Antigen epitopes of animal coronaviruses:a mini-review

Coronaviruses are widespread in nature and can infect mammals and poultry,making them a public health concern.Globally,prevention and control of emerging and re-emerging animal coronaviruses is a great challenge.The mecha-nisms of virus-mediated immune responses have important implications for research on virus prevention and control.The antigenic epitope is a chemical group capable of stimulating the production of antibodies or sensitized lympho-cytes,playing an important role in antiviral immune responses.Thus,it can shed light on the development of diagnos-tic methods and novel vaccines.Here,we have reviewed advances in animal coronavirus antigenic epitope research,aiming to provide a reference for the prevention and control of animal and human coronaviruses.

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.

An immunoglobulin Y that specifically binds to an in silico-predicted unique epitope of Zika virus non-structural 1 antigen

Objective:To identify unique immunogenic epitopes of Zika virus non-structural 1(NS1)antigen and produce immunoglobulin Y(IgY)for potential use in he diagnosis of of Zika virus infection.Methods:Immunogenic epitopes were identified using in silico B-cell epitope prediction.A synthetic peptide analog of the predicted epitope was used to induce antipeptide IgY production in hens which was purified using affinity chromatography.Presence of purified IgY and its binding specificity were performed by gel electrophoresis and ELISA,respectively.Results:Out of the nine continuous epitopes identified,the sequence at position 193-208(LKVREDYSLECDPAVI)was selected and used to produce anti-peptide IgY.The produced IgY was found to bind to the synthetic analog of the Zika virus NS1 immunogenic epitope but not to other flaviviruses and random peptides from other pathogens.Conclusions:In this study,we identified an immunogenic epitope unique to Zika virus that can be used to develop a serodiagnostic tool that specifically detect Zika virus infection.

An Indirect ELISA of Classical Swine Fever Virus Based on Quadruple Antigenic Epitope Peptide Expressed in E.coli

In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.

Helicobacter pylori specific immune response induced by conservative flagellin linear B-cell epitope

AIM:To testify the immunogenicity of a conservative B-cell linear epitope of Helicobacter pylori ( H pylori) flagellin A. METHODS: Different programs were used to analyze the secondary structure, molecular hydropathy, and surface accessibility of Hpyloriflagellin A. Linear B-cell epitopes were estimated based on the structural and physiochemical information. Analysis of residue divergence was proposed to screen a conservative linear epitope. The 29-peptide (Pep29mer) synthesized by chemical method, including the predicted conservative B-cell epitope and a known K^2d compatible T-cell epitope, was used to immunize mice, and then H pylori-specific antibodies were detected by ELISA. RESULTS: Based on the analyses of divergent amino acid residues, structural and physiochemical characteristics, it was strongly suggested that the short fragment NDSDGR was the core of a conservative linear epitope in flagellin A. Animals immunized by Pep29mer acquired efficient immune response. In detail, serum Hpylori-specific IgA and IgGl increased significantly in immunized group, while IgG2a only had an insignificant change. Hpylori-specific IgA in gastrointestinal flushing fluid also increased significantly. CONCLUSION: The conservative short fragment NDSDGR is the core of a linear B-cell epitope of flagellin A.

Prokaryotic Expression and Immunogenicity of Major Antigen Epitope of Rabbit Haemorrhagic Disease Virus VP60

[ Objective] To investigate the immunogenicity of main antigen epitope of RHDV （ Rabbit haemorrhagic disease virus） VP60 expressed in a prokaryotic system. [ Method] The major antigen epitope gene of RHDV VP60 was amplified by RT-PCR. It was cloned into pET-28b （ ＋ ） and expressed in E. coli Rosetta strain. The recombinant protein was detected by Western blot. The pudfied recombinant protein was used to immunize rabbits in order to observe its immunogenicity. [ Result] Western blot analysis revealed a clear band at approximately 24.0 kDa. The purified recom- binant protein reacted with the purified RHDV in ELISA. [ Conclusion] The prokaryotically expressed main antigen epitope of RHDV VP60 shows good immunogenicity.

