摘要
目的 构建携带人组织纤溶酶原激活物 (t PA)和血管内皮细胞生长因子 16 5 (VEGF16 5 )基因的真核表达质粒 p Bud CE4 .1/ t PA - VEGF16 5 ,并观察其在血管平滑肌细胞 (VSMC)中的表达。方法 从人心脏组织中克隆 t PA和 VEGF16 5基因。将 t PA和 VEGF16 5基因克隆至真核表达质粒 p Bud CE4 .1中 ,构建共表达质粒 p Bud-CE4 .1/ t PA - VEGF16 5。将 p Bud CE4 .1/ t PA- VEGF16 5转染 VSMC,用 RT- PCR法检测转染的 VSMC中 t PA和VEGF16 5 m RNA的表达 ,用 EL ISA法检测转染的 VSMC培养液中 t PA和 VEGF16 5蛋白质。结果 人 t PA和VEGF16 5基因的 RT- PCR产物分别为 1.9kb和 5 76 bp。经酶切鉴定证实 ,t PA和 VEGF16 5基因已克隆至真核表达质粒 p Bud CE4 .1中。以其转染 VSMC后 ,经 RT- PCR和 EL ISA检测发现 ,t PA和 VEGF16 5在 m RNA和蛋白质水平均有表达。结论 成功地构建了真核共表达质粒 p Bud CE4 .1/ t PA- VEGF16 5 ,并在 VSMC中表达 t PA和VEGF16 5。
Objective To construct the eukaryotic co-expression plasmid of tissue-plasminogen activator(tPA) genes and vascular endothelia growth factor165(VEGF165) and observe its expression in vascular smooth muscle cells(VSMCs). Methods The tPA and VEGF165 genes were cloned from hominal heart tissue with RT-PCR, and then the tPA and VEGF165 genes were cloned into eukaryotic expression plasmid pBudCE4.1 to construct the eukaryotic co-expression plasmid pBudCE4.1/tPA-VEGF165. The pBudCE4.1/tPA-VEGF165 was transfected into VSMCs via lipofection mediation. The expression levels of tPA and VEGF165 mRNAs in the transformed VSMCs were detected by RT-PCR and the expression levels of tPA and VEGF165 proteins were detected by ELISA. Results The size of RT-PCR product of tPA and VEGF165 genes was 1.9 kb and 576 bp respectively. Restrictive enzyme digestion analysis showed that recombinant co-expression plasmid pBudCE4.1/tPA-VEGF165 had been constructed successfully. The expression of tPA and VEGF165 at mRNA and protein levels in the transformed VSMCs and cells culture supernatant were detected respectively by RT-PCR and ELISA. Conclusion The recombinant eukaryotic co-expression plasmid pBudCE4.1/tPA-VEGF165 has been successfully constructed. The tPA and VEGF165 are expressed in transformed VSMCs.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2005年第2期161-164,共4页
Journal of Sichuan University(Medical Sciences)
基金
国家 973计划 (项目编号 2 0 0 3 CB5 15 5 0 1)
国家自然科学基金 (批准号 3 0 2 70 5 14 )资助
关键词
质粒
组织纤溶酶原激活物
血管内皮生长因子
血管平滑肌细胞
Plasmid Tissue-plasminogen activator Vascular endothelia growth factor Vascular smooth muscle cells
