Expression and refolding of OLA Ⅰ protein with peptides derived from sheeppox virus / 生物工程学报

The aim of this study was to refold the OvisAries leukocyte antigen (OLA) class Ⅰ protein with peptides derived from sheeppox virus (SPPV) to identify SPPV T cell epitopes. Two pairs of primers were designed based on the published sequence of a sheep major histocompatibility complex Ⅰ to amplify the heavy chain gene of OLA Ⅰ α-BSP and the light chain gene of OLA Ⅰ-β2m. Both genes were cloned into a pET-28a(+) expression vector, respectively, and induced with ITPG for protein expression. After purification, the heavy chain and light chain proteins as well as peptides derived from SPPV were refolded at a ratio of 1:1:1 using a gradual dilution method. Molecular exclusion chromatography was used to test whether these peptides bind to the OLA Ⅰ complex. T-cell responses were assessed using freshly isolated PBMCs from immunized sheep through IFN-γ ELISPOT with peptides derived from SPPV protein. The results showed that the cloned heavy chain and light chain expressed sufficiently, with a molecular weight of 36.3 kDa and 16.7 kDa, respectively. The protein separated via a SuperdexTM 200 increase 10/300 GL column was collected and verified by SDS-PAGE after refolding. One SPPV CTL epitope was identified after combined refolding and functional studies based on T-cell epitopes derived from SPPV. An OLA Ⅰ/peptide complex was refolded correctly, which is necessary for the structural characterization. This study may contribute to the development of sheep vaccine based on peptides.

Study on prediction of CTL Epitope for Yersinia pestis antigens / 中国地方病学杂志

Objective To establish a method predicting CTL epitopes of antigens in Yersinia pestis,which provides an effective research plan for epitnmics in Yersinia pestis.Methods CTL epitopes were predicted by nHLAPred.BIMAS and SYFPEITHI.Results CTL epitopes of LcrV antigen were detected as V8-16 NPQHFIEDL,V311-319 KYDSVMQRL and V258-264 SYNKDNNEL.Conclusions Prediction of CTL epitope can be used to make epitope mapping because of its hiigh accuracy.It may provide a reference for the epitope mapping plan.

Selection and identification of HLA-A2-restricted CTL epitopes within heparanase / 中华微生物学和免疫学杂志

Objective To select and identify human leukocyte antigen(HLA)-A2-restricted T-cell epitope within heparanase(Hpa),and to provide aeademic bases for peptide-based immunotherapy with malignant tumor.Methods The Hpa sequence was scanned for prediction of the immunogenic peptides-based CTL epitopes using the HLA-binding prediction software SYFPEITHI and BIMAS.Ten the affinity of candidate epitopes to HLA-A2 was evaluated by T2 binding test.Acfivation of T cell was assessed by ELIS-POT and cytotoxictiy by lactate dehydrogenase(LDH)release assay.Results of the six predicted nona-peptides,Hpa(310-318,FLNPDVLDI)showed high affinity of binding to HLA-A2 and led to IFN-γ secre-tion in vitro.Furthermore,Hpa(310-318) FLNPDVILDI could induce PBMC from a HLA-A2 positive healthy donor to lyse specifically HCC-LM6 and SW-480 cells which expressing both Hpa and HLA-A2.Conclusion The nona-peptide Hpa(310-318)FLNPDVLDI may be all HLA-A2-restricted CTL epitope.which is capable of inducing Hpa-specific CTL in vitro.

Effects of cen-penetrating peptide on the presentation of MHC classⅠ-restricted cytotoxic T lympho-cyte epitope of human papillomavirus 16E7 / 中华皮肤科杂志

0.05).At the late stage (8-48 h) of incubation,the presence time of E7_(49-57)/K~b was significantly pro- longed on the surface of Tat-E7_(49-57)-incubated cells than that on the surface of other peptides-incubated cells (all P

Replacing CTL epitope of recombinant HBsAg by SOEing PCR / 第三军医大学学报

Objective To replace the endogenous CTL epitope LLD in HBsAg with EYILSLEEL of glypican(GPC3)in order to prepare a GPC3-HBsAg multiple peptides vaccine for HBV infection.Methods The HBsAg/GPC3 DNA was amplified by gene splicing by overlap extension(SOEing)PCR from pcHBsAg plasmid and then inserted into pBSSK+ vector to construct a pBSSK/GPC3 vector.The vector was then identified by PCR,double digesting and sequencing.The fragment encoding GPC3 CTL epitope EYILSLEEL was obtained by double digesting the plasmid pBSSK/GPC3 and then inserted into pcDNA3.1+ vector.Results Sequencing and restrict endonulease digestion indicated that eukaryotic expression vector pcDNA-HBsAg/GPC3 was constructed successfully.Conclusion The endogenous CTL epitope LLD of HBsAg is replaced by the EYILSLEEL,and an eukaryotic expression vector pcDNA-HBsAg/GPC3 is constructed.