Hepatitis B surface antigen-negative but hepatitis B envelope antigen-positive false occult hepatitis B virus infection:A case report

BACKGROUND Occult hepatitis B infection(OBI)is characterized by the detection of hepatitis B virus(HBV)DNA in serum(usually HBV DNA<200 IU/mL)or the liver but negativity for hepatitis B surface antigen(HBsAg).The diagnosis of OBI relies on the sensitivity of assays used in the detection of HBV DNA and HBsAg.HBsAg assays with inadequate sensitivity or inability to detect HBV S variants may lead to misdiagnosis of OBI in people with overt HBV infection.CASE SUMMARY We report a HBsAg-negative but hepatitis B envelope antigen-positive patient who had a significant HBV DNA level.The patient was initially diagnosed as having OBI.However,sequence analysis revealed a unique insertion of amino acid residues at positions 120-124 in the S protein,which affects the formation of a disulfide bond that is associated with the formation of a loop.It is well known that there is an overlap between the S protein and Pol protein.We found that this new insertion site occurred in polymerase/reverse transcriptase domain,indi-cating that this insertion might be involved in HBV pathogenicity.The patient was finally diagnosed with a false OBI.CONCLUSION An insertion of amino acid residues at positions 120-124 of the S protein affects the formation of immunodominant epitopes and results in negative HBsAg levels.

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.

Hepatocellular carcinoma-specific immunotherapy with synthesized α1,3-galactosyl epitope-ulsed dendritic cells and cytokine-induced killer cells

AIM： To evaluate the safety and clinical efficacy of a new immunotherapy using both α-Gal epitope-pulsed dendritic cells （DCs） and cytokine-induced killer cells. METHODS： Freshly collected hepatocellular carcinoma （HCC） tumor tissues were incubated with a mixture of neuraminidase and recombinant αl,3-galactosyltrans- ferase （αI,3GT） to synthesize α-Gal epitopes on car- bohydrate chains of the glycoproteins of tumor mem- branes. The subsequent incubation of the processed membranes in the presence of human natural anti-Gal IgG resulted in the effective phagocytosis to the tumor membrane by DCs. Eighteen patients aged 38-78 years with stage 111 primary HCC were randomly chosen for the study; 9 patients served as controls, and 9 patients were enrolled in the study group.RESULTS： The evaluation demonstrated that the pro- cedure was safe; no serious side effects or autoimmune diseases were observed. The therapy significantly pro- longed the survival of treated patients as compared with the controls （17.1 ± 2.01 mo vs 10.1 ±4.5 mo, P = 0.00121）. After treatment, all patients in the study group had positive delayed hypersensitivity and robust systemic cytotoxicity in response to tumor lysate as measured by interferon-y-expression in peripheral blood mononuclear cells using enzyme-linked immunosorbent spot assay. They also displayed increased numbers of CD8-, CD45RO- and CD56-positive cells in the peripheral blood and decreased α-fetoprotein level in the se- rum. CONCLUSION： This new tumor-specific immunotherapy is safe, effective and has a great potential for the treat- ment of tumors.

Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

AIM： To insert the constructed TGF-β1 epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1 antigenicity in its prokaryotic expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1 vaccine. METHODS： The TGF-β1 encoding epitope gene （the mature TGF-β1 from 78-109 amino acid residues, TGF-β1^32） was amplified by polymerase chain reaction from the recombinant pGEM-7z/ TGF-β1 ^32 vector. The HBcAg gene fragments （encoding HBcAg from 1-71 and 89-144 amino acid residues） were amplified from PYTAI- HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1^32 and HBcAg89-144 into restrictive endonuclease enzyme and ligated with T4 Ugase. The fusion gene fragments HBc.Ag1-71-TGF-β1^32 HBcAg89-144 were recloned to pET28a（＋） and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a （＋）/ CTC was transformed and expressed in E. coli BL21 （DE3） under induction of IPTG. After purification with Ni^＋2 NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope. RESULTS： Enzyme digestion analysis and sequencing showed that TGF-β1 epitope gene was inserted into the el loop of C-terminus of truncated hepatitis B core antigen. SDS-PAGE analysis showed that relative molecular mass （Mr） of the expressed product by pET28a （＋）/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1 polyclonal IgG but not with anti-HBcAg. The purity of protein was about 90 % and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1 antigenicity and could be used as anti-TGF-β1 vaccine. CONCLUSION： A recombinant prokaryotic expression system with high expression efficiency of the target TGF-β1 epitope gene was successfully established. The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1. The expressed TGF-β1 epitope gene shows good immunogenicity and antigenicity.

Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide &quot;ALAHGVRAL (core 150-158)&quot;. The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.

乙型肝炎病毒前S2抗原决定簇（HBVPreS2epitope）与乙型肝炎病毒核心抗原（HBcAg）的基因融合与表达

乙肝前Ｓ２（ＨＢＶＰｒｅＳ２）肽段由５５个氨基酸组成，其Ｎ端肽段含Ｔｈ和Ｂ细胞抗原决定簇。我们将化学合成的ＰｒｅＳ２ｅｐｉｔｏｐｅ（１２０－１４５）基因与ＨＢｃＡｇ基因不同位点进行融合，融合基因在大肠杆菌中获得表达，并对融合蛋白进行了纯化。经ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ实验表明，融合蛋白具有ＰｒｅＳ２和ＨＢｃＡｇ两者的抗原性。此外，研究还表明，强启动子能使表达水平有一定提高。

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.

