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目的 :克隆犬胰岛素样生长因子 (IGF -I)编码区基因 ,构建重组真核表达载体。方法 :从犬左心室组织中提取总RNA ,通过RT -PCR获取IGF -1cDNA编码区全序列 ,将其与 pcDNA3 .1(+ )质粒载体连接 ,构建重组真核表达载体pcDNA3 .1(+ ) /IGF -1,转化大肠杆菌DH5α后 ,随机挑选数个克隆 ,提取质粒DNA ,通过限制性酶切和核苷酸序列鉴定阳性克隆。结果 :重组质粒pcDNA3 .1(+ ) /IGF -1的酶切图谱和序列分析结果与国外文献报道一致。结论 :克隆到IGF -1编码区基因 ,成功构建重组真核表达载体 pcDNA3 .1(+ ) /IGF -1。
Objective: To clone the gene encoding coding region of insulin-like growth factor (IGF-I) and construct recombinant eukaryotic expression vector. Methods: The total RNA was extracted from the canine left ventricular tissue and the full-length cDNA of IGF-1 cDNA was obtained by RT-PCR. The recombinant plasmid pcDNA3 .1 (+) was ligated with pcDNA3. 1 (+) plasmid to construct recombinant eukaryotic expression vector pcDNA3. / IGF-1. After transformation of E. coli DH5α, several clones were randomly selected and plasmid DNA was extracted. The positive clones were identified by restriction enzyme digestion and nucleotide sequencing. Results: The results of restriction enzyme digestion and sequence analysis of recombinant plasmid pcDNA3 .1 (+) / IGF-1 were consistent with those reported in foreign literature. Conclusion: The gene encoding IGF-1 was cloned and the recombinant eukaryotic expression vector pcDNA3. 1 (+) / IGF-1 was successfully constructed.