【摘要】 目的通过重组技术,对胰岛素样生长因子IGF-Ⅰ基因进行缺失、突变以获得最有利于高水平表达的IGF-ⅠcDNA。构建原核表达载体,并进行表达,以获得能够高水平表达高活性产物的基因工程菌珠。方法提取人肝脏组织总RNA,RT-PCR后琼脂糖电泳初步鉴定产物。将cDNA回收、补平后插入克隆质粒KS,酶切鉴定后,对IGF-Ⅰ基因进行序列分析。采用PCR法从克隆质粒中扩增IGF-Ⅰ片段,亚克隆至pET-DsbA原核表达载体,酶切鉴定,并进行序列测定。转化到大肠杆菌BL21(DE3)PLysS中,IPTG诱导表达,聚丙稀酰胺凝胶电泳、WesternBlot分析目的蛋白。结果成功构建原核表达载体pET-DsbA-IGF-Ⅰ,测序结果与预期序列完全一致,并在大肠杆菌BL21(DE3)plysS表达出IGF-Ⅰ融合蛋白。结论IGF-Ⅰ融合蛋白在大肠杆菌BL21(DE3)PLysS中的表达,对进一步研究IGF-Ⅰ的功能及开发其生物制品具有重要意义。更多还原

【Abstract】 Objective To clone the human insulin like growth factorⅠgene, construct its expression vector and expression it in E.coli.Method The human insulin like growth factorⅠwas amplified from human liver cDNA by RT-PCR and then inserted into the KS vector.IGF-Ⅰ was inserted into pET-DsbA.The recombinant plasmid was identified by enzyme digestion, sequencing and expressed in E.coli.Result IGF-Ⅰ gene in plasmid pET-DsbA-IGF-Ⅰ was successfully expressed in BL21(DE3)plysS.Proteins were identified by SDS-PAGE and Western Blot.Conclusion The recombinant IGF-Ⅰ protein expressed with pET-DsbA-IGF-Ⅰ could be used for the development of its biological products.更多还原