Mass Spectrometric Analysis Of HBV Polypeptides Presented By B Lymphoblastic Cell Lines Carrying Specific HLA-DR Genes

Background and PurposeHBV infection is a serious infectious disease that seriously endangers human health.A large number of domestic and foreign studies have shown that HLAⅡpresented antigenic peptide can effectively clear the infected virus in the process of inducing specific lymphocyte reaction,but the exact role in the process of chronic hepatitis B is not completely clear.The epidemiological observation of our research group on acute,chronic and self-limited HBV infection population in the early stage found that,HBV subtype C2 was the main HBV infection in Heilongjiang,and the chronic outcome of hepatitis B was positively correlated with the frequency of HLA-DR12.In specific experimental peptide-induced peripheral blood mononuclear cells（PBMCs）proliferation and HLA-DR antibody inhibition experiments,compared with protective HLA-DR13,HLA-DR12 presents a broad spectrum of HBV antigenic peptides with low specificity.It is suggested that the clinical outcome of HBV infection in a population with HLA-DR genetic background may be realized by the preference of HLA-DR binding HBV antigen peptide spectrum,which stimulates different polarization directions of CD4⁺T.The aim of this research is to combine co-immunoprecipitation（Co-IP）with mass spectrometry（MS）and immunobioinformatics methods were used to investigate the characteristics of HBV-specific antigenic peptides presented by HLA-DR associated with HBV infection progression.MethodsThe PBMCs of HBV-immunized healthy persons were transformed by EBV to construct BLCLs carrying specific HLAⅠandⅡ.CD19 was detected by flow cytometry,and HLA-A/B/C,HLA-DP/DR/DQ were classified by PCR-SBT.After transient transfection of p AAV/HBV1.2_C2plasmid into BLCLs at 18,24,and 48h,HBV load,HBs Ag,and HBe Ag levels in supernatant were detected by q RT-PCR and ELISA,respectively.The relative expression level of HLA-DR m RNA was detected by q RT-PCR.Transfected cells were collected for 48h,and Co-IP was used to enrich peptide/HLA-DR complex（p HLA）.At the same time,HLA-DR level was detected by WB,p HLA was sequenced by LC-MS/MS,and the results were performed by software comparison and statistical analysis.Results1.Five strains of BLCLs carrying specific HLAⅠandⅡwere constructed,and the cell surface of each strain showed CD19 high pattern（85.5～96.4%）.They were named HLA-DR12/12,HLA-DR07/12,HLA-DR09/09,HLA-DR14/15,and HLA-DR13/15,respectively.2.BLCLs was co-transfected with p AAV/HBV1.2_C2plasmid and pmax GFP plasmid,and the transfection efficiency reached 65%in 48h.At 18,24 and 48h after transfection,HBV DNA load decreased gradually.HBs Ag levels in HLA-DR12/12and HLA-DR14/15 strains were significantly higher at 48h than at 24h after transfection（P<0.01）;HBe Ag levels in HLA-DR12/12,HLA-DR07/12 and HLA-DR13/15 strains were significantly higher at 48h with 24h after transfection（P<0.01）.At the same time,the relative expression of HLA-DR m RNA reached the peak at 24h after transfection with HLA-DR07/12 and HLA-DR12/12 strains.The expression levels of HLA-DR09/09,HLA-DR14/15 and HLA-DR13/15 strains peaked at 18h after transfection.3.Co-IP showed that the relative expression level of HLA-DR in five BLCLs transfected with recombinant plasmid was higher than that before transfection（P<0.01）,and p HLA was successfully prepared.A total of 36 HBV antigen peptides（5,12,5,8 and 6）were detected from HLA-DR12/12,HLA-DR07/12,HLA-DR09/09,HLA-DR14/15 and HLA-DR13/15 cell lines by LC-MS/MS.Net MHCⅡpan-4.0algorithm predicted 2 strong binding p HLA and 10 weak binding p HLA.At the same time,the obtained peptide spectrum was matched with the reported peptides,and of these,25 peptides were reported as common,overlapping or containing sequences,and 13 were consistent with alleles carried by the corresponding cell lines.Conclusions1.Five strains of BLCLs were constructed and named HLA-DR12/12,HLA-DR07/12,HLA-DR09/09,HLA-DR14/15,and HLA-DR13/15,respectively.2.A total of 36 HLA-DR restricted HBV antigen peptides were preliminarily screened in the five BLCLs,and 23 new antigen peptides may be found.