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1.
传染性胰腺坏死病毒VP2COE蛋白单克隆抗体的制备及与初步应用 总被引：2，自引：2，他引：0
为制备抗IPNV VP2蛋白的单克隆抗体,对其基本特性进行鉴定并进行初步应用。实验利用Ni-NTA亲和层析纯化的IPNV VP2 COE重组蛋白作为免疫原,免疫8周龄的雌性BALB/c小鼠,经3次免疫后,将免疫小鼠的脾细胞与骨髓瘤SP2/0细胞融合。采用间接ELISA和有限稀释法筛选杂交瘤细胞,共得到2株能稳定分泌特异性抗体的阳性细胞株,分别命名为5G10和5F3,亚类鉴定均为IgG1亚类。2株杂交瘤细胞的染色体数目在75～120之间。间接ELISA检测5G10和5F3细胞培养上清的效价分别为1∶105、1∶102,腹水效价分别为1∶108、1∶104。Western-blotting和间接免疫荧光鉴定结果显示,2株单抗均能特异性地识别IPNV。间接ELISA表明,2株单抗不与IHNV、VHSV、SVCV、HRV等病毒反应,说明获得的单抗具有高度的特异性。相加ELISA实验结果显示,2株单克隆抗体分别识别IPNV VP2蛋白上不同的抗原位点。应用间接免疫荧光方法对临床确定为患有IPN虹鳟肝组织进行检测,结果证实该2株单克隆抗体可用于后续实验。 相似文献
2.
利用RT-PCR方法扩增出IPNV-ZYX分离株主要结构蛋白VP2的抗原表位区基因(616 bp), 命名为IPNV VP2 COE, 将其克隆到pCold TF表达载体中构建重组质粒pCold TF-VP2 COE, 在大肠杆菌BL21(DH5α)感受态表达, 经SDS-PAGE电泳分析, 表达蛋白约78 ku, 用镍离子亲和层析柱纯化该蛋白, 制备抗血清, 间接ELISA结果显示, IPNV (ATCC VR-1318)细胞培养物与鼠抗VP2 COE蛋白血清发生特异性反应, 效价为1∶12 800; 间接免疫荧光结果显示, 鼠抗VP2 COE血清可与黑龙江某渔场已知感染IPNV虹鳟肝组织产生特异性的荧光, 以上两项结果表明, 表达IPNV VP2 COE蛋白具有良好的免疫原性和免疫反应性, 为IPNV检测方法的建立及疫苗的制备提供理论依据 相似文献
3.
制备抗大鲵虹彩病毒(CGSIV)MCP COE蛋白的单克隆抗体,并对其基本特性进行鉴定及开展初步应用。用纯化的重组CGSIV MCP COE蛋白免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA法检测阳性孔,经有限稀释法亚克隆,筛选单克隆杂交瘤细胞株。采用间接ELISA法、免疫印迹法和单克隆抗体亚型检测试剂盒等鉴定单抗的稳定性、特异性和亚型;采用腹水诱生法制备单抗腹水,饱和硫酸铵法纯化腹水单抗;间接ELISA法分别测定单抗杂交瘤细胞上清液和腹水单抗的效价;利用间接免疫荧光法观察CGSIV的增殖。结果显示,细胞融合克隆率为98.68%,阳性率为20.52%,得到1株能够稳定分泌特异性抗体的阳性杂交瘤细胞,命名为1M6。单克隆抗体的亚型属于Ig G2b,κ链;免疫印迹法和间接ELISA法测定结果显示,单抗具有很好的特异性;杂交瘤细胞株培养上清液的效价为1∶1600,腹水单抗效价为1∶2×106,亲和常数为2×105。间接免疫荧光显示CGSIV感染EPC细胞24 h后在宿主细胞质中可以观察到成熟的病毒粒子形成的包涵体,而且在宿主细胞核内也能发现病毒粒子。抗CGSIV MCP COE蛋白单克隆抗体的成功制备为CGSIV免疫学检测方法的建立和其他相关研究的开展奠定了基础。 相似文献
4.
