共查询到18条相似文献，搜索用时 234 毫秒
1.
为制备猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus, PHEV)N蛋白的单克隆抗体，本研究构建了pET-32a-N重组质粒，原核表达并纯化重组N蛋白，免疫BALB/c小鼠，细胞融合后利用间接ELISA进行筛选，获得了3株稳定分泌的阳性杂交瘤细胞株，分别命名为2C3、4F3、5C6。Western blot与间接免疫荧光试验表明，3株单抗均能与293T细胞中表达的N蛋白产生特异性反应。经测定，3株单抗的效价均为1×106,重链类型均为IgG1,轻链类型均为κ。利用一系列表达的部分重叠的N截短蛋白片段，经Western blot鉴定单抗所识别的抗原表位，结果显示，267KPRQK271为单抗2C3、4F3、5C6所识别的表位。本研究为PHEV临床检测方法的建立和表位疫苗的研发奠定了基础。 相似文献
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为鉴定禽传染性支气管炎病毒(IBV)刺突(S1)蛋白的中和抗原表位，本研究通过IBV全病毒免疫BALB/c小鼠，经融合、亚克隆筛选获得了4株稳定分泌抗IBV S1蛋白抗体的杂交瘤细胞株4A11、1B11、5E5和7C9。经鉴定制备的单克隆抗体腹水抗体ELISA效价为106以上，Western blot和间接免疫荧光试验分析显示该单克隆抗体反应性和特异性良好。随后用8种基于S1分型的IBV毒株为试验毒株，同实验室已有的8株IBV S1单抗(1E9、1H1、1E4、3C6、3C7、2F3、2E5、4F9)共12株单抗进行气管环中和活性检测，发现S1蛋白单抗与8种S1亚型的毒株均有不同程度的中和作用。随后，在抗原表位和Western blot分析的基础上，鉴定出单克隆抗体识别的抗原表位区域，获得了6个中和抗原表位，其中除416IQTRTEP422表位，其他5个表位仍未有相关报道。本研究成功制备出4株特异性识别S1蛋白且具有中和活性的单克隆抗体，不仅丰富了IBV的单克隆抗体库，为今后研究IBV S1蛋白分子结构提供了关键生物材料... 相似文献
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为获得抗牛支原体NM001株单克隆抗体,并评价其特性,以牛支原体分离株NM001作为抗原免疫6周龄BALB/c小鼠,利用杂交瘤技术和间接ELISA方法筛选到2株能稳定分泌抗牛支原体的单克隆抗体细胞,命名为2C5和7G3。其细胞上清的间接ELISA抗体效价分别为6.4×103和1.2×104。经亚型测定,单抗2C5和7G3均属于IgG1类,轻链均是λ型。制备腹水并对单抗进行纯化和特性鉴定,两株单抗的间接ELISA抗体效价分别为1.02×105和4.09×105,且两株单抗与无乳支原体、山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、绵羊肺炎支原体、牛巴氏杆菌均无交叉反应。Western Blot结果显示,2株单抗均能特异性识别牛支原体全菌蛋白中的相应蛋白。试验表明,单抗2C5和7G3能够与牛支原体发生特异性反应,从而为牛支原体血清学检测提供一定的物质基础。 相似文献
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以原核系统表达的非洲猪瘟病毒p54重组蛋白为免疫原接种BALB/c小鼠,经过细胞融合、间接ELISA筛选和多次亚克隆获得了3株能够稳定分泌抗p54蛋白单克隆抗体的杂交瘤细胞系,分别命名为2A4、5F6和RH15。3株单克隆抗体的重链均属于IgG1亚类,轻链均为Kappa型。Western blot和免疫荧光试验(IFA)证明3株单克隆抗体具有良好的反应性,可以用于靶抗原的特异性检测。通过噬菌体展示肽库筛选,确定了单克隆抗体2A4、5F6识别的抗原表位为157NTASQ161,RH15识别的抗原表位为170RQRNTYTHKDL180,进一步完善了靶抗原的B细胞线性抗原表位信息。 相似文献
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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。 相似文献
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1株非洲猪瘟病毒p30蛋白单克隆抗体识别的B细胞抗原表位的鉴定 总被引：1，自引：1，他引：0
旨在研究非洲猪瘟病毒(ASFV) p30蛋白的B细胞抗原表位,本研究制备了p30蛋白的单克隆抗体(mAb),并以该单克隆抗体为工具进行B细胞抗原表位定位。首先,通过原核表达及Ni柱亲和纯化获得p30蛋白,将纯化蛋白免疫BALB/c小鼠进行杂交瘤细胞制备,通过间接酶联免疫吸附试验(iELISA)筛选出阳性杂交瘤细胞,并以细胞表达的方法制备单克隆抗体;采用间接免疫荧光试验(IFA)和蛋白质免疫印迹(Western blot)对单克隆抗体的特异性进行鉴定。利用IEDB表位预测软件对p30蛋白B细胞抗原表位进行预测,根据预测结果对CP204L基因进行截短表达,利用IFA、Western blot和iELISA对其抗原表位进行鉴定。最后,利用噬菌体十二肽库对制备的p30单克隆抗体进行4轮生物淘选,筛选多肽表位,并与上述基因截短表达筛选方法进行比对。特异性鉴定结果显示,该单克隆抗体能成功识别感染猪肺泡巨噬细胞的ASFV;基因截短表达筛选结果表明,其识别的抗原表位区域为84M～K142;噬菌体淘选试验结果表明,116TSSFETLFE124为本试验制备的单克隆抗体所识别的p30蛋白抗原表位核心序列,该结果进一步缩小了表位分布范围。本研究制备了p30蛋白的1株单克隆抗体,并对其抗原表位进行鉴定,为血清学诊断试剂的研发和p30蛋白功能研究奠定基础。 相似文献
