人IGFBP-1基因真核表达载体的构建及其对胃癌细胞BGC-823增殖的影响

Construction of Eukaryotic Expression Vector Containing IGFBP-1 Gene and Its Effect on BGC-823 Cell Proliferation

目的构建IGFBP-1真核表达载体,观察IGFBP-1基因真核表达载体转染胃癌细胞株BGC-823后基因的表达及其对BGC-823细胞增殖的影响.方法以GES-1细胞基因组为模板,PCR技术扩增人IGFBP-1的基因片段,克隆人pc DNA3-His-Flag真核表达载体,重组载体pc DNA3-IGFBP-1-His-Flag,并通过PCR、双酶切和测序鉴定.重组质粒经阳离子脂质体法转染BGC-823细胞,RT-PCR和Western blot检测细胞IGFBP-1的表达情况,并通过CCK8法观察IGFBP-1过表达对BGC-823细胞增殖的影响.结果经PCR、双酶切和测序鉴定证明IGFBP-1真核表达载体构建成功,并在胃癌细胞株BGC-823高效表达.CCK8实验显示:IGFBP-1过表达对BGC-823细胞增殖无影响.结论成功构建IGFBP-1真核表达载体,IGFBP-1过表达对BGC-823细胞的增殖无影响,可为进一步研究IGFBP-1生物学功能奠定基础.

Objective To investigate the effect of IGFBP-1 gene on BGC-823 cell proliferation,an eukaryotic expression vector containing IGFBP-1 gene was constructed. Method With GES-1 cell genome as a template,IGFBP-1 gene was amplified by PCR and inserted into the eukaryotic expression plasmid pcDNA3-His-Flag,and then the recombinant plasmid pcDNA3-IGFBP-1-His-Flag was identified by PCR,restriction enzymes digestion and DNA sequencing.Recombinant plasmids pcDNA3-IGFBP-1-His-Flag were transfected into BGC-823 cells by using cationic liposomes. Results RT-PCR and Western blot detected the expression of IGFBP-1 mRNA and protein,and then observed the effect of IGFBP-1 on BGC-823 cells proliferation by CCK8. CCK8 assay showed that IGFBP-1 has no influence on BGC-823 cells proliferation.Conclusion The results indicated that IGFBP-1 eukaryotic expression vector was successfully constructed and IGFBP-1 genes were highly expressed in transfected BGC-823 cells. IGFBP-1 has no influence on BGC-823 cells proliferation. The results provide a basis for the further study of biological functions of IGFBP-1.