摘要
通过PCR技术和体外DNA重组技术将绿色荧光蛋白cDNA和酸性成纤维细胞生长因子cDNA构建成融合基因,克隆到表达载体pET3c中,构建成表达菌株BL21(DE3)/pET3c-GAF。经IPTG诱导表达,融合蛋白的表达量占菌体总蛋白的25%;DNA测序结果表明设计合成的融合基因与预期相符。Westernblot结果表明重组蛋白具有aFGF的免疫原性。经IPTG诱导后的菌体及蛋白粗提液在荧光显微镜下可观察到强烈的绿色荧光。融合基因在大肠杆菌中实现了表达,用MTT法测得纯化融合蛋白与野生型aFGF促Blab/c3T3细胞增殖活性相当,为利用绿色荧光分子探针研究aFGF的在活体内的作用机制建立了新的方法。
With polymerase chain reaction （PCR） and methods of DNA recombination in vitro, constructed the GAF fusion gene, which the green fluoresent protein gene （GFP） cDNA was connected with 5＇end of the cDNA of the acidic Fibroblast Growth Factor （aFGF）. The GAF fusion gene was cloned into pET3c expression vector. The recombinant plasmid was transformed into BL21 （ DE3 ） for expressing the aim protein. The expression of GAF was induced by IPTG and its expression level was 25% of the total cellular protein. It was confirmed that the DNA sequence of the synthetic fusion gene was identical with that of the designed gene by DNA sequencing. Western blot analysis show that the recombinant protein has the immunocompetence of aFGF. Fusion protein and the cell after inducing can emit the green fluorescence. The recombinant was expressed in BL21 （DE3） successfully. The mitogenic activity of the purified GAF assessed by MTT was comparable to that of the wild - type aFGF. It affords a new approach to study the function of aFGF in vivo with the GFP tag.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2005年第10期7-11,共5页
China Biotechnology
基金
国家"863"计划资助项目(2001AA215131
2002AA2Z3318)
国家自然基金重点资助项目(30230370)
