中文摘要：

目的：构建人成纤维细胞生长因子7的重组原核表达质粒pGEX-4T-3-FGF7,并诱导其基因在大肠杆菌DH5α中大量表达,纯化蛋白并检验生物学活性。 方法：实验于2006-12在本实验室完成。采用逆转录-聚合酶链反应技术,从人皮肤成纤维细胞内扩增编码人成纤维细胞生长因子7的cDNA序列,以DNA重组技术将获得的目的基因片段连接至原核表达载体pGEX-4T-3上,经抗生素筛选挑取阳性克隆扩增后,提取DNA进行酶切鉴定和测序。同时应用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白免疫印迹方法对其表达产物进行特异性鉴定。超滤纯化蛋白后以四甲基偶氮唑盐法测定其在不同浓度作用下对人表皮角化细胞系生长的影响。 结果：克隆出的成纤维细胞生长因子7基因的cDNA包括翻译起始密码子、编码序列和终止密码子等部分,含有成纤维细胞生长因子7基因的cDNA的表达质粒pGEX-4T-3-hFGF7在DH5α细菌体内能够表达,并且随着诱导时间的延长,成纤维细胞生长因子7基因的表达量增加,重组人成纤维细胞生长因子7蛋白主要存在于包涵体中。在所测浓度范围内该蛋白可以剂量依赖模式促进人表皮角化细胞系细胞在体外分裂增殖。 结论：人成纤维细胞生长因子7基因可以在原核细胞中获得表达并具有促角质细胞增殖的生物活性,为深入研究该蛋白在皮肤创伤愈合中的具体作用奠定了基础。

英文摘要：

AIM：To construct the recombinant prokaryotic expression plasmid pGEX-4T-3-fibroblast growth factor （FGF）-7, and induce human FGF-7 protein to abundantly express in E.coli DH5α, then to purify the protein and test its bioactivity. METHODS： The experiment was conducted in the laboratory in December 2006. RT-PCR technology was used to amplify cDNA fragment coding for human FGF-7 in vitro. DNA recombination method was adopted to insert the fragment into the multiple cloning sites of the expression vector pGEX-4T-3. Recombinant plasmid was then transfected into E, coli DH5α and the positive clones were subsequently selected by antibiotic. Sequence of the insert was identified by digestion of the recombinant plasmid with restriction endonuclease and analyzed by sequencing. The FGF-7 fusion protein expressed by E.coli was analyzed by SDS-PAGE and Western blotting. After purification with ultrafiltration, the influence of protein on the regeneration of human epidermal keratinocytes （HEK） was tested with MTT method. RESULTS： The cDNA fragment coding for human FGF-7 gene was composed of translation initiation codon, coding sequence and termination codon; the expression plasmid pGEX-4T-3-FGF-7 could express FGF-7 fusion protein after transfected into DH5α, and the protein content increased along with the inducing time. The fusion protein was mainly located in inclusion bodies. During the tested concentration range, the purified protein could stimulate the proliferation of HEK in a dose-dependent manner in vitro. CONCLUSION： The prokaryotic expression plasmid pGEX-4T-3-FGF-7 is successfully constructed, and the purified protein has the bioactivity to stimulate the proliferation of HEK, which lays a foundation for the further research of the role of FGF-7 in skin wound healing.