GST／RIPX融合蛋白表达载体的构建及其在大肠埃希菌中的表达

Reconstruction of pGEX-4T-2-RIPX and Its Expressions in E.coli

目的构建GST/RIPX融合蛋白基因表达载体,并在大肠埃希菌(E.coli)中诱导表达。方法以质粒pcDNA3.1-RIPX为模板,通过BamH1和Xho1酶切位点将RIPX定向插入pGEX-4T-2中,构建原核表达质粒pGEX-4T-2-RIPX,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,转入化E.coli BL21中,异丙基硫代β-D半乳糖苷诱导表达,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-4T-2-RIPX,并用SDS-PAGE方法证实了GST/RIPX融合蛋白的表达。结论成功构建了RIPX原核表达载体,并证实了融合蛋白的表达,为进一步纯化RIPX蛋白和研究RIPX的生物学功能奠定了基础。

Objective To construct GST/RIPX fusion protein expression vector and induce its expression in Escherichia coli（E.coli）.Methods The coding sequence of RIPX was amplified from the plasmid pcDNA3.1-RIPX by PCR and inserted into pGEX-4T-2 by BamHI and Xho1.The positive recombinants were identified by restriction endonuclease digestion and DNA sequencing.Then they were transformed into E.coli BL21,induced by IPTG and identified by SDS-PAGE.Results Enzyme digestion and DNA sequencing results indicated that the prokaryotic expression plasmid pGEX-4T-2-RIPX was successfully constructed and confirmed.The desired GST/RIPX fusion protein was detected by SDS-PAGE.Conclusion The prokaryotic expression plasmid of RIPX is successfully constructed and the expression of fusion proteins is confirmed.This study provides the basis for the further purification of RIPX and research on the biological function of RIPX.