以人载脂蛋白B为靶点的基因表达下调剂筛选模型的建立与应用

Establishment and application of a high-throughput screening model for identifying down-regulators of human apoB gene expression

目的构建以人ApoB为靶点的基因表达调节剂的高通量筛选模型,筛选apoB基因表达下调剂。方法以人基因组DNA为模板,通过PCR扩增apoB基因上游的启动子序列,将扩增的DNA片段克隆至荧光素酶报告基因质粒pGL4.17,采用脂质体介导的方法将重组质粒转染人肝癌细胞HepG2,经G418抗性筛选,获得稳定转染细胞株。应用槲皮素对筛选模型进行评价,进而基于检测荧光素酶的表达活性变化筛选人apoB基因表达下调剂。结果PCR扩增得到人apoB基因上游的启动子序列,构建完成相应的重组荧光素酶报告基因质粒pGL4-apoB-P,建立了稳定转染细胞株ApoB-Luc HepG2。以槲皮素为阳性化合物进行模型评价,Z'因子为0.77,符合高通量筛选的要求。应用该模型对5688个微生物发酵液粗提物进行筛选,获得了2株阳性菌株。结论建立了人apoB基因表达下调剂筛选模型,并应用于微生物来源样品的筛选。

Objective In this study,a high-throughput screening model of gene expression regulators targeting human ApoB was constructed to screen down-regulators of apoB gene expression.Methods Human genomic DNA was used as a template to amplify the promoter sequence of apoB gene by PCR.The amplified promoter fragment was cloned into the luciferase reporter gene plasmid of pGL4.17,and then the recombinant plasmids were transfected into HepG2 cell line using the liposome-mediated method.A stable transfected cell line was obtained after antibiotic G418 resistance screening.Quercetin was applied to evaluate the effectiveness of the screening model.The down-regulator of human apoB gene expression was identified by assessing the changes in the expression activity of luciferase.Results The promoter sequence of human apoB gene was amplified and validated.The reporter plasmid named pGL4-apoB was obtained by inserting the promoter sequence upstream of the luciferase reporter gene of pGL4.17,and the stable transfected ApoB-Luc HepG2 cell line was then established.The drug screening model was evaluated using quercetin as a positive compound.The result shows that Z'-factor is above 0.5,which is suitable for high-throughput screening.Then 5,688 fermentation extracts of microorganisms were screened in this model,and two positive strains were identified.Conclusion The screening model for down-regulators of human apoB gene expression was established and is suitable for the screening of microbial samples.