人肝细胞核因子-1β基因cDNA全长的克隆与表达

Cloning and expression of human HNF-1β in E.Coli

目的构建人肝细胞核因子-1β(hHNF-1β)基因cD-NA全长的原核表达质粒[pET-28a(+)-hHNF-1β]并在大肠杆菌(E.Coli)中高效表达。方法提取正常成人肝脏组织的总RNA,RT-PCR后的扩增产物回收,构建克隆载体pMD-HNF-1β,设计带酶切位点BamHⅠ和HindⅢ的引物,PCR后酶切连入pET-28a(+)中,转化DE3,测序后IPTG诱导表达,Western blot检测其特异表达,并对重组hHNF-1β(rhH-NF-1β)诱导表达时IPTG的浓度、时间和温度等条件进行了优化。结果测序证实所得的hHNF-1βcDNA序列与其在GenBank中的序列一致,重组质粒pET-28a(+)-hHNF-1β的双酶切结果与预期大小完全一致。IPTG诱导的最佳浓度是0.04 mmol/L,时间为6 h,温度为37℃。结论成功克隆和构建了hHNF-1β基因的原核表达载体并对诱导表达的条件进行了优化,为进一步的功能分析奠定基础。

Objective To construct human hepatocyte nuclear factor-1β（hHNF-1β） full length cDNA sequence of the recombinant plasmid pET-28a（＋）-hHNF-1β,to express the hHNF-1β protein effectively in E.Coli.Methods Total RNA were isolated from the normal liver tissue of human,then full length cDNA of hHNF-1β was amplified by two-step RT-PCR.The cloning plasmid pMD-hHNF-1β was constructed after retrieval of the amplification products,then hHNF-1β was subcloned with primers containing restriction endonucleases recognition sites of BamHⅠ and Hind Ⅲ,ligated into pET-28a（＋）,transducted into BL21（DE3）.After sequenced,the bacteria were induced with IPTG and proteins were expressed,identified with Western blot.Then induction conditions of high level expression were experimented.Results The sequencing result proved that the cloned hHNF-1β cDNA sequence was uniform with its sequence in GenBank.The double enzyme cutting result of recombinant plasmid pET-28a（＋）-hHNF-1β was coincident as we expected.rhHNF-1β was highly expressed in the form of insoluble inclusion body in E.Coli.Western blot result showed the highly expressed protein was really hHNF-1β.The best induction concentration of IPTG was 0.04 mmol/L,the best induction time was 6 h and the best induction temperature was 37℃.Conclusion The prokaryotic expression plasmid of rhHNF-1β was obtained and its expression conditions were optimized,thus established a basis for further function analysis.