为了建立鳜传染性脾肾坏死病毒(ISKNV)疫苗抗原含量的ELISA检测方法,制备了3株抗ISKNV主衣壳蛋白(MCP)的单克隆抗体,鉴定了其生物学特性。将大肠杆菌表达的重组MCP纯化复性后,连续3次免疫BALB/c小鼠,然后将免疫小鼠的脾细胞与SP2/0细胞融合,经过克隆、筛选,获得3株能稳定分泌抗ISKNV MCP蛋白的单克隆抗体阳性细胞株,分别命名为5F1、3D9和5B4,均为Ig G1亚型。间接ELISA实验表明,3株单抗可特异性识别ISKNV,与鳜弹状病毒、大鲵虹彩病毒等无交叉反应。将5F1株免疫小鼠后制备腹水,以重组MCP和ISKNV细胞培养物上清液为检测抗原,ELISA检测腹水效价分别为1∶51 200和1∶400。间接免疫荧光(IFA)和Western Blotting鉴定结果显示,5F1能够与ISKNV病毒发生特异性反应,并初步确定5F1单抗株制备的腹水用于IFA的使用浓度为1∶200、Western Blotting的使用浓度为1∶1000。结果证实,成功制备了抗ISKNV MCP的单克隆抗体,可特异性识别ISKNV病毒粒子和MCP蛋白,为建立ISKNV疫苗抗原含量检测方法奠定了基础。 相似文献
5.
草鱼呼肠孤病毒HZ08株VP4蛋白单克隆抗体的制备及鉴定 总被引：4，自引：3，他引：1
为了建立针对草鱼呼肠孤病毒(GCRV)流行株的血清学检测方法,实验构建了能高效表达GCRV HZ08株主要衣壳蛋白VP4的重组表达载体pET32a-S6,利用纯化的VP4重组蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞与SP2/O细胞融合,经过克隆和筛选,获得3株能稳定分泌抗VP4重组蛋白单克隆抗体(monoclonal antibody,MAb)的杂交瘤细胞,分别命名为2C2、2F3和5E5.抗体经亚型鉴定均为IgG1,轻链为K链;间接ELISA试验证明,3株杂交瘤细胞分泌的MAb可特异性识别GCRV-HZ08,与GSRV,ISKNV,IHNV均无交叉反应.选择2C2作为腹水生产细胞株,免疫小鼠后腹水ELISA效价为1∶720 000;IFA和Western-blotting结果显示,这株杂交瘤细胞分泌的MAb能够特异性识别GCRV-HZ08病毒粒子.本实验制备的MAb具有良好的生物学特性,为GCRV流行株血清学检测方法的建立及VP4蛋白相关功能研究奠定了基础. 相似文献
6.
中华鳖虹彩病毒单克隆抗体的制备及其抗原表位的初步分析 总被引：1，自引：0，他引：1
以纯化的中华鳖虹彩病毒为抗原免疫Balb/C小鼠,取免疫鼠脾细胞经杂交融合获得了8个能稳定分泌抗中华鳖虹彩病毒特异性单克隆抗体的杂交瘤细胞株。单克隆抗体亚级份分析结果表明,Mab2A4属IgA,Mab8E1是IgG2a,其他的6株单抗Mab1D3、Mab2H1、Mab3A1、Mab4B5、Mab5E1和Mab6F2均为IgG1。酶联免疫吸附剂测定（ELISA）分析表明,8株单抗均能特异性地识别中华鳖虹彩病毒,与EPC、CO、FHM等宿主细胞不产生交叉反应,腹水抗体的ELISA效价在105～106。IFA分析表明,8株单抗中仅Mab5E1没有免疫荧光反应特性,其余7株单抗均能对染毒病灶产生特异性的荧光染色。中和试验结果证实8株单抗均没有中和病毒的特性。应用Western-blotting进行中华鳖虹彩病毒的抗原表位初步分析,结果显示：Mab1D3和Mab2A4分别识别分子量为84 ku和35 ku中华鳖虹彩病毒结构蛋白,Mab3A1能够同时识别分子量分别为14 ku和16 ku的两条多肽,说明这3株单抗结合位点是非构象依赖性抗原决定簇,其余单抗不具备Western-blotting反应特性。这些结果提示上述单抗对中华鳖虹彩病毒抗原特异、灵敏,可用于中华鳖虹彩病毒的检测和结构蛋白分析。 相似文献
7.
