猪伪狂犬病病毒gE抗体胶体金免疫层析检测方法的建立及应用

以纯化的抗人红细胞单链抗体(ScFv)-猪伪狂犬病病毒(PRV)gE蛋白双功能融合蛋白为诊断抗原和胶体金标记物,以羊抗猪IgG包被硝酸纤维膜作为质控带,制作检测猪伪狂犬病毒gE抗体的双抗原胶体金试纸条。利用方阵滴定试验筛选出金标抗原最佳工作浓度为17.6μg,检测线诊断抗原最佳标记量为1.76μg,血清最佳稀释度为1∶10,作用时间15 min,与猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪乙型脑炎病毒(JEV)、猪布鲁菌(Brucella)阳性血清和PRVgE缺失疫苗接种的猪免疫血清检测线均不出现红色条带,与PRV标准阳性血清反应检测线出现红色条带。试纸条操作简单,肉眼于15 min内可判定结果;试纸条在室温保存6个月,其特异性和敏感性没有明显变化;与美国IDEXX和法国LSI gE-ELISA抗体检测诊断试剂盒检测结果比较,1 164份猪血清的符合率均为90.55%。制备的胶体金试纸条具有操作简便、敏感性和特异性较高的特点,可用于PRV野毒感染的快速筛查。     

牛传染性鼻气管炎病毒抗体胶体金检测试纸条的制备

采用SF 9昆虫细胞-杆状病毒系统重组表达牛传染性鼻气管炎病毒gD蛋白抗原,经纯化鉴定后将gD蛋白用胶体金标记作为示踪抗原,未标记的gD抗原作为捕获抗原,并以羊抗牛IgG抗体作为质控抗体,建立了牛传染性鼻气管炎病毒双抗原夹心法胶体金检测试纸条。试验结果表明,该试纸条检测灵敏度高,特异性良好,与牛口蹄疫病毒、牛病毒性腹泻病毒等阳性血清无交叉反应。使用建立的胶体金试纸条和IDEXX牛传染性鼻气管炎病毒抗体ELISA检测试剂盒同时检测112份牛血清,阳性符合率为93.5%,阴性符合率为96.0%,总符合率为94.6%。说明该试纸条可以应用于临床牛传染性鼻气管炎病毒抗体的诊断和检测。     

牛传染性鼻气管炎病毒抗体检测胶体金免疫层析试纸的研制

将gD蛋白用胶体金标记并包被于结合垫作为检测探针,未标记的gD蛋白包被NC膜作为检测线,以金标记鼠IgG与羊抗鼠IgG作为独立质控系统,利用双抗原夹心法建立检测牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus, IBRV)抗体的胶体金免疫层析试纸,并对该试纸进行了性能评价。结果显示,该试纸在检测副结核分枝杆菌(MAP)、赤羽病病毒(AKAV)、牛病毒性腹泻病毒(BVDV)、牛支原体(M.bovis)、小反刍兽疫病毒(PPRV)、布氏杆菌(Brucella)、口蹄疫病毒(FMDV)的标准阳性血清时均为阴性反应;该试纸在检测IBRV标准阳性血清时敏感性为1∶16;将该试纸与进口商品化ELISA试剂盒共同检测60份待检牛血清,两者的阳性符合率为93.3%,阴性符合率为100%,总符合率为96.7%。结果表明,本试验建立的胶体金免疫层析试纸可在10～15 min完成检测,具有快速、准确、简便等特点,为临床定性检测及现场诊断牛传染性鼻气管炎提供了有效工具。     