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为获得牛支原体（Mycoplasma bovis,M.bovis）VspX蛋白单克隆抗体,将编码该蛋白的基因克隆、表达并纯化,作为免疫原,以QuickAntibody-Mouse 5W为免疫佐剂,免疫BALB/c小鼠。经3次免疫后,将小鼠脾细胞与SP2/0骨髓瘤细胞融合,经3次亚克隆筛选后,共获得5株能稳定分泌抗VspX蛋白抗体的杂交瘤细胞株,分别命名为1A8、3A3、3C12、3H9及4D11。亚型鉴定表明,3C12重链为IgG2b,其余4株为IgG1,轻链均为κ链。间接ELISA结果表明,5株细胞培养上清的抗体效价在1：1×104~1：2×105,腹水效价在1：1×105~1：8×105。选其中两株杂交瘤细胞株3H9和4D11的腹水纯化,进行亲和力测定,解离常数分别为6.3×109和7.8×109,属高亲和力抗体。Western blotting结果显示,5株单抗均能与牛支原体发生特异性反应,而单抗4D11与羊无乳支原体标准株PG2和丝状支原体丝状亚种标准株PG3均不反应。流式细胞术结果表明,单抗4D11与牛支原体表面的VspX的结合呈剂量依赖性。间接免疫荧光结果表明,单抗4D11可以识别黏附到胚胎牛肺细胞上的重组VspX蛋白。本试验成功制备的单克隆抗体为VspX蛋白功能的研究奠定基础。 相似文献
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为获得分泌抗大豆凝集素(SBA)单克隆抗体的杂交瘤细胞,以纯化的SBA为抗原,免疫BALB/c小鼠,加强免疫3 d后取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合,应用有限稀释法和间接ELISA 方法克隆筛选出2株分泌抗SBA单克隆抗体的杂交瘤细胞系A7、F12。细胞上清抗体效价均在1∶2×103以上,腹水抗体效价均为1∶1×106,单抗亚型鉴定结果均为IgG2b型,分子质量为189.6 ku,亲和常数为7.1×107 mol/L,Western blotting结果表明,2株单抗具有较高的特异性。该McAb的制备为建立SBA定量检测方法奠定了基础。 相似文献
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为获得猪戊型肝炎病毒(Hepatitis E virus,HEV)Ⅳ型衣壳蛋白单克隆抗体,将猪HEV衣壳蛋白的C端267(408—675)个氨基酸基因序列克隆入原核表达载体pET-28a(+),构建重组质粒pET-28a-ORF2-C,转化E.coli Rosetta(BL21)感受态细胞进行诱导表达,SDS-PAGE和Western blot鉴定,纯化后免疫小鼠。取免疫小鼠的脾脏与鼠骨髓瘤细胞SP2/0融合制备单克隆抗体。通过间接ELISA和竞争ELISA方法筛选并鉴定单抗。结果表明蛋白得到正确、高效表达,获得3株识别不同的抗原表位区的单克隆抗体,分别命名为Mab-1E4(IgG1)、Mab-2C7(IgG1)和Mab-2G9(IgG2b),其中1E4和2G9能阻断临床阳性猪血清,提示该2株单克隆抗体识别的抗原表位是猪HEVⅣ型衣壳蛋白上重要的抗原表位区,而单抗Mab-2C7不能阻断。本研究为猪HEVⅣ型的诊断及研究提供重要工具。 相似文献
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[目的] 纯化猪塞尼卡谷病毒（Seneca Valley virus，SVV）SVV-CH-HB2016毒株，并制备其结构蛋白VP1、VP2和VP3的单克隆抗体。[方法] 以蔗糖密度梯度离心法纯化的SVV-CH-HB2016病毒颗粒作为抗原，免疫BALB/c小鼠，取脾细胞与骨髓瘤细胞（SP2/0）进行细胞融合。通过间接免疫荧光试验（IFA）结合间接ELISA筛选阳性细胞株，制备能特异性分泌针对结构蛋白的杂交瘤细胞株。采用Western blotting和IFA方法分别检测单克隆抗体与重组表达蛋白及天然结构蛋白的反应性，并对单克隆抗体的病毒中和保护效果进行测定。利用空斑试验和实时荧光定量PCR方法探究中和性单克隆抗体对SVV-CH-HB2016毒株吸附过程的影响，最后用抗体相加试验来分析14株单克隆抗体的抗原表位。[结果] 在蔗糖密度梯度为5%~45%（W/V）时获得了纯度较好、浓度较高的SVV-CH-HB2016毒株结构蛋白，免疫小鼠血清抗体效价均达到了1：12 800，成功制备了17株能稳定分泌特异性单克隆抗体的杂交瘤细胞株。经验证14株单克隆抗体能与重组结构蛋白发生Western blotting反应，17株单克隆抗体能与病毒发生IFA作用；2G6、4A3和4C11 3株单克隆抗体对SVV-CH-HB2016毒株感染的BHK-21细胞具有明显中和保护作用，也能有效抑制SVV-CH-HB2016毒株对293T细胞的吸附。经分析发现，除1F5与2E1、4B8与4F11外，其余10株单克隆抗体分别针对不同抗原表位。[结论] 本研究初步建立了SVV的纯化方法，制备了17株特异性针对SVV-CH-HB2016毒株的单克隆抗体，为后期进一步开展SVV全病毒灭活疫苗的研发、ELISA检测方法的建立及保护性抗原表位的鉴定奠定了基础。 相似文献
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XUE Fei YANG Zichun XU Yanhui WANG Yaling YU Ting FAN Maodi LIU Juanhua GAO Song LIU Xiufan 《畜牧兽医学报》1956,51(12):3122-3132
