猪口蹄疫病毒抗原位点及其抗原性差异分析

为了分析 3株猪口蹄疫病毒 ( FMDV- B、D、S)的抗原位点 ,采用 5株具有 EL ISA反应特性的抗猪口蹄疫病毒的单克隆抗体 ( Mc Ab- A6、F9、G17、G5 2、S2 5 ) ,通过 EL ISA试验分别测定 3个抗原的最适包被浓度以及 5株单抗对各抗原的饱和工作浓度。 EL ISA叠加试验的增值结果表明 ,5株 Mc Ab分别针对 4个不同的抗原位点 ,其中 Mc Ab- A6和 F9识别同一个抗原位点。FMDV- B株含有这 4个不同抗原位点 ,而 FMDV- D、S株只有 3个抗原位点 ,没有 Mc Ab-A6、F9识别的抗原位点。根据抗原位点差异 ,可以将 3个毒株分成 2个不同组     

猪戊型肝炎病毒单克隆抗体的抗原识别位点分析

采用ELISA叠加试验,对6株具有ELISA反应特性的猪戊型肝炎病毒(SHEV)单克隆抗体(McAb-αC11、αC12、γH1、γF8、BC4和CH8)的抗原识别位点进行检测。抗体反应增值结果表明,6株单抗分别针对4个不同抗原位点,McAb-γH1,BC4,CH8识别的位点各不相同,其中McAb-γH1、BC4识别的位点有部分重叠,McAb-αC11、αC12,γF8识别同一位点,位于γH1,BC4识别位点的重叠区内。     

猪繁殖与呼吸综合征病毒SX-1株N蛋白单克隆抗体的制备

用纯化的重组HIS标签N蛋白免疫BALB／c小鼠,采用杂交瘤技术制备抗PRRSVN蛋白的单克隆抗体（McAb）,经细胞融合获得5株可分泌特异性McAb的抗PRRSVN蛋白的杂交瘤细胞1A12,3F11,5A3,5F9,4E4。其中1A12,3F11,5A3,4E4杂交瘤上清与HIS-蛋白与GST—N蛋白两种抗原包被的ELIA反应均为阳性。5F9与HIS—N蛋白包被的ELISA反应为阳性,而与GST—N蛋白包被的ELISA反应呈阴性。单抗1A12,3F11,4E4,5A3与HIS-N与GST—N蛋白的Western blotting反应均为阳性,而5F9只与HIS-N反应。IFA检测显示1A12,3F11,5A3,4E4株单抗都有明显的荧光,说明其与PRRSV呈阳性反应。同时,ELISA检测又有很好的特异性。     

相思子毒素-a单克隆抗体的制备与鉴定

用含1%甲醛的磷酸盐缓冲液对相思子毒素-a(abrin-a)减毒处理制备类毒素,以类毒素为免疫抗原分4次免疫3只BALB/c小鼠。采用聚乙二醇(PEG)法,取免疫脾细胞与小鼠骨髓瘤细胞(SP2/0)融合,筛选分泌抗abrin-a的单克隆抗体(McAb)杂交瘤细胞株。将阳性细胞株接种BALB/c小鼠制备McAb腹水,根据亚类鉴定结果,分别采用蛋白A或蛋白G亲和层析柱纯化腹水,间接酶联免疫吸附试验(ELISA)检测McAb特异性,Western-blot法分析McAb抗原识别位点。结果获得1G5、3C2和4G1共3株McAb杂交瘤细胞株,均为抗abrin-a的特异性McAb,与相思子凝集素、蓖麻毒素(ricin)和蓖麻凝集素均无交叉反应,其中4G1为特异性结合abrin-a A链的McAb,1G5和3C2两株为特异性结合abrin-a B链的McAb;制备的McAb可用于abrin-a含量的检测。     