鱼类病毒性出血性败血症病毒诊断胶体金免疫层析方法的建立

为建立一种快速检测鱼类病毒性出血性败血症病毒(Viral haemorrhagic septicaemia virus,VHSV)的胶体金免疫层析方法(GICA),采用柠檬酸三钠还原法制备胶体金颗粒,以标记纯化的VHSV G蛋白单克隆抗体(MAb)10A7为捕捉抗体,将纯化的抗VHSV N蛋白的MAb 10EN单克隆抗体和羊抗鼠IgG抗体包被在硝酸纤维素膜上,作为检测带与质控带,经试验条件优化,组装形成胶体金免疫层析试纸条。用本试验研制的胶体金试纸条检测VHSV感染的CHSE细胞培养物。结果显示:在检测线和质控线均呈现红色条带,而健康的CHSE细胞培养物对照仅在质控线呈现红色条带;试纸条检出病毒培养物的病毒量最低限为10~(4.0) TCID_(50);随机挑取3个不同批次的试纸条,对阳性样品和阴性样品进行重复试验,未发现差异。特异性试验表明,该试纸条与传染性造血器官坏死病毒(IHNV)、传染性胰坏死病毒(IPNV)、鲤春病毒血症病毒(SVCV)、牙鲆弹状病毒(HRV)、草鱼呼肠孤病毒(GCRV)无交叉反应。将同一批次的试纸条在4℃条件下保存不同时间后进行检测,发现保存6个月的试纸条仍具有良好的稳定性。本试验研发的胶体金诊断试纸条具有良好的特异性、灵敏性和重复性,在快速辅助检测方面具有推广应用价值。     

检测猪瘟病毒野毒株胶体金免疫层析方法的建立

为建立快速、简便检测猪瘟病毒(CSFV)野毒的胶体金免疫层析方法(GICA),本研究采用柠檬酸三钠还原法制备胶体金颗粒,标记纯化的抗CSFVE2蛋白的单克隆抗体(MAb)6E10作为捕捉抗体,将纯化的抗CSFVE2蛋白的MAbHQ06和兔抗鼠IgG抗体包被在硝酸纤维素膜上,分别作为检测线和质控线,优化反应条件,组装成胶体金免疫层析试纸条。结果表明,所制备的试纸条用于检测CSFV野毒感染的PK-15细胞培养物,在检测线和质控线处均呈现红色条带,健康PK-15细胞培养物对照仅在质控线呈现红色条带;试纸条检出病毒培养物的最低限为103.5 TCID50;用不同批次的试纸条重复检测,结果无差异;该试纸条不与猪瘟兔化弱毒(HCLV)、牛病毒性腹泻病毒(BVDV)、猪繁殖与呼吸综合征病毒(PRRSV)、传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)、猪轮状病毒(PRV)、伪狂犬病病毒(PrV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV2)反应。所制备的试纸条具有良好的特异性、敏感性和重复性,初步达到了区分检测CSFV野毒株和弱毒株的目的。     

牛副结核胶体金免疫层析试纸条的研制

将提纯的牛副结核分支杆菌重组蛋白MAP0862-2154c作为硝酸纤维素膜检测线的包被抗原,兔抗羊IgG作为硝酸纤维素膜质控线的包被抗体,金标羊抗牛IgG点喷到玻璃纤维素膜上,制成用于检测牛副结核抗体胶体金免疫层析试纸条。用牛的副结核标准阳性血清与检测试纸条反应,在检测线处出现红色反应带,而滴加阴性血清的试纸条检测线处未出现反应条带,上述2种血清在质控线处均出现红色反应条带;试纸条可检出牛副结核抗体的最低抗原包被浓度为400μg/mL;试纸条不与牛结核病、牛布鲁氏菌病的阳性血清发生反应;试纸条37℃保存9d后的检验结果与4℃保存的检验结果相同。所制备的试纸条具有灵敏、特异的优点,稳定性良好;10min左右即出结果,操作简便,可用于临床诊断。     