Chidamide and sintilimab combination in diffuse large B-cell lymphoma progressing after chimeric antigen receptor T therapy

BACKGROUND Diffuse large B-cell lymphoma(DLBCL)is curable with first-line chemoimmunotherapy but patients with relapsed/refractory(R/R)DLBCL still face a poor prognosis.For patients with R/R DLBCL,the complete response rate to traditional next-line therapy is only 7%and the median overall survival is 6.3 mo.Recently,CD19-targeting chimeric antigen receptor T cells(CAR-T)have shown promise in clinical trials.However,approximately 50%of patients treated with CAR-T cells ultimately progress and few salvage therapies are effective.CASE SUMMARY Here,we report on 7 patients with R/R DLBCL whose disease progressed after CAR-T infusion.They received a PD-1 inhibitor(sintilimab)and a histone deacetylase inhibitor(chidamide).Five of the 7 patients tolerated the treatment without any serious adverse events.Two patients discontinued the treatment due to lung infection and rash.At the 20-mo follow-up,the median overall survival of these 7 patients was 6 mo.Of note,there were 2 complete response rates(CRs)and 2 partial response rates(PRs)during this novel therapy,with an overall response rate(ORR)of 57.1%,and one patient had a durable CR that lasted at least 20 mo.CONCLUSION In conclusion,chidamide combined with sintilimab may be a choice for DLBCL patients progressing after CD19-targeting CAR-T therapy.

为提升IPTR患者血小板输注后CCI值建立分级规避HLA抗体对应抗原方法及HLAMatchmaker的应用研究

目的:建立分级规避HLA抗体MFI阈值对应抗原方法,联合应用HLAMatchmaker表位计算法,选择供患者表位最小错配评分值,评估两种方法为免疫性血小板输注无效(Immune platelet transfusion refractoriness,IPTR)患者选择HLA相容性血小板供者,在提升血小板输注后校正增加值(CCI)的应用价值。方法:采用SPRCA法完成51例IPTR患者的7807次血小板交叉配型实验,判断其免疫反应阴/阳性结果。采用Luminex单抗原流式微珠法检测患者的HLA-I类抗体,获得不同特异性抗体对应HLA-I类抗原MFI值,并将其分组及分级,强阳性组(MFI>4000,1级)、中阳性组(1000中阳性组>弱阳性组)。强阳性和中阳性组与阴性对照组之间的SPRCA实验免疫反应阳性结果检出数存在统计学差异(P<0.001),弱阳性位组和阴性对照组之间的SPRCA实验免疫反应阳性结果检出数无统计学差异(P>0.05)。设置强阳性组为相应特异性HLA位点对应抗原1级规避阈值,中阳性组为2级规避阈值,弱阳性组为3级规避阈值,在供者血小板紧缺情况下,可以不需要规避弱阳性组。规避1和2级HLA-I类抗体对应供者抗原及选择HLAMatchmaker表位错配评分数≤7血小板供者策略,24 h内CCI值均>4.5×109/L,均可获得临床血小板输注有效。结论:在为IPTR患者选择HLA-I类相容性供者时,分级规避HLA-I类抗体对应供者抗原,综合选择供受者HLAMatchmaker表位错配评分数≤7,经血小板交叉配型实验确认为阴性结果的供者选择策略,对提升IPTR患者血小板计数具有一定实际应用价值。

