Clinical and experimental investigations into the immune modulatory effects of Qianjinba polysaccharides on a rheumatoid arthritis mouse model:in vivo and in vitro studies

Qianjinba is derived from the dried root of a legume vine and is known for its plant polysaccharide,which has demonstrated anti-inflammatory,analgesic,and neuroprotective properties.Despite these known effects,its immunomodulatory potential remains underexplored.This study aimed to investigate the immunoregulatory effects of Qianjinba polysaccharide(QJBDT)in a murine model of rheumatoid arthritis(RA)and elucidate its mechanism of action in inhibiting the disease.To assess the immunomodulatory effects,RAW264.7 macrophage cells were stimulated with bacterial lipopolysaccharide(LPS)and subsequently treated with varying concentrations of QJBDT or total glucoside of paeonic acid(TGP).Cell proliferation and the expression levels of mitogen-activated protein kinase p38 and nuclear factor NF-κB proteins were evaluated.Following the successful modeling of RA in mice,different doses of QJBDT and TGP were administered via intragastric administration once daily for 21 d.Mice were divided into normal,model,QJBDT low-,medium-,and high-dose groups,along with a positive control group(TGP group),each comprising 10 mice.Organ coefficients were calculated,immune indexes in blood cells were determined,and alterations in T helper cell 17(Th17)and regulatory T cells(Treg)were analyzed using flow cytometry.Compared to the normal group,cell proliferation rates significantly increased across all groups(P<0.05).Elevated expressions of p38 and NF-κB proteins were observed in the model group,QJBDT low-and medium-dose groups(P<0.05).Various systemic immune inflammation indexes,including systemic immune inflammation index(SII),total inflammation systemic index(AISI),and systemic inflammation response index(SIRI),platelet-to-lymphocyte ratio(PLR),neutrophil-to-lymphocyte ratio(NLR),monocyte-to-lymphocyte ratio(MLR),showed increments(P<0.05).Additionally,body weight and organ coefficients of the thymus,spleen,and liver decreased in both the model and low-dose groups(P<0.05).Moreover,Th17 levels increased while Treg levels decreased across all groups(P<0.05),resulting in a heightened Th17 to Treg ratio in the model and low-dose groups(P<0.05).Notably,the QJBDT medium-dose,high-dose,and positive control groups demonstrated significant inhibitory effects on cell proliferation compared to the model group(P<0.05),along with reduced expression levels of p38 and NF-κB(P<0.05).Furthermore,various immune indicators from routine blood tests exhibited different degrees of decrease(P<0.05),while immune indices displayed increases(P<0.05).The low-,medium-,and high-dose groups of QJBDT,as well as the positive control group,showed decreased Th17 levels,elevated Treg levels,and diminished Th17 to Treg ratios(P<0.05).Additionally,there was no statistically significant difference in efficacy between the QJBDT high-dose and TGP groups(P>0.05).In conclusion,this study demonstrated that QJBDT exerted potent immunomodulatory effects on RA in mice in a dose-dependent manner.Its mechanism of action might involve the inhibition of NF-κB activation by suppressing the p38 MAPK signaling pathway.

Bioinformatics analysis of the structure and linear B-cell epitopes of aquaporin-3 from Schistosoma japonicum

Objective:To analyze the structure of aquaporins-3(AQP-3) from Schistosoma japonicum(SJAQP-3) using bioinformalical methods,and to provid of references for vaccine targets research.Methods:Protparam,BepiPred,TMHMM Server,MLRC,Geno3d,DNA star software packages were used to predict the physical and chemical properties,hydrophilicity plot, flexibility regions,antigenic index,surface probability plot,secondary structure,and tertiary structure of amino acid sequence of SJAQP-3.Results:SJAQP-3 had six transmembrane regions and two half-spanning helices that form a central channel.The half-spanning helices fold into the centre of the channel.Either of the half-spanning helix had a conserved motif of NPA common to all aquaporins.Predicted linear B-Cell epitopes were most likely at the N-terminal amino acid residues of Saa-7aa,59aa- 62aa,225aa-230aa,282aa -288aa,294aa -29Saa and 305aa -307aa area.59aa- 62aa,22Saa-230aa located outside the membrane,the others located inside the cell.Conclusions:SJAQP-3 is a integral membrane protein in Schistosoma japonicum tegument.There are six potential epitopes in SJ AQP-3.It might be a potential molecular target for the development of vaccines.