鸡毒支原体胶体金免疫层析试纸条的研制和初步应用

为研制一种用于快速检测鸡毒支原体(MG)的胶体金免疫层析试纸条,采用直径20nm的胶体金颗粒标记纯化的MG多克隆抗体,硝酸纤维素膜检测线和质检线分别喷加纯化的MG多克隆抗体和兔抗鸡IgG抗体,制作胶体金试纸条。试纸条检测MG阳性显示两条红色反应条带;阴性为一条红色反应带。本试纸条可检测到1∶1 280倍稀释的MG抗原,即颜色变化单位为8×104 CCU/mL,具有高度的灵敏性。用试纸条检测衣原体、滑液支原体、猪肺炎支原体、鸡大肠埃希菌和鸡白痢沙门菌等均呈阴性,表明该试纸条具有较好的特异性。对试纸条检测呈MG阳性的38只病鸡进行MG培养验证,所得结果一致;同时对MG培养检测的另外95份病鸡样品进行试纸条测试比对,符合率为96.84%,证明该试纸条具有很高的准确性。该胶体金免疫层析试纸条检测MG操作简单,灵敏度、准确度、特异性等均符合要求,可以用于养殖场批量MG的检测。     

牛赤羽病胶体金抗体检测试纸条的研制

研究旨在制备一种现场快速筛查牛赤羽病的免疫层析试纸条。通过胶体金标记赤羽病病毒N蛋白,将兔抗牛IgG和兔抗N蛋白多克隆抗体分别喷涂于NC膜上作为检测线（T）和质控线（C）。结果显示,牛赤羽病毒阳性抗体血清稀释度为1∶512时,检测线仍显色;且与牛病毒性腹泻病毒、牛结核病、布鲁氏菌病、牛巴氏杆菌病、地方流行性牛白血病毒和蓝舌病毒的阳性血清均无交叉反应;使用试纸条和商品化ELISA试剂盒同时检测40份田间牛血清样品的结果符合率为92.6%。研究表明,试纸条适用于基层养殖户对牛赤羽病的筛查。     

鸡传染性法氏囊病病毒VP2基因的融合表达及兔抗血清的制备

以鸡传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)Gt株基因组RNA为模板,通过RT-PCR扩增VP2基因,经EcoR Ⅰ/Sal Ⅰ酶双酶切处理后与经相同酶切处理的pET30a原核表达载体连接,将酶切、PCR及序列鉴定正确的阳性重组子转化E. coli DE3菌,经IPTG诱导,表达的VP2蛋白通过Ni-NTA树脂纯化后,免疫新西兰白兔,制备兔抗血清,并对其进行间接ELISA、IFA和Western blot分析.SDS-PAGE分析表明,在IVFG诱导下成功表达了约为50 kDa的His-VP2融合蛋白;间接ELISA检测制备的兔抗血清效价在1:25 600以上;IFA及Western-blot分析结果表明,其可特异性结合真核表达的VP2蛋白以及IBDV全病毒.研究结果提示,免抗VP2血清可用于VP2蛋白及病毒的检测,为进一步研究IBDV分子生物学功能提供了物质基础.     

犬瘟热病毒核蛋白基因的原核表达及其间接ELISA检测方法的建立

应用RT-PCR技术特异扩增出犬瘟热病毒(Canine distemper virus,CDV)核衣壳蛋白基因(Nucleocapsid pro-tein,N)高度保守序列,克隆至质粒pMD18-T中获得重组质粒pMD18-T-N,测序结果证实了重组质粒的可靠性。将目的片段克隆至大肠杆菌表达载体pGEX-4T-1中谷胱甘肽转移酶(GST)基因的下游,重组质粒转化大肠杆菌BL21株,经1 mmol/L IPTG诱导,N基因融合蛋白获得高效表达。SDS-PAGE电泳分析表明,表达产物的相对分子质量与预期的55 000相符。Western-blot检测表明,表达产物与CDV标准阳性血清呈阳性反应。在此基础上,初步建立了以纯化的N蛋白为包被抗原的间接ELISA检测方法。结果表明,大肠杆菌中表达的CDV N蛋白在免疫原性上与天然N蛋白具有较高相似性,可作为诊断用抗原,从而为进一步开发犬瘟热抗体检测试剂盒以及单克隆抗体、胶体金试纸条奠定了基础。     

Development of a one-step strip test for the diagnosis of chicken infectious bursal disease