14株H9N2亚型禽流感病毒分离株的HA和NA基因序列分析

为了解浙江省H9N2亚型禽流感病毒(AIV)血凝素(HA)和神经氨酸酶(NA)基因的分子特征和遗传变异情况，本试验对2012—2021年分离到的14株H9N2亚型AIV分离株进行HA和NA基因序列测定和分析。结果显示：14株分离株的HA和NA基因均属于Y280分支，HA核苷酸同源性为91.5%～99.4%,NA核苷酸同源性为89.0%～98.7%,其与疫苗株CK/SS/94、CK/F/98、CK/6/96和CK/1/00的HA核苷酸同源性为86.0%～91.1%。14株分离株HA蛋白裂解位点氨基酸组成均为RSSR↓GLF;受体结合位点主要表现为K141N、A142T、T155N、H183N和V190T突变，其中第226位和第228位全部为L和G,可与α-2,6唾液酸受体结合，具有感染人的特性；蛋白潜在糖基化位点均为6个，主要变异表现在第200～202位1个位点缺失和第295～297位1个位点增加；抗原相关位点主要表现为G82E、S137D、D145G、N159G、A160D、T192R和N193D突变，此外，有5株出现D160N突变。14株分离株NA蛋白存在6个潜在糖基化位点，其中1株...     

相思子毒素-α单克隆抗体的制备与鉴定

用含1%甲醛的磷酸盐缓冲液对相思子毒素-a(abrin-a)减毒处理制备类毒素,以类毒素为免疫抗原分4次免疫3只BALB/c小鼠.采用聚乙二醇(PEG)法,取免疫脾细胞与小鼠骨髓瘤细胞(SP2/0)融合,筛选分泌抗abrin-a的单克隆抗体(MeAb)杂交瘤细胞株.将阳性细胞株接种BAI.B/c小鼠制备McAb腹水,根据亚类鉴定结果,分别采用蛋白A或蛋白G亲和层析柱纯化腹水,间接酶联免疫吸附试验(ELISA)检测McAb特异性,Western-blot法分析McAb抗原识别位点.结果获得1G5、3C2和4G1共3株McAb杂交瘤细胞株,均为抗abrin-a的特异性McAb,与相思子凝集素、蓖麻毒素(ricin)和蓖麻凝集素均无交叉反应,其中4G1为特异性结合abrin-a A链的MCAb,1G5和3C2两株为特异性结合abrin-a B链的MCAb;制备的MCAb可用于abrin-a含量的检测.     

糖化酶单抗的研制及其抗原识别位点的分析

为建立掺糖造假蜂蜜中残留糖化酶的检测方法,用从黑曲霉中提取的糖化酶作为免疫原,免疫BALB/c小鼠,利用杂交瘤技术获得了12株稳定分泌针对糖化酶抗体的杂交瘤细胞株。单克隆抗体亚型鉴定结果显示,10株为IgG1,2株为IgG2b,轻链均为κ轻链。Western blotting分析结果表明,12株抗体均可特异性结合糖化酶。其中6株单抗(McAb-2H4F9、6H9D8、8F2F11、8F2E9、1A8G6、1C4D5)细胞株采用体内诱生法制备的腹水效价均1∶1×10⁴以上。采用抗体叠加试验对这6株抗糖化酶单抗的抗原识别位点进行检测,反应增殖结果表明,6株单抗分别针对4类不同抗原位点,McAb-6H9D8和McAb-8F2F11针对第Ⅰ种抗原决定簇;McAb-1A8G6和McAb-1C4D5针对第Ⅱ种抗原决定簇;McAb-8F2E9针对第Ⅲ种抗原决定簇;McAb-2H4F9针对第Ⅳ种抗原决定簇。制备的抗体针对不同的抗原表位,为双抗夹心ELISA方法的建立提供前提。     

抗猪囊虫循环抗原杂交瘤细胞系的建立和鉴定

建立了8株抗猪囊虫循环抗原(CA)的杂交病细胞系。其中6F12和2E7为抗囊虫特异McAb;McAb 1C6、1C7、1B5、4D9、2B9和8D8与囊虫、(?)球蚴、细颈囊尾蚴抗原均可发生反应。这些McAb的腹水ELISA效价为105～107,细胞培养上清液效价为102,并均可与病猪血清中的囊虫CA反应形成沉淀线。用1B5、6F12和8D8分别致敏血球,以反向间接血凝试验检测98份囊虫病猪血清,检出率分别为70.41%(69/98)、6.12%(6/98)和7.14%(7/98)。本研究制备的McAb可用于猪囊虫循环抗原检测。     