Prediction of T cell and B cell epitopes of the 22-, 47-, 56-, and 58-kDa proteins of Orientia tsutsugamushi

Objective:To predict B cell and T cell epitopes of 22-kDa,47-kDa,56-kDa and 58-kDa proteins.Methods:The sequences of 22-kDa,47-kDa,56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA,DNAstar,Bcepred,ABCpred,NetMHC,NetMHCⅡand IEDB.The 58-kDa tertiary structure model was built by MODELLER9.17.Results:The 22-kDa B-cell epitopes were located at positions 194-200,20-26 and 143-154,whereas the T-cell epitopes were located at positions 154-174,95-107,17-25 and 57-65.The 47-kD a protein B-cell epitopes were at positions 413-434,150-161 and 283-322,whereas the T-cell epitopes were located at positions 129-147,259-267,412-420 and 80-88.The 56-kDa protein B-cell epitopes were at positions 167-173,410-419 and 101-108,whereas the T-cell epitopes were located at positions 88-104,429-439,232-240 and 194-202.The 58-kDa protein B-cell epitopes were at positions 312-317,540-548 and 35-55,whereas the T-cell epitopes were located at positions 415-434,66-84 and 214-230.Conclusions:We identified candidate epitopes of 22-kDa,47-kDa,56-kDa and 58-kDa proteins from Orientia tsutsugamushi.In the case of 58-kDa,the dominant antigen is displayed on tertiary structure by homology modeling.Our findings will help target additional recombinant antigens with strong specificity,high sensitivity,and stable expression and will aid in their isolation and purification.

The Prediction of B Cell Epitopes for VP73 Protein of African Fever Virus

[Objective] The B cell epitopes for VP73 protein of African swine fever virus was predicted. [ Method] Based on the analysis of the amino acid sequence and the flexible regions of VP73 protein, the B cell epitopes for VP73 protein of African swine fever virus were predicted by method of Kyte-Doolittie, Emini and Jameson-Wolf. [Result] The B cell epitopes were located at or adjacent to the N-terminal No. 11 - 18,26 -48,73 -82,136 - 150,159 - 174,181 - 189,191 - 210,247 - 276,279 - 295,313 - 323 and 382 - 392. [Conclusion] The multi-parameters analytic method was adopted to predict the B cell epitopes for VP73 protein of African swine fever virus, which laid solid foundation for further characterizing the protein of VP73 and researching epitope vaccine.

Ribavirin and IFN-α combination therapy induces CD4+ T-cell proliferation and Th1 cytokine secretion in patients with chronic hepatitis B

AIM: To investigate the anti-viral mechanism of combination therapy of interferon (IFN)-α and ribavirin in patients with chronic hepatitis B. METHODS: Twenty patients were assigned to receive either IFN-α plus ribavirin (group A,n = 14) or no treatment as a control (group B,n = 6). Patients were analyzed for T-cell proliferative responses specific for hepatitis B virus (HBV)-antigen and cytokine production by peripheral blood mononuclear cells (PBMCs). RESULTS: Combination therapy induced HBV-antigen specific CD4+ T-cell proliferative responses in four patients (28.6%). Production of high levels of HBV-specific IFN-γ,tumor necrosis factor (TNF)-α,interleukin (IL)-12 by PBMCs was found in five patients (35.7%),who showed significantly lower HBV DNA levels in serum at 12 mo after treatment ended (P = 0.038) and at 24 mo of follow-up (P = 0.004) than those without high levels of cytokine production. CONCLUSION: HBV-antigen specific CD4+ T cells may directly control HBV replication and secretion of anti-viral T helper 1 (Th1) cytokines by PBMCs during combination therapy of chronic hepatitis B with ribavirin and IFN-α.