传染性胰腺坏死病毒VP3蛋白的原核表达及抗原性分析 总被引：1，自引：0，他引：1
应用RT-PCR方法扩增了IPNV编码内衣壳VP3蛋白的基因615bp,将VP3基因克隆至原核表达载体pET30b,并在大肠杆菌BL21中得到了表达。通过SDS-PAGE分析表明,重组菌诱导后得到了预期大小约30ku的VP3蛋白,与理论值相符,经薄层扫描分析表明目的蛋白表达量可占菌体总蛋白的30%。用镍离子亲和层析柱纯化可溶性的VP3蛋白,并制备抗血清。Western-blotting结果显示,VP3蛋白可被兔抗IPNV阳性血清识别;间接ELISA结果显示,IPNV细胞培养物作为抗原,兔抗VP3蛋白高免血清稀释度为1∶25600时,P/N>2,抗血清可与IPNV全病毒发生反应,以上两项结果说明,表达的VP3蛋白与天然的IPNVVP3蛋白一样具有相同的抗原性。试验利用原核表达系统成功地高效表达了IPNVVP3蛋白,融合蛋白以可溶性形式存在,并制备了高效价的抗血清。 相似文献
8.
凝集素是无脊椎动物非特异性免疫系统的重要组成成份,可凝集外来病原菌、进行非己识别、调理和介导血细胞吞噬。开展了锯缘青蟹血清凝集素的提取、单克隆抗体制备及凝集素定位研究,以N-乙酰基葡萄糖胺（N-Acetyl-D-glucosamine,GlcNac）为配基制备了Sepharose 4B亲和层析柱,可选择性结合青蟹血清凝集素;青蟹血清通过亲和层析柱后,以0.45 mol/L NaCl洗脱,得到有较高凝集活性的单一洗脱峰,该洗脱峰凝集素比活为血清凝集活性的32.6倍,SDSPAGE显示存在87 ku和79 ku主要蛋白成分;以纯化青蟹凝集素免疫BALB/c小鼠,经细胞融合、单抗筛选获得BF82等7株可稳定分泌抗凝集素单抗的小鼠杂交瘤细胞株,单抗腹水的ELISA效价为1∶104～1∶105;单抗可特异性抑制青蟹凝集素的血凝,血凝抑制效价为1∶32～1∶64,亚型鉴定表明7株单抗均为IgG1型,免疫印迹表明单抗可特异性结合87 ku、79 ku蛋白;采用凝集素单抗间接免疫荧光法进行了青蟹血淋巴细胞染色,结果显示凝集素主要分布于颗粒细胞的细胞质颗粒和透明细胞的细胞膜上。 相似文献
9.
草鱼呼肠孤病毒Ⅱ型VP6蛋白多克隆抗体制备及其特异性分析 总被引：1，自引：0，他引：1
为了制备高效的草鱼呼肠孤病毒Ⅱ型VP6蛋白多克隆抗体并对其特异性进行鉴定,实验以草鱼呼肠孤病毒Ⅱ型HZ08株为模板,采用PCR方法扩增S9基因,将该S9基因与p ET-32a(+)载体连接构建p ET-32a-S9原核表达载体,转化到大肠杆菌BL21(DE3)后用IPTG诱导表达;纯化后的重组VP6蛋白免疫新西兰兔,制备多克隆抗体,使用间接ELISA方法测定抗体效价,Western blot和IFA试验鉴定抗VP6蛋白多克隆抗体特异性。结果显示草鱼呼肠孤病毒Ⅱ型的S9基因在原核表达载体中能够正确地表达VP6蛋白,纯化的重组蛋白免疫新西兰兔制备的抗VP6多克隆抗体,经间接ELISA方法测定其效价约为1∶105,Western blot和IFA试验结果显示制备的多克隆抗体能特异性识别GCRVⅡ型毒株,而不能识别GCRV I型、Ⅲ型以及其它病毒,表明该多克隆抗体具有较高的特异性。研究表明制备的抗VP6蛋白多克隆抗体能够特异性识别GCRVⅡ型病毒,为GCRVⅡ型病原学研究及草鱼出血病临床诊断奠定基础。 相似文献
10.
为建立针对Ⅱ型草鱼呼肠孤病毒(GCRV)的血清学检测方法,分别构建了GCRV JX02株外衣壳蛋白VP4、VP35的原核重组表达质粒PGEX-4T-3-S6、PGEX-4T-3-S11,用纯化的重组蛋白r VP4、r VP35分别免疫小鼠制得相应的多克隆抗体,用间接ELISA方法测定2种抗体的效价,用Western Blot鉴定抗体的特异性。SDS-PAGE分析细菌表达的r VP4、r VP35大小分别约为98ku和61ku,且都主要以包涵体的形式存在;间接ELISA方法测定制备的抗体效价分别约为1:4×105和1:106;Western Blot结果显示,制备的2种多克隆抗体都既能够识别原核表达的重组蛋白,又能够识别JX02毒株上的对应蛋白,并且发现感染JX02的草鱼血清中存在结合VP4、VP35的相应抗体。本研究制备的2种多克隆抗体都具有良好的生物学特性,并且这2种重组蛋白作为相应抗体捕获原可以用于通过检测抗病毒抗体来确诊草鱼是否感染Ⅱ型GCRV。本研究将为GCRV主要流行株血清学检测方法的建立以及VP4、VP35蛋白相关功能研究奠定基础。 相似文献
11.