禽流感病毒单克隆抗体的研制及初步应用

接种H5N1亚型禽流感病毒(AIV)鸡胚尿囊液，经甲醛灭活后，差速离心提纯病毒，三次免疫BALB／c小鼠，取免疫小鼠脾细胞与骨髓细胞SP2／0-Ag-14融合。用间接ELISA和血凝抑制(HI)试验筛选阳性杂交瘤细胞株。本研究共获14株能够稳定分泌特异性单克隆抗体(McAbs)的杂交瘤细胞株，其中McAb A6具有HI特性，并可以与重组鸡痘病毒表达的H5亚型AIV HA蛋白反应。用间接EHSA试验测定McAbs与125株AIV分离株的反应性。用反应谱广的McAbs(483和F2)进行交叉反应实验，确定了McAbs可用于夹心ELISA的最佳包被和酶标方式。以McAb483为基础建立的夹心ELISA对175株各亚型的AIV的阳性检出率在90％以上。     

施马伦贝格病毒核衣壳蛋白的表达及单克隆抗体制备

为表达与纯化具有天然构象的施马伦贝格病毒(Schmallenberg virus,SBV)核衣壳(N)蛋白,并制备其单抗(McAb),本研究在SBV-N基因的N端加入6个组氨酸(6×His)标签后,将其克隆至杆状病毒表达载体pFastBac~(TM)1中,构建重组供体质粒pFastBac-His-SBV-N。将pFastBac-His-SBV-N转化DH10Bac E.coli,通过蓝白斑筛选获得重组杆粒rBacmid-His-SBV-N。将rBacmid-His-SBV-N转染Sf9昆虫细胞制备表达His-SBV-N融合蛋白的重组杆状病毒,借助Ni-NTA琼脂糖纯化重组杆状病毒感染Sf9细胞中的His-SBV-N蛋白。以纯化的HisSBV-N免疫BALB/c小鼠制备McAb,利用ELISA叠加试验检测McAb抗原识别位点的异同,并采用高碘酸钠法对识别不同抗原表位的McAb进行辣根过氧化物酶(HRP)标记。最终获得了4株识别不同抗原表位的McAb(2A11、2E1、4H11和6E12)并进行了HRP标记;亚型鉴定表明,2A11为IgG1亚型,2E1、4H11和6E12均为IgG2b亚型;间接免疫荧光试验证实,4株McAb均能够识别稳定表达SBV-N蛋白的BHK-21细胞系;Western blot进一步表明,HRP标记的4株McAb均与His-SBV-N蛋白发生特异性反应。His-SBV-N融合蛋白及其McAb的成功制备,为施马伦贝格病血清学检测方法的建立提供了良好的生物材料。     

Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus

Two foot-and-mouth disease virus (FMDV) monoclonal antibodies (mAbs) were produced from mice immunized with either FMDV serotype A, subunit (12S) or FMDV serotype O, whole virus (140S). Both mAbs (F1412SA and F21140SO) recognized all seven serotypes of FMDV in a double antibody sandwich (DAS) ELISA, suggesting that the binding epitopes of the two mAbs are conserved between serotypes. These mAbs are IgG1 isotype and contain kappa light chains. In order to define the mAb binding epitopes, the reactivity of these mAbs against trypsin-treated and denatured FMDV were examined using an indirect ELISA. The binding site of the mAb, F1412SA is trypsin sensitive and the epitope is linear. Both ELISA and Western blot results suggested that the polypeptide VP2 contributed to the immunodominant site. This mAb showed reactivity to VP2 peptide (DKKTEETTILEDRIL). The mAb, F21140SO, recognized an epitope which is trypsin resistant and discontinuous. This mAb binding to FMDV is dependent on conformational structures of intact viral (140S) or subunit (12S) particle, since it failed to recognize any viral protein in Western blot. This conformational and highly conserved epitope is the first identified epitope among all seven FMDV serotypes. Because the use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal antisera, these two mAbs with different specificities are suitable for type-independent diagnosis of FMDV, such as DAS ELISA, or could be adapted to immuno-chromatographic or flow-through rapid test.     

Production and characterization of monoclonal antibodies to (poly100)S1 protein of avian infectious bronchitis virus

Fragments within S1 genes ((poly100)S1) of infectious bronchitis virus (IBV) strains ZJ971, M41 and SC021202 (SC) were subcloned into a prokaryotic expression vector and expressed in Escherichia coli. Monoclonal antibodies (mAbs) against the recombinant (poly100)S1 proteins were produced, characterized and used to analyse epitopes on the S1 subunit of IBV. Nine mAbs raising from the three (poly100)S1 proteins recognized five different epitopes of the S1 subunit, designated as S1-A, B, C, D and E. Epitopes S1-C and S1-D are common for the three IBV strains, while S1-A and S1-B exist on ZJ971 and M41 strains, and S1-E was a strain-specific epitope for SC strain. Immunocytochemistry indicated that all the mAbs to the (poly100)S1 proteins can react with the homologous S1 glycoprotein expressed in Vero cells. Moreover neutralization test demonstrated that only mAbs 6E2, 4F9 and 6G4 had neutralization activity for the homologous IBV. These mAbs to (poly100)S1 protein were potential candidates for detecting and distinguishing IBV strains, and also used to examine antigenic variation of the S1 protein.     