基于幽门螺杆菌hp0169基因三维结构的生物信息学分析

目的:克隆幽门螺杆菌(Hp) hp0169基因并进行晶体学研究,分析其二级结构和三维结构。方法:由UniProt数据库中检索Hp NCTC26695菌株hp0169基因及其编码蛋白序列,采用生物信息学方法分析Hp重组胶原蛋白酶(HpPrtC)蛋白理化性质,SOPMA和DNAStrar软件预测HpPrtC蛋白二级结构特征,SWISS-MOPEL软件构建HpPrtC蛋白三维结构,IEDB和ABCpred软件预测HpPrtC蛋白B淋巴细胞抗原表位,SYFPEITHI网站预测T淋巴细胞抗原表位,专家库(EP)算法和随机森林(RF)算法预测HpPrtC蛋白可结晶性。原核表达HpPrtC重组蛋白,经Ni^(2+)亲和层析和分子筛技术纯化蛋白,结晶试剂盒筛选HpPrtC的结晶条件。结果:hp0169基因共包含1 269个碱基配对,编码蛋白全长422个氨基酸,理论等电点为7.64,相对分子质量为47 300。HpPrtC蛋白为亲水性、可溶性蛋白。HpPrtC蛋白α螺旋的氨基酸数量占全部氨基酸数量百分率为35.78%,β片层为18.72%,β转角为6.87%,无规则卷曲为38.63%。抗原表位分析,HpPrtC蛋白含有B淋巴细胞的5个优势线性表位和3个构象表位及多个T淋巴细胞潜在优势抗原表位。同源建模,HpPrtC蛋白呈二聚体,单体由β折叠围成桶状结构,周围被α螺旋和无规则卷曲围绕。HpPrtC蛋白为中等难度结晶,且无信号肽和跨膜螺旋,在0.2 mol·L^(-1)氯化镁、 0.1 mol·L^(-1)三羟甲基氨基甲烷(Tris)、3.4 mol·L^(-1)己二醇和pH 8.5条件下出现细小成簇的针状晶体。结论:HpPrtC是一种亲水型蛋白,呈二聚体构造,在适宜条件下呈细小成簇针状晶体,其具有T淋巴细胞和B淋巴细胞优势抗原表位,可作为Hp疫苗设计的抗原,用于构建多价融合疫苗或多表位疫苗,本研究结果为Hp的防治提供了实验基础。

猪细小病毒4型单克隆抗体制备及抗原表位鉴定

为了制备猪细小病毒(PPV)4型Cap蛋白单克隆抗体(mAb)并鉴定其抗原表位,试验采用原核表达的Cap重组蛋白免疫BALB/c小鼠,将其脾细胞与SP2/0细胞进行融合,通过酶联免疫吸附测定(ELISA)、血凝抑制(HI)和免疫过氧化物酶单层细胞试验(IPMA)筛选阳性克隆,最终获得2E7、4D6和5C9这3株特异性杂交瘤细胞株;然后用mAb对Cap蛋白多肽进行ELISA检测,鉴定分析其抗原表位。结果显示:4D6和5C9 mAb识别表位为线性表位,分别为387RRQDN^(391)和578QRKE^(581),而2E7 mAb识别的表位为构象表位;通过对Cap蛋白3D结构定位分析,387RRQDN^(391)和578QRKE^(581)抗原表位位于蛋白表面,且在PPV4亚型中高度保守,而与猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪乙型脑炎病毒(JEV)和猪博卡病毒(PBoV)基因序列同源性低。本研究为PPV4诊断试剂的开发和新型疫苗的研制提供了参考。

猪细小病毒病研究进展

猪细小病毒(Porcine parvovirus,PPV)是一种无囊膜DNA病毒,主要引起母猪繁殖障碍。该病毒在全世界广泛流行,我国猪场PPV感染率高达90%以上,给养猪业带来巨大经济损失。PPV经口、鼻传播,主要侵染猪的生殖器官,引起母猪的死胎、木乃伊胎和公猪的精液质量下降。临床采用接种疫苗的方式进行防控,起到了一定的效果。论文对病毒的基因组和蛋白特征、流行病学和疫苗研究进行综述,以期为PPV的基础研究和疫苗开发提供参考。