A total of 31 antibody-secreting hybridoma cells against the yellowtail ascites virus (YAV) were established. These monoclonal antibodies (MAbs) reacted with the cytoplasm of YAV-infected CHSE-214 cells, but not with uninfected CHSE-214 cells in an immunofluorescence test. Using these MAbs, two classes of polypeptides (VP2 and VP3) were characterized by immunoprecipitation followed by SDS-PAGE, although one MAb did not react with either polypeptide. Fifteen out of the 17 MAbs that were reactive with VP2 polypeptides neutralized virus infectivity, but all 13 MAbs that were reactive with VP3 did not neutralize infectivity. In the immunofluorescence test, 29 out of the 31 MAbs obtained showed the same reaction pattern to 12 YAV isolates from yellowtail, Seriola quinqueradiata, goldstriped amberjack, Seriola aureovittata, and threeline grunt, Parapristipoma trilineatum, from different geographical regions. The remaining two MAbs showed slightly different reaction patterns to the YAV isolates. The reaction patterns of the MAbs to the VR-299, Sp and Ab strains of IPNV were also investigated. Fourteen MAbs reacted to all three IPNV strains. The other 17 MAbs showed a negative reaction with at least one strain. 相似文献
12.
13.
A collection of infectious pancreatic necrosis virus (IPNV) isolated in five provinces of eastern Canada was analysed by an antigen-coated immunosorbent assay and by neutralization tests using selected monoclonal and polyclonal antibodies. Relevant antigenic sites of the two major capsid proteins, VP2 and VP3, were simultaneously compared. The A1 serotype was predominant and no significant variations of VP2 epitopes were observed. However, two subtypes could be distinguished on the basis of one or two epitopes on VP3. Other serotypes such as A6, A7 or A8 have been detected in piscicultures of New Brunswick and Nova Scotia. The antigenic characterization of IPNV strains appears of interest for epidemiological studies. 相似文献
14.
Characterization and application of monoclonal antibodies against white spot syndrome virus 总被引：1，自引：1，他引：1
Three hybridoma clones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with white spot syndrome virus (WSSV) isolated and purified from Penaeus monodon (Fabricius), collected from north-eastern Taiwan. By sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), the protein profile of this isolate contained four major proteins with sizes of approximately 35 (VP35), 28 (VP28), 24 (VP24), and 19 kDa (VP19). Western blot analysis revealed that two MAbs (1D7 and 6E1) recognized epitopes on VP28 and one MAb (3E8) recognized an epitope on VP19. The MAb 6E1 isotyped to the IgG1 class was used in both an indirect immunofluorescence assay (IFA) and in an immunochemical staining protocol for successful identification and localization of WSSV in infected shrimp tissues. Antigenic similarity of isolates from Indonesia and Malaysia to the Taiwan isolate was illustrated by IFA with MAb 6E1. A MAb (2F6) which bound specifically to two shrimp proteins, 75 and 72 kDa, and reacted to the healthy and non-target tissues of WSSV in infected shrimp, such as hepatopancreas, is also described here and shows the necessity for specific identification of antibodies. 相似文献
15.
