pPET-SUMF2各亚型的原核质粒构建及蛋白表达

Construction of subtypes of pPET-SUMF2 and its protein expression in prokaryotic plasmid

构建人硫酸酯酶修饰因子2(SUMF2)基因各亚型的融合表达载体并在大肠杆菌中诱导表达pPET-SUMF2各亚型的融合蛋白。用EcoRI和XhoI双酶切人SUMF2各种亚型基因及质粒pPET-30a;将2种酶切产物按常规方法连接、转化至大肠杆菌DH5α。挑取菌落培养,提取质粒进行验证。将构建成功的pPET-SUMF2各亚型重组表达质粒转化至BL21(DE3)中,经IPTG诱导后,SDS-PAGE电泳分析以及Western blot检测SUMF2各亚型的融合蛋白表达。成功构建了pPET-SUMF2各亚型的重组表达质粒,并成功诱导SUMF2各亚型融合蛋白的表达,Western blot结果显示在预期位置有特异蛋白条带表达,并在BL21(DE3)内表达了SUMF2各亚型的融合蛋白,为进一步研究SUMF2的功能以及SUMF2与哮喘发病的关系奠定了基础。

This study was to construct the prokaryotic expression plasmid containing the subtypes of human sulfatase modifying factor 2(SUMF2)gene,and detect its expression in prokaryotic plasmid.The subtypes of SUMF2 gene fragment were inserted into pPET-30 ato conduct a prokaryotic plasmid,then sequenced and identified by restrictive endonuc lease digestion.The constructed recombinant plasmid was finally induced by IPTG and the protein was tested by western blot and SDS-PAGE.The results show that the recombinant expression plasmid PET/SUMF2-V2,PET/SUMF2-V3,PET/SUMF2-V4,PET/SUMF2-V5,PET/SUMF2-V7 are successfully constructed and the fusion protein expression of the subtypes of SUMF2 is successfully induced.The expression of protein is observed by western blot and SDS-PAGE.We successfully constructed the subtypes of pPETSUMF2 and got their protein expression in prokaryotic plasmid.The findings in this yesearch builds a foundation for further research on the biological function of SUMF2 and the relationship between SUMF2 and asthma.