uPA和PAI-1基因克隆及真核表达载体的构建及鉴定

目的：克隆人卵巢上皮癌组织uPA和PAI-1基因cDNA全长,构建表达uPA和PAI-1基因的真核表达载体。方法：采用RT-PCR技术从人卵巢上皮癌组织总RNA中逆转录uPA和PAI-1基因的cDNA全长,克隆到pGEM-TEasy Vector上,通过酶切和测序进行鉴定;酶切后与真核表达载体pcDNA3.1/myc-his（-）B连接,酶切及测序再次鉴定;采用脂质体法将重组质粒DNA转染至卵巢癌上皮细胞SKOV3细胞;采用有限稀释法和G418分别加压筛选并培养SKOV3-uPA和SKOV3-PAI-1细胞及对照细胞;Western blot法检测转染前后SKOV3细胞uPA和PAI-1基因的表达。结果：成功扩增出uPA和PAI-1基因的cDNA的全长,并克隆入T载体,DNA测序证实序列正确,通过连接成功构建含正确目的基因的表达载体pcDNA3.1-uPA和pcDNA3.1-PAI-1,Western blot能检测到uPA和PAI-1基因蛋白分别在SKOV3-uPA和SKOV3-PAI-1细胞中的表达。结论：成功构建了uPA和PAI-1表达基因的真核表达载体,为进一步研究uPA和PAI-1基因在卵巢癌侵袭转移中的作用提供了实验基础。

THE CONSTRUCTION OF uPA AND PAI-1 EUKARYOTIC EXPRESSIVE VECTOR

Objective：To clone plasminogen activator （uPA） and plasminogen activator inhibitor-1（PAI-1） gene from ovarian cancer tissue and construct the eukaryotic expressive vector for uPA and PAI-1 gene.Methods：The uPA and PAI-1 ORF were amplified using RT-PCR method from ovarian cancer tissue and clone to T vector,and linked with pcDNA3.1/myc-his（-）B plasmid after enzymation. It was tested by the enzymation and DNA sequencing. The pcDNA3.1-uPA and pcDNA3.1-PAI-1 were transfected into SKOV3 cells by using liposomes method. The limited dilution and G418 steady pressure screening were used to incubate SKOV3-uPA and SKOV3-PAI-1 and control cells. The uPA and PAI-1 protein expression of the transfected and control cell lines were detected by Western blot method respectively.Result：Gene was cloned and the eukaryotic expressive vector containing uPA and PAI-1 was successfully constructed by confirming of the enzymation and DNA sequencing. Western blot test results showed that the positive expression of uPA and PAI-1 gene in SKOV3 cell lines could be inducted by transfected the plasmid DNA of pcDNA3.1-uPA and pcDNA3.1-PAI-1 into cells.Conclusion：The successful construction of expressive vectors containing uPA and PAI-1 and provides an experimental basis to explore the role of uPA and PAI-1 gene in ovarian cancer.