Development and characterization of monoclonal antibodies against FMD virus type Asia-1 and determination of antigenic variations in the field strains

Twelve mouse monoclonal antibodies (MAbs) were developed against an Indian vaccine strain of foot and mouth disease virus (FMDV) type Asia-1 WBN 117/85. The MAbs were tested for their ability to bind to whole virus particle, trypsin-treated 146S (TT-146S) virus particle, sub-viral (12S and disrupted virus) antigens by ELISA and to neutralize virus infectivity in cell culture. Extensive characterization of MAbs revealed the existence of three different groups based on the binding of non-overlapping epitopes. Eight type Asia-1 specific MAbs (RF7, RF8, RD10, RE11, RC11, RC10/O, RB11 and RC10/M), which formed group 1 (G1), were found to bind a neutralizing, trypsin-sensitive (TS) and conformational epitope. Two MAbs (WB8 and WC3) in group 2 (G2) were found to bind a non-neutralizing, trypsin-resistant, conformational and 12S-specific epitope, which was intertypically conserved in all the four serotypes of FMDV (O, A, C and Asia-1) prevalent in India. Two MAbs (KG10 and KF10), which formed group 3 (G3), were found to be against a non-neutralizing, TS and conformational epitope, common to types Asia-1 and A. Members of G1 were IgG2a isotype, while those of G2 and G3 were IgG1 and IgG2b isotypes, respectively. Antigenic analysis of 31 FMDV type Asia-1 field isolates and two vaccine strains, using a panel of type Asia-1-specific MAbs, revealed antigenic similarity of the virus isolates tested and non-existence of neutralization escape mutants. The developed MAbs have practical utility, especially in the manufacture of FMD vaccine, diagnosis and FMDV characterization.     

Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies.

A panel (26) of monoclonal antibodies (MAbs) was elicited against three distinct isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1. Each MAb was characterized according to the location of its epitope: Class I, restricted to the intact virion (140S); Class II, restricted to 140S and the virion protein subunit (12Sps); Class III, available on 140S, 12Sps and virus protein 1; Class IV, restricted to 12Sps. In addition, the MAbs were further categorized by isotype, neutralization of viral infectivity, capacity to bind in radioimmunoassay and precipitation in the Ouchterlony reaction. Neutralization of FMDV infectivity by a MAb of the IgA isotype is reported for the first time. A minimum of seven distinct neutralization epitopes were described on FMDV Asia-1. Some of the neutralizing MAbs bound FMDVs in addition to those that they neutralized. The MAbs defined epitopes common to FMDV serotypes Asia-1, A, O1 and C but neutralizing capacity was restricted to serotype Asia-1. Class IV MAbs defined epitopes highly conserved throughout the FMDV serotypes. Identification of FMDV neutralization epitopes makes possible the direct selection of optimal FMDV strains for vaccine fabrication. In addition, these data are crucial to the design of future synthetic vaccines.     

Antigenic variation of Mycoplasma gallisepticum, as detected by use of monoclonal antibodies.

A panel of monoclonal antibodies (MAb) developed against Mycoplasma gallisepticum strain PG31 was used to probe the antigenic profiles of 5 recognized strains (PG31, R, S6, F, A5969) and 6 field isolates of M gallisepticum. Monoclonal antibody G9 predominantly recognized antigens at apparent molecular mass positions of 90 to 98 kDA. The MAb reacted with all strains and isolates, but the molecular mass position of the antigens varied among some mycoplasmas. Monoclonal antibody G12 reacted with all strains and isolates of M gallisepticum and had an identical banding pattern. However, MAb G10 and G11 reacted selectively only with a limited number of strains and/or isolates. Surface distribution of the MAb-recognized antigens was revealed by immunoelectron microscopy. Partial physicochemical characterization of MAb G9-recognized antigens identified glycopeptide characteristics. Monoclonal antibody G9 reacted with surface antigens and, hence, participated in agglutination of M gallisepticum. However, the degree of agglutination varied among the various strains and isolates, indicating a quantitative or conformational limitation or an alteration in the anomeric expression of the epitopes. Antigenic variation in M gallisepticum may be mediated by immunologic selective pressures, or a proclivity for habit niche in the host.     

