人乳腺癌细胞HYAL1基因真核表达载体的构建及其在MCF-7和ZR-75-30细胞中的表达

目的 构建人乳腺癌HYAL1基因的真核表达载体并稳定转染HYAL1基因低表达的乳腺癌MCF-7和ZR-75-30细胞株,为进一步探讨HYAL1基因在乳腺癌的侵袭和转移中的作用奠定基础.方法 采用RT-PCR技术从高表达HYAL1的人乳腺癌细胞株MDA-MB-435S中扩增透明质酸酶HYLAL1基因,并将其插入真核表达载体pcDNA3.1/V5-His-TOPO中,构建的重组表达质粒经脂质体介导转染HYAL1基因低表达的乳腺癌MCF-7和ZR-75-30细胞.结果 用RT-PCR成功地扩增出1条1 332 bp的DNA片段,经限制性内切酶酶切分析和DNA测序证明已经成功构建了人乳腺癌HY-AL1基因的真核表达载体.RT-PCR证明转染了重组质粒的乳腺癌MCF-7和ZR-75-30细胞可以高效稳定的表达HYAL1基因,且其穿透细胞外基质的能力增强.结论 成功构建了人乳腺癌HYAL1基因的真核表达载体,获得了稳定表达人HYAL1基因的MCF-7和ZR-75-30细胞克隆,且其侵袭能力增强.

Construction of eukaryotic expression vector of human HYAL1 gene and its expression in MCF-7 and ZR-75-30 cells

ObjectiveTo construct the eukaryotic expression vector of human HYAL1 gene and obtain MCF-7 and ZR-75-30 cell clones expressing HYAL1 gene stably.MethodsThe cDNA encoding HYAL1 gene of human breast cancer was amplified by RT-PCR from the total RNA isolated from human MDA-MB-435S cells and inserted into pcDNA3.1/V5-His-TOPO vector.The recombinant plasmid was transferred into MCF-7 and ZR-75-30 cells.ResultsA 1332-bp DNA fragment was successfully amplified from human MDA-MB-435S cell.Restriction enzyme digestion analysis and DNA sequencing showed that HYAL1 gene was inserted into recombinant vector.RT-PCR analysis revealed that HYAL1 gene could be expressed stably in the transfected MCF-7 and ZR-75-30 and it had strong invasive potential.ConclusionThe eukaryotic expression vector of human HYAL1 gene was successfully constructed.MCF-7 and ZR-75-30 cell clones that can express HYAL1 gene were obtained and can promote the invasion.