Prediction of B Cell Epitope of DMRT Protein in Oreochromis niloticus

[Objective] The research aimed to predict the B cell epitope of DMRT protein in Oreochromis niloticus. [Method] The secondary structure of amino acid sequence of DMRT protein was revealed by Garnier-Robson, Chou-Fasman and Karplus-Schulz methods. The hydrophilicity plot, surface probability and antigenic index were obtained by Kyte-Doolittle, Emini and Jameson-Wolf methods, respectively. Based on the above results, the B cell epitopes for DMRT were predicted. [Result] Both the prediction results from Garnier-Robson, Chou-Fasman methods indicated that the α-helix centers of DMRT protein in O. niloticus were in the N terminal No. 31-56, 68-75, 110-116, 209-211 and 239-243; the β-sheet centers of DMRT protein in O. niloticus were in the N terminal No. 95-99, 177-183, 225-234 and 251-254. With the assistant of Kyte-Doolittle, Emini and Jameson-Wolf methods, the B cell epitopes for DMRT were located in or nearby the N terminal No. 13-16, 35-38, 47-54,84-93, 101-109, 127-156, 166-177 and 198-201. [Conclusion] These results are helpful for preparing the antibody of DMRT protein and revealing the sex determination mechanism of O. niloticus.

Theoretical Study of Continuous B-Cell Epitopes with Developed BP Neural Network

In order to identify continuous B-cell epitopes effectively and to increase the success rate of experimental identification, the modified Back Propagation artificial neural network (BP neural network) was used to predict the continuous B-cell epitopes, and finally the predictive model for the B-cells epitopes was established. Comparing with the other predictive models, the prediction performance of this model is more excellent (AUC = 0.723). For the purpose of verifying the performance of the model, the prediction to the SWISS PROT NUMBER: P08677 was carried on, and the satisfying results were obtained.

Correlation of peripheral blood Th17 cell surface CCR4, CCR6 and CXCR3 expression with the illness in patients with chronic hepatitis B

Objective:To study the correlation of peripheral blood Th17 cell surface CCR4, CCR6 and CXCR3 expression with the illness in patients with chronic hepatitis b (CHB).Methods: A total of 48 patients who were diagnosed with chronic hepatitis b in Jianyang People's Hospital between July 2014 and January 2017 were selected as the CHB group of the research, 66 healthy volunteers who received physical examination during the same period were selected as control group, the peripheral blood was collected to determine Th17 cell surface CCR4, CCR6 and CXCR3 expression, and serum was collected to determine the levels of Th17 cytokines, liver function indexes and liver fibrosis indexes.Results: Peripheral blood Th17 cell surface CCR4, CCR6 and CXCR3 fluorescence intensity of CHB group were significantly higher than those of control group;serum IL-17, IL-21, IL-22, ALT, AST, GLB, HA, PC-Ⅲ, LN and C-Ⅳ levels of CHB group were significantly higher than those of control group and positively correlated with peripheral blood Th17 cell surface CCR4, CCR6 and CXCR3 fluorescence intensity while serum ALB level was significantly lower than that of control group and negatively correlated with peripheral blood Th17 cell surface CCR4, CCR6 and CXCR3 fluorescence intensity.Conclusion: The high expression of peripheral blood Th17 cell surface CCR4, CCR6 and CXCR3 in patients with CHB can result in increased secretion of Th17 cytokines, liver function injury and liver fibrosis.

Bioinformatics Analysis on B cell Epitopes of Rice Allergen RAG1

[Objective] To predict the secondary structure and B cell epitopes of the rice major allergen RAG1. [Method] The amino acid sequence of rice allergen RAG1 was acquired from Expasy protein database. The secondary structure of RAG1 was predicted by DNAStar Protean software with Gamier-Robson program, Chou-Fasman program and Karplus-Schulz program; the B cell epitopes of RAG1 was predicted with the Kyte Doolittle hydrophilic program, Emini surface accessibility program and Jameson-Wolf antigenic index program. [Result] The predictions on secondary structure and B cell epitopes showed that the regions of 33-44, 119-129, 155-163 were the dominant B cell epitopes. [Conclusion] This study predicted the potential dominant B cell epitopes in rice allergen RAG1 by comprehensive use of multi-methods and multi-parameters, and provided a theoretical basis for further researches on identification, antigen modification and epitope vaccine design of RAG1 B cell epitopes.