抗Asia1型口蹄疫病毒单克隆抗体的制备及生物学特性鉴定

用纯化的Asia1型口蹄疫病毒免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,经间接ELISA和间接免疫荧光(IFA)筛选,有限稀释法克隆,获得了2株稳定分泌单克隆抗体的杂交瘤细胞株,分别命名为3H6、5G3,其细胞培养上清效价分别为1:64和1:128,小鼠腹水效价分别为1×10~(-4)和8×10~(-3);ELISA和IFA结果显示,2株单抗仅与Asial型口蹄疫病毒反应,不与O型口蹄疫病毒反应,表明它们均为抗Asial型口蹄疫病毒的型特异性单克隆抗体。westem blot结果显示,2株单克隆抗体均不与全病毒抗原反应,表明它们所针对的抗原表位均为构象表位。相加ELISA试验表明,两株单抗识别不同的抗原表位。经硫氰酸盐洗脱法测定,3H6和5G3的相对亲和力指数分别为1.0 mol/L和1.5 mol/L。这2株单抗的获得为建立口蹄疫病毒检测方法提供了强有力的工具。     

Identification of B-cell epitopes in the NSP1 protein of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) was divided into North American and European genotypes. NSP1 was an important non-structural protein of PRRSV, which was auto-cleaved from the replicase polyprotein into NSP1α and NSP1β subunits and played an important role in the immune suppression. In this study, six monoclonal antibodies (MAbs) against the recombinant PRRSV NSP1, expressed in Escherichia coli system, were screened out and identified. Western blot and IFA results indicated that 4 out of 6 MAbs recognized the recombinant NSP1α and 2 MAbs recognized NSP1β. Epitope mapping results indicated that MAb 4H2 recognized the linear epitopes E(54)EPLRW(59) in NSP1α, MAbs (2G5, 3E11 and 4D4) recognized the epitopes H(157)VLTNLP(163) in NSP1α, and MAbs 3C7 and 1H7 reacted with the epitopes 185aa to 232aa in NSP1β. Protein sequence alignment of NSP1 indicated E(54)EPLRW(59) was conserved in all North American PRRSV strains, whereas European type strains has variable amino acids in this region. The epitope H(157)VLTNLP(163) was relatively conserved among all PRRSV strains, except for a L162→S162 change in European type strains. The epitope 185-232aa was variable among North American PRRSV strains. These results may facilitate future investigations into the function of NSP1 of PRRSV and diagnostic methods for PRRSV infection.     

Identification of a variable epitope on the Newcastle disease virus hemagglutinin–neuraminidase protein

Fifteen virulent Newcastle disease viruses (NDVs) were isolated from diseased birds in Eastern China in 2005. To investigate the antigenic variation in the epitopes on NDV hemagglutinin–neuraminidase (HN) protein, these isolates, together with six reference strains, were subjected to the hemagglutination inhibition (HI) tests using five HI-positive monoclonal antibodies (MAbs) against velogenic NDV strain ZJ1. The MAbs 2G5, 3A4, 3B5 and 6B1 recognized 12 of the 15 NDV isolates, and exhibited HI activity towards the six reference strains. However, these MAbs did not react with three local isolates, JS-02/05, JS-06/05 and JS-10/05. HN gene sequence analysis of all NDV strains revealed that these MAb-resistant NDV isolates possessed residue K at position 347 of the HN protein, whereas all remaining strains possessed E or G at the same site. To determine the contribution of the residue at position 347 to antigenic epitope formation, we generated by reverse genetics two recombinant viruses, ZJ1HNK with an E347K mutation on ZJ1 HN, and JSHNE with a K347E mutation on JS-06/05 HN. The HI test demonstrated that ZJ1HNK lost reactivity with MAbs 2G5, 3A4, 3B5 and 6B1, whereas JSHNE did react with these MAbs. Further verification by immunofluorescent assay demonstrated that residue 347 was a critical determinant for formation of the antigenic epitope (residues 345–353) on the HN protein.