Prediction of HLA-A~*0201-restricted CTL epitopes in SARS-CoV E protein / 第三军医大学学报

Objective To predict the HLA A *0201 restricted CTL epitopes (nonamers) in SARS coronavirus (SARS CoV) E protein. Methods The CTL epitopes of E protein were predicted by using supermotif method combined with quantitative matrix method. Results Thirteen HLA A *0201 restricted CTL epitope candidates were predicted in SARS CoV E protein. Conclusion The prediction of the CTL epitopes in SARS CoV E protein will benefit the identification of CTL epitopes by experiment. Those results are of importance for immune recognition and vaccine design for SARS CoV.

Identification a HLA-A2-restricted CTL epitope of breast cancer differentiation antigen NY-BR-1 / 中国免疫学杂志

Objective:To identify HLA-A2-restricted CTL epitope of breast cancer differentiation antigen NY-BR-1.Methods:The HLA-A2-restricted CTL epitope of breast cancer differentiation antigen NY-BR-1 is predicted by combination quantitative motif method and the molecular dynamics.The three epitope were assayed their affinity to HLA-A2.Results:The affinity to HLA-A2 of NY-BR-1_ 1043-1051 is a best.Conclusion:NY-BR-1_ 1043-1051 is a HLA-A2 restricted epitope.

Polylatic acid microspheres-induced CTL cytotoxicity against hepatocellular carcinoma cell lines / 中国肿瘤生物治疗杂志

Objective: To investigate the in vitro efficiency of two kinds of polylatic acid (PLA) immunomicrospheres: M1(hAFP158~166)and M2(hAFP218~226) in specific inducement of CTL and the cytotoxicity of CTL against hepatocellular carcinoma cells. Methods: Pripheral blood monocytes (PBMC) of HLA-A2+ healthy volunteers stimulated in vitro by peptide-loading microspheres were taken as effectors. The experiments were divided into three groups: control group, hAFP+ tumor cells group, and hAFP- tumor cells group. The specific cytolysis of target cells was assessed by 51Cr-release assay. Results: Both M1 and M2 induced proliferation of HLA-A2+ PBMC, forming visible clones. The effector cells induced by M1 and M2 both had an cytolysis rate over 75% for hAFP+ tumor cells, which was significantly higher than that for hAFP- tumor cells (P0.05). Conclusion: The two kinds of microspheres can both induce specific CTL in vitro, which can effectively kill hAFP+ tumor cells.

A CTL epitope of respiratory syncytial virus fused with G protein fragment regulates specific immunoresponses / 中国免疫学杂志

Objective: To investigate the regulation of respiratory syncytial virus CTL epitope fused with G protein antigen fragment G1 to the specific immunoresponses. Methods: The recombinant plasmid pET-DsbA-G1 or pET-DsbA-G1F/M2 was transferred into E.coli BL21(DE3) and the fusion protein DsbA-G1F/M2 or DsbA-G1 was expressed.The expressing product was induced and purified by affinity chromatography. The two proteins were used to immunized BALB/c mice i.p, respectively. Serum and spleen cells were collected regularly. RSV-specific CTL responses were measured by MTT, IgG and IgG1 and IgG2a antibodies by ELISA, neutralizing antibodies by plaque reduction assay. Results: The recombinant proteins were expressed successfully and the purity was over 90% after purified by affinity chromatography. The protein DsbA-G1F/M2 induced significant RSV-specific CTL responses, while DsbA-G1 without CTL epitope did not induce detctable CTL responses. Strong IgG antibody responses were elicited,indicated by both. IgG1 and IgG2a titers induced by DsbA-G1F/M2, while only IgG1 was induced by DsbA-G1.The ratio of IgG1/IgG2a was downregulated significantly. Both antigens induced high level of neutralizing antibodies, but the latter was a little lower. Conclusion: DsbA-G1F/M2, the fusion protein of a CTL epitope and G protein fragment G1 can induce both cellular immunity and humoral immunity. The activation of CTLs downregulates the ratio of IgG1/IgG2a.The more balanced immunoresponse is advantageous for improving safety of the candidate vaccine.