Restoring the Treg cell to Th17 cell ratio may alleviate HBV-related acute-on-chronic liver failure

AIM: To investigate the role of T helper 17 cells (Th17) and regulatory T cells (Treg) in hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF).METHODS: We enrolled 79 patients with HBV infection into the study, 50 patients with HBV-related ACLF and 29 patients with chronic hepatitis B (CHB), from the First Affiliated Hospital of Medical College from January 2009 to June 2012. The ACLF patients were diagnosed according to the criteria recommended by The 19th Conference of the Asian Pacific Association for the Study of the Liver in 2009. Twenty healthy individuals with a similar gender and age structures to the two patient groups were also included as the normal controls (NC). Of the 50 ACLF patients, 28 were subsequently classified as non-survivors: 19 patients died from multiorgan failure, 3 underwent liver transplantation, and 6 discontinued therapy during follow-up because of financial reasons. The remaining 22 ACLF patients whose liver and anticoagulation function recovered to nearly normal levels within the next 6 mo were classified as survivors. The number of circulating Treg and Th17 cells was determined upon diagnosis and during the 8th week of follow-up through flow cytometry. RESULTS: The percentage of circulating Treg cells in the ACLF group was significantly higher than that in the CHB group (5.50% ± 1.15% vs 3.30% ± 1.13%, P < 0.01). The percentages of circulating Th17 cells in the ACLF and the CHB groups were significantly higher than that in the NC group (6.32% ± 2.22% vs 1.56% ± 0.44%, P < 0.01; 3.53% ± 1.65% vs 1.56% ± 0.44%, P < 0.01). No significant difference in Treg cell to Th17 cell ratio was observed between the ACLF group and the CHB group (0.98 ± 0.44 vs 1.12 ± 0.64, P = 0.991), whereas those in the two HBV infection groups were significantly lower than that in the NC group (1.85 ± 1.22; both P < 0.01). The percentage of Treg cells in the survivors during the 8th week of follow-up was significantly lower than that during peak ACLF severity [total bilirubin (TBIL) peak] (3.45% ± 0.97% vs 5.18% ± 1.02%, P < 0.01). The percentage of Th17 cells in survivors during the 8th week of follow-up was significantly lower than that during the peak TBIL (2.89% ±0.60% vs 5.24% ± 1.46%; P < 0.01). The Treg cell to Th17 cell ratio during the 8 th week of follow-up was significantly higher than that during the TBIL peak (1.22 ± 0.36 vs 1.10 ± 0.54; P < 0.05). CONCLUSION: Restoring the Treg cell to Th17 cell ratio during the follow-up phase of ACLF could maintain the immune system at a steady state, which favours good prognosis.

Mechanism of T cell hyporesponsiveness to HBcAg is associated with regulatory T cells in chronic hepatitis B

AIM： To study the mechanisms of hyporesponsiveness of HBV-specific CD4^＋ T cells by testing TH1 and TH2 commitment and regulatory T cells. METHODS： Nine patients with chronic hepatitis B were enrolled. Peripheral blood mononuclear cells were stimulated with HBcAg or HBsAg to evaluate their potential to commit to TH1 and TH2 differentiation. HBcAg-specific activity of regulatory T cells was evaluated by staining with antibodies to CD4, CD25, CTLA-4 and interleukin-10. The role of regulatory T cells was further assessed by treatment with anti-interleukin-10 antibody and depletion of CD4^＋CD25^＋ cells. RESULTS： Level of mRNAs for T-bet, IL-12R β2 and IL-4 was significantly lower in the patients than in healthy subjects with HBcAg stimulation. Although populations of CD4^＋CD25^highCTLA-4^＋ T cells were not different between the patients and healthy subjects, IL-10 secreting cells were found in CD4^＋ cells and CD4^＋CD25^＋ cells in the patients in response to HBcAg, and they were not found in cells which were stimulated with HBsAg. Addition of anti-IL-10 antibody recovered the amount of HBcAgspecific TH1 antibody compared with control antibody （P 〈 0.01, 0.34% ± 0.12% vs 0.15% ± 0.04%）. Deletion of CD4^＋CD25^＋ T cells increased the amount of HBcAgspecific TH1 antibody when compared with lymphoo/tes reconstituted using regulatory T cells （P 〈 0.01, 0.03% ± 0.02% vs 0.18% ± 0.05%）.CONCLUSION： The results indicate that the mechanism of T cell hyporesponsiveness to HBcAg includes activation of HBcAg-induced regulatory T cells in contrast to an increase in TH2-committed cells in response to HBsAg.

