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  

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

作　　者：苏新良[1] 任国胜[1] 王小毅[1] 谭金祥[1] 

机构地区：[1]重庆医科大学附属第一医院内分泌外科,重庆400016

出　　处：《第三军医大学学报》2008年第2期146-149,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30070292)~~

摘　　要：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1332bp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。Objective To construct the eukaryotic expression vector of human HYAL1 gene and obtain MCF-7 and ZR-75-30 ceil clones expressing HYAL1 gene stably. Methods The 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. Results A 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. Conclusion The 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.

关 键 词：透明质酸酶 HYAL1 真核表达 乳腺癌细胞 侵袭 

分 类 号：R394-33[医药卫生—医学遗传学] R394.2[医药卫生—基础医学]