类胰岛素生长因子I(IGF-I)基因的克隆及融合蛋白的表达

Cloning of Insulin Like Growth Factor-I(IGF-I) Gene and Expression of Fusion Protein

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 210bp of DNA fragment of insulin like growth factor-I (IGF-I) and clone it into plasmid pGEX- 1 λ T for high expression. Methods IGF-I gene was isolated from the human liver cDNA bank synthesized by an improved RT-PCR. The IGF-I cDNA amplified by PCR was digested by BamH I and EcoR I,and inserted into the pGEX-1λ T vector digested by the 2 kinds of enzyme,then trans-formed to E. coli NM522. The positive clones were screened out and identified by enzyme digestion and se-quencing. Results The somatic protein expressed under the induction of IPTG was collected by centrifugation and detected by SDS-PAGE. A significant band with a relative molecular weight of 34000 was observed. Thin layer scanning proved that the expressed product contained more than 20% of total somatic protein. Western blot showed the IGF-I antigenicity of expressed protein. Conclusion Recombinant human IGF-I was successfully ex-pressed in this paper. It laid a foundation of further study on the biological characteristics of human IGF-I.