Prediction of promiscuous T-cell epitopes in the Zika virus polyprotein:An in silico approach

Objective:To predict immunogenic promiscuous T-cell epitopes from the polyprotein of the Zika virus using a range of bioinformatics tools.To date,no epitope data are available for the Zika virus in the IEDB database.Methods:We retrieved nearly 54 full length polyprotein sequences of the Zika virus from the NCBI database belonging to different outbreaks.A consensus sequence was then used to predict the promiscuous T cell epitopes that bind MHC 1 and MHC II alleles using Propred1 and Propred immunoinformatic algorithms respectively.The antigencity predicted score was also calculated for each predicted epitope using the Vaxi Jen 2.0 tool.Results:By using Pro Pred1,23 antigenic epitopes for HLA class I and 48 antigenic epitopes for HLA class II were predicted from the consensus polyprotein sequence of Zika virus.The greatest number of MHC class I binding epitopes were projected within the NS5(21%),followed by Envelope(17%).For MHC class II,greatest number of predicted epitopes were in NS5(19%) followed by the Envelope,NS1 and NS2(17% each).A variety of epitopes with good binding affinity,promiscuity and antigenicity were predicted for both the HLA classes.Conclusion:The predicted conserved promiscuous T-cell epitopes examined in this study were reported for the first time and will contribute to the imminent design of Zika virus vaccine candidates,which will be able to induce a broad range of immune responses in a heterogeneous HLA population.However,our results can be verified and employed in future efficacious vaccine formulations only after successful experimental studies.

IFN-<i>γ</i>-, IL-4-, IL-17-, PD-1-Expressing T Cells and B Cells in Peripheral Blood from Tuberculosis Patients

Although the efficacy of tuberculosis (TB) vaccines is tightly linked to cell-mediated immunity, some functions of T and B cells in TB patients remain unclear. To address how Mycobacterium tuberculosis infection inhibits T effector responses, we assessed the proportions of T cell subsets and B cells in peripheral blood from pulmonary TB (PTB) patients, pleural TB (PLTB) patients, and healthy subjects (HS, who showed purified protein derivative (PPD)-positive reactions) with flow cytometry. Compared to HS, PTB and PLTB patients exhibited higher proportions of B cells and Th17 cells, and lower proportions of Th2 cells and ratios of Th1 to Th17 cells and of Th2 to Th17 cells. PTB patients had higher CD4+ T cells and PD-1+ CD4+ T cells than HS. Newly diagnosed PTB patients (nPTB) had higher proportions of B cells than HS;in contrast, PTB patients subjected to effective treatments (oPTB) and HS shared similar proportions of B cells. oPTB patients had higher proportions of CD4+ T cells, Th17 cells, and PD-1+ CD4+ T cells than HS, but this difference did not occur in nPTB patients. These findings suggest that shifting ratios of Th1 to Th17 cells may be beneficial for M. tuberculosis to amplify.

Exact Location of Linear B-cell Epitopes of VP3 Protein of Goose Parvovirus

Four monoclonal antibodies (MAbs) against Goose Parvovirus (GPV) VP3 protein already available were used to precisely locate linear B-cell epitopes in VP3 of GPV. The epitopes, recognized by four MAbs, had already been identified at low levels of resolution. Complementary oligonucleotides encoding ten amino acid fragments, with five amino acid overlaps were designed with suitable sticky ends for recombination with pET-32a and subsequent expression as small-fragment fusion proteins. Antigenicity of specific oligopeptides was determined by Western blotting with the MAbs. Using the same methods, amino acids were deleted one by one from the peptides of interest, enabling the two epitopes to be precisely located at amino acids 430-435 (-DRIMNP-) and 643-647 (-VFIKN-).

