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目的 扩增类胰岛素生长因子Ｉ（ＩＧＦ－Ｉ）２１０ｂｐ的ＤＮＡ片段，将其克隆到ＰＧＥＸ－１ λＴ质粒，并转化到 Ｅ．ｃｏｌｉ ＮＨ５２２中高效表达。方法 用改进的ＲＴ－ＰＣＲ法合成人肝脏ｃＤＮＡ，然后从该ｃＤＮＡ文库中扩增出ＩＧＦ－Ｉ基 因。将扩增的ＩＧＦ－Ｉ ｃＤＮＡ用 ＢａｍＨＩ和ＥｃｏＲ Ｉ双酶切后，插入到同样双酶切处理的ｐＧＥＸ－１λＴ载体中，连接转化 Ｅ． ｃｏｌｉ ＮＭ５２２，筛选克隆，酶切鉴定后，进行了核苷酸序列分析及表达。结果 将ＩＰＴＧ诱导表达的菌体离心收集，经 ＳＤＳ－ＰＡＧＥ分析，在相对分子质量３４０００处可见明显高表达带，表达量可占菌体蛋白总量的２０％以上。免疫印迹表 明重组蛋白具有ＩＧＦ－Ｉ的抗原性。结论 重组人ＩＧＦ－Ｉ的成功克隆与表达，将为进一步研究人ＩＣＦ－Ｉ的生物学特性 打下基础。
Objective To amplify a 210 bp DNA fragment of insulin-like growth factor I (IGF-I), clone it into PGEX-1 λT plasmid and transform it into E.coli. Highly expressed in E. coli NH522. METHODS: Human liver cDNA was synthesized by a modified RT-PCR method, and then the IGF-I gene was amplified from the cDNA library. The amplified IGF-I cDNA was double-digested with BamHI and EcoR I and inserted into the pGEX-1λT vector, which was also double-digested, to transform E. coli. coli NM522, screening clones, digestion and identification, the nucleotide sequence analysis and expression. Results The cells induced by IPTG were collected by centrifugation. SDS-PAGE analysis showed that the expression of IPTG was significant at a molecular weight of 34000, which was over 20% of the total bacterial proteins. Western blotting showed that the recombinant protein has the antigenicity of IGF-I. Conclusion The successful cloning and expression of recombinant human IGF-I will lay the foundation for further study on the biological characteristics of human ICF-I.