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目的 :优化采用PCR技术从PBR3 2 2 bFGF质粒扩增bFGF基因片段的条件并构bFGF真核表达载体 ,为研究bFGF基因转移在骨组织工程学中的应用提供研究基础。方法 :(1)在不同的模板量、Mg2 + 浓度、退火温度下经PCR扩增bFGF基因 ,琼脂糖凝胶电泳检测。 (2 )载体和PCR产物经EcoRI和HindⅢ酶切、纯化 ,T4连接酶连接 ,转化大肠杆菌 ,抗生素筛选重组质粒。结果 :(1)得到三个最佳参数为 :模板量为 2 μl(5 0 4μg/ml) ;Mg2 + 浓度为2 0mmol/L ;退火温度为 5 5℃。 (2 )酶切、PCR和DNA序列鉴定均证实插入片段的正确性。结论 :在此优化条件下经PCR反应得到了特异、高效和忠实的bFGF基因片段 ,并成功构建了bFGF真核表达载体。
OBJECTIVE: To optimize the conditions for amplification of bFGF gene fragment from PBR3 2 2 bFGF plasmid by PCR and to construct eukaryotic expression vector of bFGF, so as to provide the basis for studying the application of bFGF gene transfer in bone tissue engineering. Methods: (1) The bFGF gene was amplified by PCR at different concentration of template, Mg2 + concentration and annealing temperature, and detected by agarose gel electrophoresis. (2) The vector and PCR products were digested with EcoRI and HindIII, purified, and ligated with T4 ligase to transform Escherichia coli. Antibiotics were used to screen recombinant plasmids. Results: (1) Three optimal parameters were obtained: the amount of template was 2 μl (504μg / ml); the concentration of Mg2 + was 20mmol / L; the annealing temperature was 55 ℃. (2) Enzyme digestion, PCR and DNA sequence identification both confirmed the correctness of the inserted fragment. Conclusion: The specific, efficient and faithful bFGF gene fragment was obtained by PCR reaction under this optimal condition, and the eukaryotic expression vector of bFGF was successfully constructed.