2.1亚型猪瘟病毒流行株E2蛋白单克隆抗体的制备及其抗原表位的鉴定

为获得仅与我国基因2.1亚型猪瘟病毒(CSFV)流行株反应的单克隆抗体(MAb),并揭示疫苗株与流行株E2蛋白抗原氨基酸的差异,本研究利用基因2.1亚型重组质粒p FastBac1-JL23 E2通过杆状病毒表达重组E2蛋白(rE2),并采用Ni柱纯化后经SDS-PAGE检测r E2的表达及纯化效果;采用western blot鉴定r E2的反应原性。SDS-PAGE及western blot结果表明经杆状病毒表达了基因2.1亚型CSFV E2蛋白,且其纯化效果和反应原性均较好。采用杂交瘤技术制备2.1基因亚型CSFV E2蛋白的单克隆抗体(MAb),采用亲和层析法纯化该MAb,采用BCA法测定其浓度及亚类。将79株1.1、2.1、2.2和2.3基因亚型的CSFV及1.1基因亚型的HCLV株和SM株感染PK-15细胞,72 h后采用IFA检测MAb与不同基因型CSFV的反应性;采用western blot鉴定MAb与10个基因亚型共18种CSFV代表株E2蛋白的反应性。采用IFA鉴定MAb与不同基因型CSFV的中和活性。结果显示,经IFA筛选后共获得16株分泌E2蛋白MAb的杂交瘤细胞株。其中仅一株MAb TCH061与所有2.1基因亚型(2.1a、2.1b、2.1c、2.1g、2.1h、2.1i、2.1j、2.1k、2.1l、2.1m、2.1n)CSFV感染的细胞反应后出现绿色荧光,与基因1.1亚型HCLV疫苗株和SM株以及其他基因型CSFV感染的细胞反应后均无绿色荧光;western blot结果显示,该MAb能够识别2.1基因亚型CSFV E2蛋白(2.1a、2.1b、2.1c、2.1g、2.1h、2.1i、2.1j),在90 ku处出现特异性条带,而与其他基因型CSFV E2蛋白均不反应。表明TCH061为2.1基因亚型CSFV E2蛋白特异性的MAb。MAb的纯化结果显示,在50 ku和25 ku处有明显条带,其浓度为1.70μg/μL且该MAb TCH061重链为Ig G2a,轻链为κ链。中和活性结果显示,TCH061对JL^(23)株(2.1b)、GDLF1株(2.1c)有一定的中和活性,但不能中和C株。分别以GD53株E^(24)个不同区域的截短蛋白为抗原通过western blot初步确定MAb识别抗原表位的区域。利用CLC Sequence Viewer比对与MAb反应的CSFV E2蛋白氨基酸序列的差异,通过SWISS-MODEL预测MAb抗原表位的关键氨基酸位点。利用杆状病毒表达CSFV JL^(23)株各位点突变后的E2蛋白,采用western blot鉴定各突变的E2蛋白与MAb TCH061的反应性。Western blot结果显示,TCH061的抗原表位位于E2蛋白的aa1~aa90,且其识别CSFV E2蛋白P^(20)不识别L^(20)的CSFV。通过SWISS-MODEL预测E2蛋白氨基酸序列的I18、G^(19)、P^(20)、L^(21)、G^(22)、A^(23)和E^(24)在空间上比较接近;MAb TCH061与JL^(23)株I18M突变的E2蛋白反应,与G^(19)K、P^(20)L和L^(21)P突变的E2蛋白均不反应,与G^(22)K突变的E2蛋白反应性较弱,与HCLV E2蛋白不反应,但与HCLV L^(20)P突变的E2蛋白反应。证实TCH061的抗原表位为^(19)GPLG^(22),经Py MOL结构模拟确定其为空间构象表位;且除了aa^(20),其他位点在各基因型CSFV中均非常保守,表明P^(20)是2.1基因亚型CSFV流行株E2蛋白抗原表位的关键氨基酸。综上所述,本研究首次获得一株区别于CSFV疫苗株和2.1基因亚型CSFV流行株的MAb TCH061,揭示了疫苗株与流行株E2蛋白氨基酸位点的差异,为研发猪瘟疫苗和CSFV流行株感染的血清学鉴别检测方法的建立提供了重要的MAb资源。

口蹄疫病毒Asia1型猪源中和抗体的筛选与抗原表位鉴定

Asia1型口蹄疫病毒(FMDV)仍然在我国周边国家存在,对我国畜牧业造成长期威胁。本研究旨在利用单个B细胞抗体技术研制Asia1型FMDV的中和性单克隆抗体,以此为工具,鉴定Asia1型FMDV的保护性抗原位点。以FMDV Asia1/JS/05为诱饵抗原,通过流式细胞术分选免疫猪PBMCs中的抗原特异性B细胞,通过巢式PCR扩增单个B细胞IgG抗体重链与轻链可变区基因序列,分别构建IgG抗体重链与轻链表达质粒,将其共转染CHO-S细胞进行抗体表达;通过间接ELISA、间接免疫荧光试验(IFA)、病毒中和试验(VNT)验证抗体反应性和中和活性,利用免疫印迹、中和抗体逃逸突变株筛选鉴定中和抗体所识别的抗原表位类型和抗原表位关键氨基酸。结果显示:获得了5株反应性良好的Asia1型FMDV特异性抗体,其中PD3和PD7为中和抗体,两株中和抗体识别相同表位,关键氨基酸为VP2蛋白72位残基(D)。本研究首次获得Asia1型FMDV特异性猪源中和抗体,鉴定VP272D是中和表位的关键氨基酸,进一步丰富了Asia1型FMDV抗原位点信息,为FMDV Asia1型分子疫苗和新诊断检测技术研究的奠定了基础。