CD36蛋白B细胞抗原表位的生信预测及其多抗原肽的制备

目的分析CD36蛋白的结构,寻找可能的B细胞抗原表位,制备含有B细胞表位的多抗原肽(multiple antigenic peptide,MAP),为基于B细胞表位的MAP用于CD36抗体的制备提供前期实验基础。方法通过生物信息学方法分析CD36蛋白的理化性质、二级结构、潜在磷酸化及糖基化位点等,预测可能的B细胞表位。以多聚赖氨酸为核心基质,采用Fmoc方法合成含有CD36 B细胞表位的八分枝MAP,并利用反向高效液相色谱(RP-HPLC)分析MAP的纯度、质谱分析法测定MAP的分子量,对合成的CD36-MAP进行鉴定。结果CD36是1种稳定的、亲水性的碱性蛋白,二级结构以无规则卷曲为主,具有多个磷酸化、糖基化位点,抗原性较强。综合分析获得可能的优势B细胞表位4个,制备了4个含有优势B细胞表位的MAP,RP-HPLC分析表明合成的MAP的纯度均在85%以上,其中3个MAP的分子量与理论预期值相符。结论CD36具有较强的抗原性,利用预测得到的4个可能的B细胞表位合成MAP,为CD36抗体的制备和相关研究提供实验基础。

新型冠状病毒E蛋白B细胞表位的预测及鉴定

目的利用生物信息学方法预测SARS-CoV-2 E蛋白B细胞表位,并利用小鼠多抗血清、人新型冠状病毒阳性血清等进行验证,以明确SARS-CoV-2 E蛋白的优势B细胞表位。方法使用SOPMA、Expasy、SWISS-MODEL及IEDB数据库和BepiPred-2.0等软件预测SARS-CoV-2 E蛋白的结构及B细胞表位;通过大肠杆菌系统表达并纯化GST标签重组表位蛋白片段,以Western blotting和间接ELISA方法检测表位蛋白与小鼠和人SARS-CoV-2 E蛋白阳性多抗血清的反应性,以初步鉴定SARS-CoV-2 E蛋白的B细胞表位。结果表位预测结果显示,E蛋白含有线性B细胞表位Ser6-Val14和Tyr57-Pro71,构象表位涉及的氨基酸序列为Glu8-Val14、Leu39-Tyr59、Ser60-Leu65;表达并纯化含有预测表位的E蛋白片段E1(Ser6-Val14表位)、E3(Tyr57-Pro71)以及阴性对照片段E2(不含表位序列),Western blotting和间接ELISA结果均显示抗E蛋白小鼠多抗和人新型冠状病毒阳性血清只与E1、E3蛋白片段呈阳性反应而与E2蛋白片段均为阴性反应,显示E蛋白线性B细胞表位预测正确。结论本研究成功预测并初步鉴定出SARS-CoV-2 E蛋白2个线性B细胞表位,为新型冠状病毒疫苗制备和免疫应答特性分析等提供参考。

N端B细胞表位缺失的兔出血症病毒VP60蛋白的表达及其免疫原性

本研究将A3C单抗识别的VP60蛋白NTA区域作为识别标记,设计一系列编码VP60 NTA区域重叠多肽的基因序列并连接于pGEX-4T-1载体,确定A3C单抗识别的精确表位。然后构建重组质粒Bacmid-VP60△A3C,将Bacmid-VP60△A3C转染Sf9昆虫细胞,得到重组杆状病毒rBac-VP60△A3C。RT-PCR、HA、IFA和Western Blot鉴定结果表明,重组蛋白VP60△A3C在Sf9细胞中高效表达,电镜观察显示其形态和结构与兔出血症病毒VP60蛋白类似。将重组蛋白VP60△A3C以每只200μg免疫2月龄RHDV血清阴性的新西兰兔,免疫后14 d,以兔出血症病毒WF株攻毒新西兰兔。结果表明,免疫组无死亡,而对照组全部死亡。本研究结果为制备表位缺失的兔出血症亚单位疫苗奠定了基础。