To development monoclonal antibodies against cOmpT of avian pathogenic Escherichia coli (APEC), the recombinant cOmpT of APEC origin expression plasmid pET-28a-compT was employed, and cOmpT protein with a molecular weight about 36 kD in the form of inclusion bodies was obtained after induction with IPTG, and then renatured by urea gradient dialysis. BALB/c mice were immunized with the purified cOmpT. An indirect enzyme-linked immunosorbent assay (iELISA) was developed, the optimal coating concentration of the antigen was 0.625 μg·mL-1 and the optimal serum dilution was 1:6 400. After the fourth immunization, the spleen of immunized mice was collected for cell fusion, three monoclonal hybridomas that can secrete antibody specific to cOmpT were obtained after multiple screenings, named 1G8, 2C3 and 2G3 respectively. And all of their immunoglobulin subclasses were IgG2b. The titers of monoclonal antibodies in the cell culture supernatant were 1:200, 1:3 200 and 1:3 200 determined by iELISA, respectively. All three monoclonal antibodies were confirmed to react with cOmpT in Western blot, without cross reaction with other tested bacteria. The antigenic epitopes recognized by the three monoclonal antibodies were identified by using a series of E. coli strains harboring expression plasmids recombined with truncated fragments from compT gene. The results revealed that the antigenic epitope required for reactivity with the 1G8 was 83DQDWMDS89, and 90SNPGTW95, 197TFKYSGW203were recognized by 2C3 and 2G3, respectively. In this study, three monoclonal antibodies against cOmpT were successfully developed and the antigenic epitopes recognized by the antibodies were identified. The cOmpT specific monoclonal antibodies obtained in this study are potentially useful tools for both the functional study of cOmpT and the development of APEC epitope vaccines. 相似文献
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To obtain the anti-kappa casein monoclonal antibody and complete the identification of the antibody characteristics. The BALB/c mice were immunized with kappa casein using foot-pad immunization. Popliteal lymph node cells from the immunized mice were fused with SP2/0 myeloma cells in the presence of PEG. Three hybridoma strains (1C4, 3G3, 3E6) which secreted the antibody specific for kappa casein were obtained．The sub-class of the antibodies were IgG1. The ascites were purified by Protein G affinity layer absorption column. The antigenic epitope of 3G3 and 3E6 were different and it was close between 1C4 and 3E6.The titer of purified ascites(1C4) was 1.28×106 and the affinity constant was 2.89×108 mol/L. A anti-kappa casein monoclonal antibody with good affinity had been achieved，which provided foundations for the rapid detection of casein in bovine milk samples. 相似文献
