Construction And Optimized Expression Of Recombinant Human STim-3-EGFP Fusion Protein

Objective:(1)To construct the recombinant plasmid expression vector pET28a-sTim-3-EGFP of human soluble Tim-3(sTim-3)by cloning the extracellular sequence of human Tim-3 gene.(2)To investigate the expression of Recombinant plasmid in E.coli BL21(DE3)induced by IPTG,and to optimize its expression conditions to obtain more soluble EGFP-sTim-3 fusion protein.Methods:Total RNA was extracted from peripheral blood mononuclear cells from healthy people according to the Trizol Reagent Manual(Invitrogen),and a cDNA library of human Tim-3 gene was constructed according to the reverse transcription reagent(Takara).The expression of Tim-3 gene was detected by RT-PCR.The extracellular fragment of Tim-3 gene was amplified by RT-PCR.The product was ligated with pMD18-T vector(Takara)to transform thecompetent Escherichia coli DH5α and ampicillin(Amp)to select the positive clones.The PCR products were identified by restriction enzyme digestion and the positive clones were sequenced.The purified plasmid pET28a-EGFP was digested by the same endonuclease digestion and agarose gel electrophoresis.The purified sTim-3 fragment and the EGFP-pET28 a fragment were digested with the positive plasmid and the constructed prokaryotic expression vector pET28a-EGFP The pET28a-sTim-3-EGFP recombinant plasmid was constructed.The conjugated Escherichia coli DH5α was transformed into kanamycin(kan)and the positive clones were identified by PCR,double digestion and sequencing.The recombinant plasmid was transformed into E.coli BL21(DE3),and the monoclonal strain was extracted and verified by PCR and double digestion.Fluorescence microscopy was used to determine the expression of IPTG in colony culture cells.SDS-PAGE electrophoresis,Coomassie brilliant blue staining and Western blot were used to verify the target protein.The protein expression conditions were optimized by adjusting the IPTG concentration,induction time,induction temperature and induction time.Results:(1)Approximately 620 bp of the extracellular domain of Tim-3 gene was amplified by total RNA from human peripheral blood mononuclear cells(RT-PCR),ligated with vector pET28a-EGFP,The results showed that the prokaryotic expression vector pET28a-sTim-3-EGFP was successfully constructed.(2)The recombinant plasmid was transformed into competent E.coli BL21(DE3)and induced by IPTG.The results of fluorescence microscopy showed that the fluorescence intensity was enhanced by SDS-PAGE and Coomassie brilliant blue staining and Western blot analysis using His tag monoclonal antibody and EGFP labeled monoclonal antibody.The results showed that about50.0 KDa s Tim-3-EGFP fusion protein expression.(3)SDS-PAGE and Coomassie brilliant blue staining showed that the concentration of IPTG had no effect on the protein expression.The temperature had little effect on the protein expression.When the OD600 value was about0.6-0.8,Higher protein expression was obtained at 5 ℃ for 5 h.Conclusions:The expression vector pET28a-sTim-3-EGFP,which is highly expressed in soluble Tim-3,was successfully constructed.The expressed recombinant protein sTim-3-EGFP was consistent with the expected expression and was expressed as a soluble expression for further purification of the protein.And sTim-3functional research to lay a good foundation.