利用单B细胞分选技术制备猪瘟病毒E2蛋白单克隆抗体及其在ELISA中的应用

本研究旨在建立一种猪瘟病毒E2单抗竞争ELISA检测方法,用于评价猪瘟C株弱毒苗和E2亚单位疫苗的免疫效果。首先,构建猪瘟E2杆状病毒表达载体pFastBAC,通过悬浮培养SF9细胞高效表达E2蛋白;其次,用纯化的E2蛋白免疫BABL/c小鼠,通过流式分选IgM-PE-/E2-APC+型单个B细胞。然后,利用半巢式PCR分别扩增E2特异性IgG抗体的重链和轻链,测序后将抗体重链及轻链构建到pCDNA3.1载体,再将构建好的质粒共转染至HEK-293细胞,制备猪瘟E2单克隆抗体。结果表明,本研究通过单B细胞分选技术获得的两株单克隆抗体,即mAb3A9(IgG1,kappa)和mAb4F7(IgG2a,lambda),分别识别猪瘟E2蛋白B细胞线性表位25GLTTTWKEYSHDLQL^(39)和259GNTTVKVHASDERGP^(273)。此外,用上述两株单克隆抗体及E2蛋白建立的单抗竞争ELISA检测方法,在血清样本检测过程中,均展现出优异的诊断敏感性(97.49%,95.97%)及特异性(96.08%,94.38%),该研究为我国猪瘟的逐步净化提供了有利的技术支撑。

非洲猪瘟病毒T、B细胞表位的免疫信息学预测与分析

旨在通过免疫信息学方法预测非洲猪瘟病毒(ASFV)结构蛋白的T、B细胞表位,为非洲猪瘟(ASF)表位疫苗的设计研制提供参考。从NCBI和RCSB数据库获取ASFV蛋白质序列和三维结构,利用IEDB、DTU Health Tech等数据库的生物信息学工具对ASFV的5种结构蛋白p72、p17、p49、M1249L和H240R的细胞毒性T细胞表位、线性B细胞和构象B细胞表位进行鉴定。结果显示:5种蛋白质均为亲水性,二级结构以无规则卷曲为主,仅M1249L例外;预测到抗原性良好、无毒、无致敏的细胞毒性T细胞优势表位27个,线性B细胞优势表位35个;预测到仅针对p72的构象B细胞优势表位2个。结论:ASFV的5种蛋白质可能具有多个潜在T、B细胞表位,其中B细胞表位相对占优,5种蛋白质中p72和M1249L最具疫苗研发前景,可结合蛋白质相关参数信息为构建ASF表位疫苗提供参考。

HPV53进化关系分析及B细胞与T细胞抗原表位预测

目的基于全长序列分析人乳头瘤病毒(human papillomavirus,HPV)53不同分离株的进化关系,并对代表性分离株病毒蛋白(E1、E2、E4、E6、E7、L1和L2)的理化性质、二级结构及B细胞与T细胞抗原表位进行预测。方法检索美国国立生物技术信息中心(National Center for Biotechnology Information,NCBI)数据库,获取HPV53全长序列并构建进化树。采用ProtParam软件分析蛋白的理化性质,PSIPRED和SOPMA软件预测其二级结构。采用ABCpred和IEDB软件预测B、T细胞抗原表位,并结合肽段柔韧性、亲水性、表面可及性、抗原性及Vaxijen评分等参数进一步筛选潜在的优势抗原表位;最后对潜在优势抗原表位与13个高危型HPV进行同源性分析。结果检索NCBI数据库共下载54条HPV53全长序列,经去重后保留48条,来自不同国家/地区的HPV53分离株可划分为A、B、C三个主要进化分支。三个分支代表株病毒的蛋白理化性质相似,E1、E6和E7蛋白的二级结构以α螺旋为主,E2、E4、L1和L2以无规则卷曲为主。经预测和筛选后,共得到6个B细胞潜在优势抗原表位和9个T细胞潜在优势抗原表位,同源性分析发现,E4和E6区域的B细胞抗原表位TTPIRPPPPPRPWAPT和CYRCQHPLTPEEKQLH,及L2区域的T细胞抗原表位SGVHSYEEIPMQ与HPV56具有较高同源性(均>90%)。结论通过生物信息学方法分析和预测发现HPV53分离株可分为A、B、C三个主要进化分支,其理化性质相似,二级结构存在部分小差异,且病毒蛋白中含有B、T细胞抗原表位,为HPV53相关多肽形式的疫苗和抗体药物开发提供了更多理论依据。