A rapid diagnostic strip for chicken infectious bursal disease (IBD) was developed based on membrane chromatography using high-affinity monoclonal antibodies directed to chicken infectious bursal disease virus (IBDV). The diagnostic strip has high specificity for detection of chicken IBDV antigen and recognizes a variety of the virus isolates, including virulent and attenuated strains, with no cross-reactivity to other viruses, such as Newcastle disease virus, Marek's disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and egg-drop-syndrome virus. The results showed that its specificity was highly consistent with the agar-gel precipitation test (AGP). The diagnostic strip detected as low as 800 median egg lethal dose (ELD50) viruses in the IBDV BC6/85-infected sample, which was comparable with AC-ELISA (400 ELD50) and 32 times more sensitive than the AGP test (2.56 x 10(4) ELD50). In experimental infection, IBDV was detected in the bursa as early as 36 hr postinfection with the diagnostic strip before the clinical signs and gross lesions appeared. It takes only 1-2 min to do a strip test to detect chicken IBDV antigen after the specimen is grounded in a whirl pack with finger massage.     

鸡传染性法氏囊病病毒BC6/85株VP2基因的原核表达、纯化及鉴定

为获得可用于鸡传染性法氏囊病病毒(IBDV)抗体检测的重组抗原VP2蛋白,根据GenBank中发表的IBDV VP2序列设计一对特异性引物,应用RT-PCR技术克隆IBDV经典标准攻毒株(BC6/85株)的VP2基因,插入质粒pET-32a中构建重组表达质粒pET-32a-VP2,经IPTG诱导后获得了以包涵体形式表达的重组蛋白。重组蛋白纯化后,Western-blot检测表明具有良好的反应原性。本研究为下步建立IBDV抗体的间接ELISA方法及新型疫苗的研制奠定了基础。     

传染性法氏囊病病毒BC6/85株全基因组克隆及其序列分析

以传染性法氏囊病病毒（IBDV）BC6／85株基因组RNA为模板,采用RT—PCR方法扩增并克隆了IBDVBC6／85株基因组全长eDNA。序列测定结果表明：A节段全长共3260个核苷酸,与IM株的同源性最高为97．4％,与其他血清I型毒株的核苷酸同源性介于91．2％～97．1％;B节段有2827个核苷酸,与IM株同源性最高为98．6％,与其他毒株的核苷酸同源性为88．7％～98．6％。通过对编码的氨基酸序列进行分析,发现BC6／85株有21个特有氨基酸,其中15个位于多聚蛋白VP2—4—3。系统进化树分析表明,BC6／85株与经典毒株、弱毒株和变异株的关系较近,而与超强毒株相对较远。     

一株传染性法氏囊病病毒强毒株的分离与鉴定

鸡传染性法氏囊病是由传染性法氏囊病病毒引起的一种急性传染病。本研究从江苏某疑似发生传染性法氏囊病的鸡场采集病料,通过观察临床症状、病理变化、RT-PCR检测、基因测序、SPF鸡胚接种、琼脂扩散试验和雏鸡攻毒等试验,证实了该鸡群发生了传染性法氏囊病,且分离到一株传染性法氏囊病病毒(JSXY株),该病毒有较强的致病力,VP4基因比较分析发现其与变异株亲缘关系最近,序列同源性为95%。本研究为江苏地区传染性法氏囊病的防治提供了有益的参考。     

Efficacy of VP2 protein expressed in E. coli for protection against highly virulent infectious bursal disease virus

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specific-pathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.     

Virulent vaccine strains of infectious bursal disease virus not distinguishable from wild-type viruses with marker the use of a molecular

Molecular techniques have not only made timely and accurate detection of infectious bursal disease viruses (IBDVs) possible but also have allowed the identification of viral strains. Previously, we identified a genetic marker that distinguished wild-type IBDV strains from vaccine strains of the virus. The marker was an NgoM IV restriction enzyme site in the VP2 gene that was present in 10 wild-type viruses but not 16 vaccine strains of IBDV. On the basis of that study, we concluded that the NgoM IV marker could be useful in the identification of wild-type potentially pathogenic strains of this virus. Because virulent (hot) vaccine strains of IBDV are used to vaccinate commercial poultry, it was important to determine if the NgoM IV marker was present in these virulent vaccines. The infectious bursal disease Blen and Bursa Vac virulent vaccines were examined and determined to contain the marker. We concluded that the presence of this marker was not unique to wild-type strains of the virus. The absence of the NgoM IV marker, however, was consistent with some level of attenuation, and its presence appears to be consistent with virulent IBDV strains.     

