PEI介导转染COS-7细胞的最佳条件为:DNA浓度2μg/ml,N/P比值15.5,在无血清条件下转染。此条件适用于滚瓶培养细胞的转染。结论已成功构建了EGFP-PDGFRβ融合基因表达载体,并优化了经PEI介导转染COS-7细胞的条件。
Objective To construct the expression vector for enhanced green fluorescent protein-platelet-derived growth factor receptor β （EGFP-PDGFRβ） fusion gent and optimize the condition for PEI-mediated transfection of COS-7 ceils with the vector. Methods EGFP and PDGFRβ genes were amplified by PCR and inserted into plasmid pcDNA3.1 （＋）. The constructed recombinant plasmid H-E-P-pcDNA3.1 （＋） was transfected to COS-7 cells in mediation of PEI, and the condition for transfection was optimized. Results Restriction analysis and sequencing proved that recombinant plasmid H-E-P-pcDNA3.1 （＋） was constructed correctly. The optimal DNA concentration, N / P ratio and medium for transfection of COS-7 cells in mediation of PEI were 2 μg / ml, 15. 5 and serum-free DMEM respectively. The optimal condition was suitable for the cells cultured in rolling bottles. Conclusion The expression vector for EGFP-PDGFRβ fusion gene was successfully constructed, and the condition for transfection of COS-7 cells in mediation of PEI was optimized.