Eukaryotic Expression Of Human Soluble Growth-Stimulated Expression Gene 2 Protein And Preparation Of Polyclonal Antibody

Human growth stimulation expression gene 2(suppression of tumorigenicity 2,ST2)protein is a member of the IL-1 receptor family.It plays a role through the IL-33-ST2 protein signaling pathway and participates in the occurrence and development of various diseases.Soluble ST2(soluble ST2,sST2)protein can not only be used as an indicator for the diagnosis and monitoring of heart failure,but also assist in the diagnosis of autoimmune diseases,chronic obstructive pulmonary disease,allergic asthma and other diseases and assess the severity of the disease.Clinically,expensive imported ELISA reagents are used to detect the concentration of soluble ST2 protein in serum,reflecting its expression level.Objective:Construct a recombinant eukaryotic expression vector of human sST2 protein and express human sST2 in vitro;optimize the expression and purification conditions of human sST2 protein to obtain high-purity human sST2 recombinant protein;prepare murine polyclonal antibodies.Methods:Stably cultivate human umbilical vein endothelial cells(HUVEC)with ECM medium,design primers based on the human sST2 gene sequence on NCBI,and obtain human sST2 gene using RT-PCR technology;identified by agarose gel electrophoresis,DNA gel recovery,and connection T Vector,gene sequencing,plasmid recovery,enzyme digestion and other steps to construct human sST2 / pcDNA3.1-his(-)recombinant eukaryotic expression vector;lipo 2000 liposome method transfected the expression vector into African green monkey kidney cells(COS7),RT-PCR method was used to observe the effect of different transfection reagent concentration and different transfection time on the expression of human sST2 recombinant protein,and the recombinant protein was purified by nickel column affinity chromatography;human sST2 recombinant protein was tested by western blot and ELISA Identification.The purified human sST2 recombinant protein was concentrated by sucrose to increase the concentration;it was fully ground and emulsified with an equal volume of adjuvant,and immunized with Balb / c mice by subcutaneous injection.The orbital blood of the mice was collected regularly,and the serum was separated,and the antibody was measured by ELISA potency.Results:The HUVEC was successfully cultured in ECM medium,the total RNA of the cells was extracted by the TRIZOL method,and the reverse transcription PCR amplification product was analyzed by agarose gel electrophoresis.The results showed that the product length was as expected,about 1 000 bp;the results of homology comparison analysis showed that,The human sST2(1 002 bp)gene was successfully inserted into the PGH-T vector;the sST2 / pcDNA3.1-his(-)recombinant eukaryotic expression vector was subjected to gel electrophoresis analysis after endonuclease BamH? and Hind ?,The results showed that the product electrophoresis position was as expected.The recombinant expression vector was transfected into COS7 cells with lipo 2000 reagent,and the target gene was amplified by RT-PCR.The results of agarose gel electrophoresis showed that within 24 hours,the transfection of lipo 2000 reagent in 24 well plates was transfected at 2 ul / well.The efficiency is the highest,and at this time,there will not be a large number of cell deaths due to the excessive concentration of the transfection reagent.The cell culture supernatant was collected,and the human sST2 recombinant protein was purified by nickel column affinity chromatography.SDS-PAGE electrophoresis showed a clear band at about 65 KDa,which was consistent with the expected protein position;western blot results showed human sST2 The recombinant protein has a His tag,and the sST2 ELISA reagent test results show that the human sST2 recombinant protein has a specific epitope that binds to the anti-sST2 antibody.Balb / c mice were immunized with human sST2 recombinant protein.After three immunizations,the serum antibody efficacy of the five mice was all> 1:32 000.Conclusions:Successfully constructed human sST2 / PGH-T recombinant cloning vector and human sST2 / pcDNA3.1-his(-)recombinant eukaryotic expression vector.Recombinant human sST2 protein is present in the cell culture supernatant in a soluble form,and it can be successfully purified by nickel column affinity chromatography.Human sST2 recombinant protein has strong immunogenicity,and can obtain highly effective mousederived polyclonal anti-human sST2 protein antibody.