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WANG Leming WANG Yurui ZENG Zixuan RAO Guoshun WU Zhengjiao JIN Weikun WANG Dongying 《中国畜牧兽医》2022,49(2):677-686
【Objective】 This study was intend to obtain cathepsin L1(rFgCat L1) specific monoclonal antibody and construct the double antibody sandwich ELISA.【Method】 Five BALB/c mice were immunized with 1 mg/mL rFgCat L1 protein for four times.Mouse splenocytes were isolated and fused with SP2/0 cells to construct hybridoma cells.Strong positive hybridoma cell lines were screened, 1×106 cells were injected intraperitoneally per mouse to prepare monoclonal antibodies.Antibody titer and antigenic epitope were detected using ELISA method, antibody subtype and specificity were identified using Western blotting method.The double antibody sandwich ELISA was constructed by combining the anti-rFgCat L1 polyclonal antibody, and its sensitivity and specificity were tested.The positive and negative critical value was screened by 20 negative sera with positive control, and the constructed double antibody sandwich ELISA was verified by 47 goat positive sera and 47 dairy cow positive sera.【Result】 After immunization, the antibody titers in serum of 4 mice were all more than 104.After isolated mouse with the highest immune response spleen cells were fused with SP2/0 cells total of 8 of them were positive cell lines were obtained after selective culture.5D5 and 7G6 were identified as strong positive strains with stable antibody secretion.After multiple subcloning screens and subcultures, the antibodies secreted in the cell supernatant were stable, with titers of 29 and 210 respectively, with ascites titers of 107 and 108.Western blotting and antibody subtype identification kits identified that the two antibodies were IgG1 type and the light chain was kappa type, both of which could specifically bind FgESP.According to the same antigen site was recognized by the two kinds of antibodies, the antigen titer of the two monoclonal antibodies were comparied, 7G6 was used as the coating antibody, and anti-rFgCat L1 was used as the enzyme-labeled secondary antibody.The optimized condition of method was that 7G6 was coated at a concentration of 2 μg/mL, the dilution concentration of anti-rFgCat L1 polyclonal antibody was 25 μg/mL, the dilution of Don-HRP-conjugated was 1∶4 000, 5% skimmed milk powder was selected as the blocking solution and the color development time was 25 min.The method was proved that could recognize the lowest antigen concentration of 0.625 μg/mL, also could specifically recognize antigen of Fasciola fasciatus.The constructed sandwich ELISA method was used for antigen detection of 47 dairy cow positive serum and 47 goat positive serum infective samples kept in the laboratory and the positive antigen rate were 72.3% and 78.7%, respectively.