Construction Of Prokaryotic Expression Vector Of MHSF2-pET-28a And Protein Expression、Purification And Functional Study

Objective:Firstly the prokaryotic expression vector mHSF2-pET-28a was constructed successfuLly.Then,mHSF2 was purified with His-selected nickel affinity gel and studied whether it had biological activity in vitro and whether it couLd enter the cells to exert biological functions.Methods:Using mHSF2 as a template,the fuLl-length mHSF2 coding sequence was amplified by PCR.The purified product was digested with BamHI and xhol,and the mHSF2 fragment was inserted into the prokaryotic expression vector pET-28 a using gene recombination technology.After the pET-28a-mHSF2 plasmid sequence was tested correct ly,the pET-28a-mHSF2 recombinant plasmid was transferred into BL21,and the host strain was induced to express mHSF2 by IPTG.The recombinant protein was identified by Coomassie brilliant blue staining and Western Blot;the recombinant protein was separated by Ni-NTA affinity chromatography,and EMS A was conducted to identify whether the purified recombinant protein had biological activity in vito.The recombinant protein was used to interfered Raw246.7 cells,and extracted total celluLar protein,and Western Blot was applyed to assesses whether the recombinant protein can enter the cell to exert its biological function.ResuLts:The fuLl-length mouse HSF2 coding sequence was cloned into a prokaryotic expression vector.The fragment identified by BamHI and xhol was about 1500 bp,and the mHSF2-pET-28a recombinant plasmid was sequenced correctly.After the recombinant plasmid was transfected into BL21 E.coli,mHSF2 couLd be expressed under the induction of IPTG.Prokaryotic expression of the recombinant protein can specifically bind His antibody and HSF2 antibody,indicating that the recombinant protein is mHSF2;EMSA detection of mHSF2 and HSE binding found that prokaryotic expression of mHSF2 had biological activity;The interaction of Raw246.7 cells with recombinant protein revealed that mHSF2 could enter into cells and play function.Conclusion:The pET-28a-mHSF2 prokaryotic expression vector was constructed using gene engineering technology and expressed soluble mHSF2.The prokaryotic expression of mHSF2 has biological functions and can enter into cells,laying the experimental foundation for subsequent research on HSF2.