C／EBPα不同截短功能域GST融合蛋白表达载体的构建及表达的研究

Construction of GST-C/EBPα Fusion Protein Expression Vectors and the Expressions in Escherichia coli

目的构建GST-C/EBPα融合蛋白表达载体,并在大肠埃希菌(E.coli)中诱导表达。方法以质粒pcDNA3.1-C/EBPα为模板,用PCR法扩增C/EBPα全长及各个截短片段,通过EcoRI和XhoI酶切位点将C/EBPα全长及各个截短突变体定向插入pGEX-5X-1中,构建原核表达质粒pGEX-5X-1-C/EBPα及其截短突变体,并转化E.coli DH5α,筛选阳性重组子,限制性内切酶酶切鉴定和DNA序列测定正确后,转入E.coli BL21中,异丙基硫代β-D半乳糖苷诱导表达,SDS-PAGE和SDS-PAGE鉴定。结果酶切及测序结果证明,成功构建了原核表达质粒pGEX-5X-1-C/EBPα及其截短突变体,并用SDS-PAGE方法证实了GST-C/EBPα全长及各个截短突变体融合蛋白的表达。结论成功构建了C/EBPα原核表达载体及其截短突变体,并证实了融合蛋白的表达,为进一步纯化C/EBPα蛋白和研究C/EBPα的生物学功能奠定了基础。

Objective To construct GST-C/EBPc~ fusion protein expression vector and induce its expression in Escheffchia coli （E.coli）. Methods The coding sequence of CCAAT-enhancer binding proteinα （C/EBPα）and its truncation fragments were amplified fi＇om the plas- mid pcDNA3.1-C/EBPα by PCR and inserted into pGEX-5X-1 by EcoRI and XhoI. The positive recombinants were identified with restriction endonuclease digestion and DNA sequencing. Then they were transformed into E.coli BI21 ,induced with IPTG and identified with SDS- PAGE and SDS-PAGE. Results The prokaryotic expression plasmid pGEX-5X-1-C/EBPα and its truncation mutants were successfully constructed and confirmed by enzyme digestion and sequencing. The desired GST-C/EBPα fusion proteins were expressed and confirmed by SDS-PAGE. Conclusion The prokaryofic expression plasrnid of C/EBPα and its truncation mutants were successfully constructed and the expression of fusion proteins was confirmed. This study provides the basis for the further research on purifying C/EBPα protein and the biological function of C/EBPα.