人纤溶酶原K5区基因真核表达载体的构建和活性鉴定

Human Plasminogen Kringle 5 cDNA Clone with Eukaryotic System and Identification of Its Expression Product

目的 构建含人纤溶酶原K5区基因的真核表达载体 ,转染人乳腺癌细胞株MDA MB 2 31,观察阳性克隆表达的K5蛋白对人脐静脉内皮细胞株ECV30 4和MDA MB 2 31细胞增殖的影响。方法 应用PCR将人纤溶酶原信号肽序列引入K5cDNA ,所得的目的片段与真核表达载体 pcDNA3重组 ,构建重组质粒pcDNA3K5 ,脂质体法将其转染MDA MB 2 31,G4 18筛选阳性克隆 ,PCR鉴定 ,RT PCR和Westernblot检测K5的表达。将鉴定正确的阳性克隆的培养上清作用于ECV30 4细胞 ,MTT法检测其增殖情况 ,并用MTT法检测转染 pcD NA3K5对MDA MB 2 31细胞增殖的影响。结果 构建的重组质粒 pcDNA3K5经酶切鉴定、测序正确 ,将其转染MDA MB 2 31后挑取的阳性克隆有 3个经PCR鉴定正确 ,并经RT PCR和Westernblot检测证实K5的表达。阳性克隆的培养上清作用于ECV30 4细胞后 ,其存活率降低 ;转染 pcDNA3K5对MDA MB 2 31细胞增殖无明显影响。结论 应用脂质体法将带有人纤溶酶原信号肽序列的K5cDNA转染MDA MB 2 31细胞后 ,其分泌产生的有生物学活性的K5 ,呈现对ECV30 4细胞增殖的抑制作用 ,而对MDA MB 2 31细胞的生长则无影响。

Purpose: To construct eukaryotic expression vector coding human plasminogen kringle 5 cDNA and investigate its inhibitory effects on human umbilical vein endothelial cell line(ECV304) as well as human breast adenocarcinoma cell line (MDA-MB-231) by liposome-mediated gene transfer. Methods: The K5 cDNA was fused in-frame with human plasminogen signal sequence by PCR and it was inserted into an eukaryotic expression vector pcDNA3, the recombinant plasmid named pcDNA3K5 was assayed by restriction endonucleases and sequenced. Subconfluent human breast carcinoma cells (MDA-MB-231) were transfected with pcDNA3K5 and pcDNA3 by liposome method. Transfected cells were selected at 1 mg/mL G418 in DMEM containing 10% FBS for 2 weeks. Single cell clones were picked and expanded in the presence of G418. The positive clones were verified by PCR. The expression of K5 mRNA and protein was assayed by RT-PCR and Western blot. The 72 h conditioned media of the positive clones were applied to ECV304 testing their antiproliferation effects and assayed by MTT. The proliferation of pcDNA3K5 transfected MDA-MB-231 was also assayed by MTT. Results: The recombinant plasmid pcDNA3K5 was verified by restriction endonuclease analysis and sequencing. After it was transfected into MDA-MB-231, three positive clones were verified by PCR. RT-PCR and Western blot detected the expression of K5 mRNA and protein. The conditioned media of the positive clones inhibited the proliferation of ECV304. In contrast, the proliferation of pcDNA3K5 transfected MDA-MB-231 was not affected. Conclusions: The biologically active K5 secreted by pcDNA3K5 transfected MDA-MB-231 showed inhibitory effects on the proliferation of ECV304 but had no effects on the proliferation of MDA-MB-231.