原核表达载体GST-RUNX3的构建及其在大肠杆菌中的表达

目的构建GST/RUNX3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-RUNX3为模板,通过Kpn1和Xho1酶切位点将RUNX3定向插入pGEX-4T-2中,构建原核表达质粒pGEX-4T-2-RUNX3,并转化E.coliDH5α,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.coliBL21中,异丙基硫代β-D半乳糖苷诱导表达,鉴定。结果双酶切获得1300bp片段,证明RUNX3片段被成功导入pGEX-4T-2,并在测序后与GenBank进行同源性比对,比对进一步证实原核表达质粒pGEX-4T-2-RUNX3构建成功,并用IPTG诱导,SDS-PAGE方法证实了GST/RUNX3融合蛋白在大肠杆菌中的表达。结论成功构建了RUNX3原核表达载体,并证实了融合蛋白在大肠埃希菌中的表达,为进一步纯化RUNX3蛋白和研究RUNX3的生物学功能奠定了基础。

Construction and expression of eukaryotic prokaryotic expression vector GST/RUNX3 in E.coil

Objective To construct GST/RUNX3 fusion protein expression vector and induce its expression in Escherichia coli （E.coli） . Methods The coding sequence of RUNX3 was amplified from the plasmid pcDNA3. 1-RUNX3 by PCR and inserted into pGEX 4T-2 by KpnI and Xhol. The positive recombinants were identified by restriction endonuclease digestion and DNA sequencing, then transformed into E.coli BL21, induced by IPTG and identified by SDS PAGE. Results The length of the 1300bp fragment was identified by double enzymes digestion, improved the fragment of RUNX3 was cloned into the pGEX-4T-2plasmid, sequence was compared with that in GenBank for homology also improved the pGEX 4T-2-RUNX3 was successfully constructed. The desired GST/RUNX3 fusion protein was expressed in E.coli and confirmed by SDS-PAGE. Conclusions The prokaryotic expression plasmid of RUNX3 was successfully construeted and the expression of fusion proteins was confirmed. This study provides the basis for the further research on purifying RUNX3 protein and the biological function of RUNX3