【Conclusion】 Anti-rFgCat L1 monoclonal antibody was successfully prepared and the double-sheet sandwich ELISA method for fascioliasis was constructed, which provided a good theoretical basis and material basis for the development of low-cost and rapid diagnostic kits. 相似文献
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为了制备牛分枝杆菌的单克隆抗体,本试验利用制备的原核表达的牛分枝杆菌重要抗原蛋白MPB70作为免疫原皮下多点注射免疫BALB/c小鼠。利用细胞融合技术,经过5次亚克隆与筛选,取已免疫好的小鼠脾细胞与SP2/0骨髓瘤细胞在PEG3500的作用下进行细胞融合。筛选得到了2株分泌抗牛分枝杆菌MPB70蛋白的杂交瘤细胞,分别命名为Anti-B-MPB70：8和Anti-B-MPB70：12。采用间接ELISA方法对获得的单克隆抗体进行亲和常数检测、效价分析及亚类鉴定,通过SDS-PAGE凝胶试验对单克隆抗体进行纯度分析,并检测其浓度,通过Western blotting方法对单克隆抗体的反应特性进行鉴定。结果显示,纯化后的MPB70蛋白分子质量大小为20 ku,浓度为0.5 mg/mL。两株单克隆抗体的亲和常数分别为9.95×109和9.53×108;抗体效价分别为1∶102 400和1∶12 800;抗体亚类分别为IgG2b和IgG1,轻链类型均为κ型;纯度>90%;浓度分别为3.0和2.2 mg/mL;且具有良好的反应特性。以上研究结果表明,本试验成功制备了抗MPB70蛋白单克隆抗体,可为牛结核病病原和抗体检测技术研究奠定基础。 相似文献
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To obtain the monoclonal antibody (McAb) against VspX protein of Mycoplasma bovis (M. bovis),VspX gene was amplified, expressed and purified. Then, BALB/c mice were immunized subcutaneously three times with the purified recombinant VspX (rVspX) mixed with QuickAntibody-Mouse 5W adjuvant. Three days after the last injection, spleen cells were collected aseptically, and fused with SP2/0 myeloma cells in the presence of polyethylene glycol. By the clone selection, five stable hybridomas against VspX protein were obtained, separately named as 1A8, 3A3, 3C12, 3H9 and 4D11. Antibody titers in cell supernatant were from 1:1×104 to 1:2×105, while from 1:1×105 to 1:8×105 in ascites of mice by indirect ELISA. The subtypes were determined to be IgG1 and IgG2b class, and all light chains were κ chain. The affinity constant of McAb 3H9 and 4D11 were 6.3×109 and 7.8×109, respectively, and they belonged to high-affinity antibodies. Western blotting results showed that all of five McAbs could specifically react with M.bovis, however, McAb 4D11 could not react with Mycoplasma arginini PG2 and Mycoplasma mycoides subsp. PG3. Flow cytometry showed that McAb 4D11 reacted with surface VspX of M. bovis in a dose-dependent manner. Indirect immunofluorescence assay demonstrated that 4D11 McAb was able to detect rVspX protein binding to embryonic bovine lung cells. In the present study, McAbs against rVspX protein had been successfully prepared, which provided a basis for future researches about the function of VspX protein and the pathogenesis of M. bovis. 相似文献
17.
