碱性成纤维细胞生长因子真核表达载体的构建及表达

目的构建人碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)真核表达质粒,研究其在体内外的表达.方法通过基因克隆技术,构建并大量制备pcDNA3.1/myc-His(-)-bFGF真核表达体系,RT-PCR和细胞免疫组织化学方法检测体外瞬时表达情况;直流电脉冲介导重组质粒pcDNA3.1/myc-His(-)-bFGF和pCD2-血管内皮细胞生长因子121(vascular endothelial growth factor,VEGF121)在兔颈部肌肉瓣内转移并表达,测定其促血管生成的生物学效应.结果构建的pcDNA3.1/myc-His(-)-bFGF真核表达体系成功转染体外培养HeLa细胞,目的基因在mRNA水平和蛋白水平均有表达.pcDNA3.1/myc-His(-)-bFGF和pCD2-VEGF121重组质粒分别转染在体肌瓣,获得外源基因高水平表达.转基因肌肉发生血管增生、血流增强的生物学效应.结论构建了人bFGF真核表达质粒,并可在体内外顺利表达,为进一步组织移植或组织工程基因治疗研究奠定了基础.

CONSTRUCTION AND EXPRESSION OF BASIC FIBROBLAST GROWTH FACTOR MAMMALIAN EXPRESSION VECTOR

Objective To construct a mammalian expression vector of basic fibroblast growth factor (bFGF) and to investigate the expression of bFGF in vitro and in vivo. Methods A mammalian expression vector pcDNA3.1/myc His( )C bFGF was constructed with gene cloning technique. The mammalian expression system was prepared and purified. The expression of bFGF cDNA in cultured transfected cells was examined by RT PCR and cell immunohistochemistry. The recombinant plasmids, pcDNA3.1/myc His( )C bFGF and pCD2 VEGF 121 , were transferred into rabbit cervical muscle by direct injection of plasmid following electric pulses in vivo. The transferred gene expression and the biological effect were measured by use of histochemistry and immunohistochemistry analysis. Results The eukaryon expression system pcDNA3.1/myc His( )C bFGF could express the target protein bFGF in vitro. The recombinant plasmids, pcDNA3.1/myc His( )C bFGF and pCD2 VEGF 121 were transferred into muscles flap in vivo successfully. The active proteins bFGF and VEGF 121 were expressed at high levels. Blood vessels increased significantly in the muscles, and blood circulation was improved by local angiogenesis. Conclusion The eukaryon expression vector of bFGF is constructed and can be expressed successfully in vitro and in vivo. That is a primary preparation for the research on tissue transplantation and tissue engineering with bFGF gene therapy.