Screening And Identification Of Toxoplasma Gondii Surface Antigen 1 And Host Cell Interaction Proteins

ObjectiveToxoplasma gondii surface antigen1(SAG1)is expressed in the tachyzoite stage of T.gondii.Its main functions are known to assist tachyzoites to invade host cells and to induce host immunity.The aim of this study was to screen host cells for proteins that interact with the T.gondii surface antigen1(SAG1)and to explore the new functions of SAG1.And hope it will lay a foundation for the study of the molecular mechanism of SAG1 invasion of host cells.MethodsSpecific primers were designed based on the known SAG1 gene sequence,and the whole genome of T.gondii GT1 strain was used as the template for PCR amplification of the SAG1 gene fragment and cloned into the p MD18-T vector.After double digestion and sequencing,the correct target gene fragment was cloned into the p GEX-4T-1 vector plasmid to construct the prokaryotic expression vector p GEX-4T-1-SAG1.The correctly sequenced prokaryotic expression vector was transformed into BL21 receptor cells.And the expression was induced by IPTG.Protein purification was performed by guanidine hydrochloride denaturation and gradient dialysis renaturation.Western Blot validation was performed using GST-tagged monoclonal antibody and SAG1 monoclonal antibody as primary antibody.By GST pull-down technology,the purified recombinant protein SAG1 was used as a decoy protein to screen the proteins interacting with human lung cancer cells(A549),human liver cancer cells(Hep G2)and human colon cancer cells(HCT116)respectively,and the pull-down proteins were analyzed by mass spectrometry to screen the host cell proteins interacting with SAG1.The results of protein mass spectrometry were analyzed and the interaction proteins with important biological significance were selected for validation.The primers with Flag tags were designed.The SAG1 gene fragment was amplified by PCR,and the eukaryotic expression vector pc DNA3.1(+)-Flag-SAG1 was constructed.The plasmid was identified and sequenced by double digestion.And the correctly sequenced plasmid was transfected into human embryonic kidney epithelial cells(293T).Immunoprecipitation,GST pull-down and immunofluorescence were used for the validation of the reciprocal proteins.ResultsIn this experiment,the SAG1 gene fragment of T.gondii GT1 strain was obtained by PCR amplification with a size of 1011 bp.The prokaryotic expression vector p GEX-4T-1-SAG1 was constructed,and the positive plasmids were double digested to obtain two expected bands of about 4969 bp and 1011 bp,and the insert fragment was sequenced to 1011 bp,which were consistent with the expected results.Induced by IPTG,the recombinant protein SAG1 was successfully expressed with a molecular weight size of 63 k Da(containing GST tag protein).The purified recombinant protein was verified by Western Blot and could be specifically recognized by GST antibody and SAG1 antibody.The results of mass spectrometry showed that there were 36,24 and 28 endogenous proteins in the interaction between recombinant protein SAG1 and human lung cancer cells(A549),human hepatoma cells(Hep G2)and human colorectal cancer cells(HCT116),respectively(the number of unique peptide segments was more than 2).Using Wayne diagram statistical analysis,it was found that there were 13 interacting proteins shared by 3 host cells,including KPYM,G3 P,HS90B,TBB5,RL7,RL18,ENOA,HNRPU,LDHA,RACK1,GSTM3,GSTP1 and ATPA.Immunoprecipitation,GST pull-down and immunofluorescence were used to validate the important scaffold protein RACK1.Flag-SAG1 was transfected into293 T cells for immunoprecipitation.Western Blot validation was performed using RACK1 antibody as primary antibody.An expected RACK1 protein band at 32 k Da confirmed the interaction between SAG1 and RACK1.The existence of direct interaction between SAG1 and RACK1 was again verified by GST pull-down assay.Recombinant plasmids p EGFP-N1-SAG1 and m Cherry-RACK1 were cotransfected with 293 T cells,and SAG1 and RACK1 were co-localized in the cytoplasm as observed by fluorescence microscopy.Conclusions1.Using GST pull-down tandem mass spectrometry,36,24 and 28 endogenous proteins of human lung cancer cells(A549),human liver cancer cells(Hep G2)and human intestinal cancer cells(HCT116)were found to interact with recombinant protein SAG1,respectively(the number of unique peptide segments was more than 2).Combined with the analysis of Venn diagram statistical methods,it is found that there are 13 interacting proteins shared by the three host cells,namely KPYM,G3 P,HS90B,TBB5,RL7,RL18,ENOA,HNRPU,LDHA,RACK1,GSTM3,GSTP1 and ATPA.2.Three methods including co-immunoprecipitation,GST pull-down and immunofluorescence were used to verify the direct interaction between the T.gondii SAG1 and RACK1.