为建立H9N2亚型禽流感病毒（Avian influenza virus,AIV）免疫层析快速检测技术,本研究以差速离心法纯化H9N2亚型AIV免疫BALB/c小鼠,将免疫小鼠脾细胞与骨髓瘤细胞SP2/0进行细胞融合和HAT选择性培养;以H9N2亚型AIV感染MDCK细胞建立异源免疫过氧化物酶单层细胞试验（IPMA）的单克隆抗体检测方法,通过对杂交瘤细胞的IPMA筛选和连续克隆化筛选鉴定抗H9N2亚型AIV中和性单克隆抗体;以胶体金标记HA单克隆抗体,配对HA单克隆抗体和羊抗小鼠IgG为检测线和质控线,制备H9N2亚型AIV快速检测试纸条,测定其特异性和敏感性。结果显示,获得了11株稳定分泌抗H9N2亚型AIV单克隆抗体的杂交瘤细胞,其单克隆抗体腹水IPMA效价在1.28×10-4至2.56×10-5之间。单克隆抗体3A2、5H6、6B8、7E10和9G12血凝抑制试验（HI）显示血凝抑制活性,其（HI）效价在6log2~9log2之间。单克隆抗体3A2、6B8和9G12在病毒中和试验中对H9N2亚型AIV有显著病毒中和活性,中和效价分别1∶6 400、1∶25 600和1∶25 600。Western blotting结果提示,该中和单克隆抗体识别HA蛋白线性抗原表位。利用配对单克隆抗体3A2和9G12研制的H9N2亚型AIV检测试纸条检测H9N2亚型AIV尿囊液的效价为9log2,灵敏度与经典血凝试验（HA）相当,与其他亚型AIV （H1、H3、H5、H7）,以及新城疫病毒和鸡传染性法氏囊病病毒等相关病毒均无交叉反应。本研究制备了具有病毒中和活性的抗H9N2亚型AIV单克隆抗体,并初步研制了H9N2亚型AIV检测试纸条,为H9N2亚型AIV新型疫苗研制和快速检测奠定良好的研究基础。 相似文献
18.
为制备抗鸭1型甲肝病毒亚型（DHAV-1a）结构蛋白VP1的单克隆抗体（MAbs）,本研究以pGEX-VP1重组蛋白免疫BALB/c小鼠,同时以纯化浓缩的全病毒作为包被抗原,建立了筛选抗VP1蛋白阳性杂交瘤细胞株的间接ELISA方法,经融合、筛选制备杂交瘤细胞及鉴定MAbs的稳定性、特异性、腹水效价和中和活性等生物学活性,获得了2株持续且稳定分泌抗体的杂交瘤细胞（1A2和5G3）。MAbs腹水ELISA效价分别为1：3.2×104和1：2.0×106。亚类鉴定均为IgG1/κ型。Western blotting结果显示,2株MAbs均能与DHAV-1a VP1蛋白和DHAV-1a全病毒发生特异性反应。特异性试验结果显示2株MAbs能与鸭1型甲肝病毒（DHAV-1）和DHAV-1a发生交叉反应。中和试验结果显示5G3株具有中和活性。结果表明2株MAbs的ELISA效价高、特异性强、稳定性好,均能与DHAV-1和DHAV-1a全病毒发生特异性反应,其中一株具有中和活性。 相似文献