Abstract. A panel of 15 monoclonal antibodies (MAbs) were raised against infeetious panercretic mecrosis virus (IPNV) associated with lake trout. salvelinus namaycush (Walwaum). (LT-IPNV) in Cornwall Lake Alberta, for LT-IPNV epietope analysis and comparison with other Canadian IPNV isolates. All the MAbs reacted with IPNV VP2 polypeptide in western blot and 10 MAbs were neutralizing. Both conformation and sequence dependent epitopes were found to be present on the IPNV VP, protein. The antibodies reeognized different epitopes on VP, protein in reeiproeal bloeking ELISA. Twelve MAbs reeognized common epitopes present on LT-IPNV and IPNV from Aretic char. Salvelinus alpinus (L.), (AC-IPNV) in binding and neutralization assays. Three MAbs reacted only with LT-IPNV indicating that it has distinct epitopes, and thus clerly differentiaing it from AC-IPNV isolated from the adjacent Northwest Trritories.Only two MAbs bound to Ja and BCI-IPNV isolate and none of the MAbs neutralized these two IPNV isolates. LT-IPNV was found to be distinct isolate, more colosly related to AC-IPNV and Canda -2 than to Ja-IPNV from alberta or other isolates in Canda. Additionally, the panel of MAbs could differnciate all the propsed Canadian IPNV scrotypes, namely C1. C2. C3 and Ja. 相似文献
16.
传染性胰腺坏死病毒VP3蛋白的干酪乳杆菌表达系统构建 总被引：2，自引：1，他引：1
根据传染性胰腺坏死病毒(IPNV)VP3蛋白的全基因序列,设计并合成引物,以IPNV(ATCC VR-1318)细胞培养毒提取的核酸为模板,对传染性胰腺坏死病毒VP3蛋白的干酪乳杆菌表达系统进行了构建研究。结果显示:进行RT-PCR扩增得到截短的VP3基因约615 bp目的片段,将其克隆到pMD18-T Simple载体,经酶切、PCR扩增和序列测定后显示目的片段正确;将目的片段分别亚克隆到乳酸菌细胞表面表达型载体和分泌表达型载体,电转化于干酪乳杆菌,获得了阳性重组菌株。结果表明,通过本实验方法可构建表达传染性胰腺坏死病毒VP3蛋白的干酪乳杆菌表达系统,为实现IPNV VP3蛋白在乳酸菌中的表达及免疫原性研究奠定了基础。 相似文献
17.
Yaping Chen Mengting Guo Yanxue Wang Xiaojing Hua Shuai Gao Yuting Wang Dechuan Li Wen Shi Lijie Tang Yijing Li Min Liu 《Journal of fish diseases》2019,42(5):631-642
Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout farming worldwide. Their co‐infection is also common, which causes great economic loss in juvenile salmon species. Development of a universal virus vaccine providing broadly cross‐protective immunity will be of great importance. In this study, we generated two recombinant (r) virus (rIHNV‐N438A‐ΔNV‐EGFP and rIHNV‐N438A‐ΔNV‐VP2) replacing the NV gene of the backbone of rIHNV at the single point mutation at residue 438 with an efficient green fluorescent protein (EGFP) reporter gene and antigenic VP2 gene of IPNV. Meanwhile, we tested their efficacy against the wild‐type (wt) IHNV HLJ‐09 virus and IPNV serotype Sp virus challenge. The relative per cent survival rates of two recombinant viruses against (wt) IHNV HLJ‐09 virus challenge were 84.6% and 81.5%, respectively. Simultaneously, the relative per cent survival rate of rIHNV‐N438A‐ΔNV‐VP2 against IPNV serotype Sp virus challenge was 88.9%. It showed the two recombinant viruses had high protection rates and induced a high level of antibodies against IHNV or IPNV. Taken together, these results suggest the VP2 gene of IPNV can act as candidate gene for vaccine and attenuated multivalent live vaccines and molecular marker vaccines have potential application for viral vaccine. 相似文献
18.
以牙鲆淋巴囊肿病毒（LCDV）为抗原免疫Balb/c小鼠，而后将小鼠脾细胞与P3U1骨髓瘤细胞融合，以囊肿组织冰冻切片的免疫荧光染色筛选杂交瘤细胞，阳性结果显示特异性块状荧光信号集中在囊肿细胞的细胞质边缘部分，且多个荧光信号相连呈现链圈状，有限稀释
法克隆阳性杂交瘤细胞，三次克隆后获得4株稳定产生抗LCDV抗体的单克隆杂交瘤细胞株（1A8、1D7、2B6、2D11）。应用Western-blotting法分析单抗识别蛋白的分子量，结果显示，单抗1D7 和2B6均能特异性结合一条分子量116 kD病毒多肽；应用免疫电镜技术定位单抗识别的抗原决定簇，结果发现胶体金颗粒集中吸附在病毒粒子衣壳周围，且背景清洁，无散在的金颗粒或其他污染物。实验结果说明分子量约为116 kD的蛋白多肽为LCDV病毒衣壳蛋白，且具有线性抗原决定簇。 相似文献