表达传染性法氏囊病病毒VP2基因的重组鸡痘病毒的构建及其免疫保护性研究

以脂质体转染技术构建了表达鸡传染性法氏囊病病毒（ＩＢＤＶ）ＶＰ２基因的重组鸡痘病毒ＦＰＶ－ＶＰ２，该病毒在鸡胚成纤维细胞及鸡体内均能稳定产生子代病毒，经翅皮下５×１０５ＰＦＵ／羽免疫１日龄ＳＰＦ鸡，免疫后４周以１００ＬＤ５０／羽ＩＢＤＶ超强毒株Ｇ株攻毒，获得了５／６的保护，但不能有效预防临床发病及法氏囊受损萎缩。实验结果证明了ＶＰ２是ＩＢＤＶ的宿主保护性抗原，提示Ｔ细胞介导的免疫可能在ＩＢＤＶ的免疫中起着较为重要的作用。本研究为ＩＢＤＶ重组病毒疫苗研制进行了有益探索。     

Prokaryotic Expression and Bioinformatics Analysis of VP2 Gene of Infectious Bursal Disease Virus C4 Strain

This study was aimed to investigate the relationship between the virulence characteristics of infectious bursal disease virus(IBDV) C4 strain and its VP2 amino acid sequence. The RNA of IBDV C4 strain was extracted,and its VP2 gene was amplified by RT-PCR.VP2 nucleotide sequences and deduced amino acids of different virulent IBDV strains were compared. At the same time, prokaryotic expression vector pET-32a(+) was used to express the VP2 gene. The expression of recombinant VP2 protein was detected by SDS-PAGE and Western blotting. The results showed that the VP2 gene of IBDV C4 strain belonged to the very virulent infectious bursal disease virus (vvIBDV) in evolutionary relationship, the VP2 nucleotides homology between IBDV C4 strain and other vvIBDV strains were 98.1% to 98.7%, and there were no mutations in S-W-S-A-S-G-S (326-332 amino acids) and 222(A), 256(I), 294(I) and 299(S). The VP2 amino acid sequence of IBDV C4 strain was consistent with the characteristics of other vvIBDV strains. However, there were three differences amino acids sites at 201(D/G), 281(G/R) and 313(V/A) between the amino acids of the C4 strain and the very virulent strain UK661. And the change of 281(R) was in the small hydrophilic region of 279 to 290, which was related to the antigenicity of the virus; The recombinant VP2 protein molecular weight expressed in Escherichia coli BL21 was about 67 ku. This study provided a basis for further research on antigenic changes resulting from amino acid variation of 201(G), 281 (R) and 313(A). These results indicated that the VP2 gene of the IBDV C4 strain was consistent with the major characteristics of the vvIBDV strain VP2 gene. The difference of three amino acid sites in the vvIBDV strain C4 might be related to the evolution of virulence of IBDV strain in China.     

Failure of viral protein 3 of infectious bursal disease virus produced in prokaryotic and eukaryotic expression systems to protect chickens against the disease

In recent years, infectious bursal disease virus (IBDV) has become a serious economic problem as a result of the emergence of new and very virulent strains. Most of the antibodies produced against IBDV are for the structural proteins viral protein (VP) 2 (VP2) and VP3. The purpose of this study was to test the potential of recombinant VP3 to induce protective antibodies. The gene for VP3 was isolated from a virulent strain of the virus and cloned into prokaryotic (Escherichia coli) and eukaryotic (baculovirus) expression systems. The protein expressed by both systems was of the expected size (32 kD) and was detected by anti-IBDV antibodies. Following partial purification, the polypeptides were injected into intact birds and induced the production of high levels of anti-IBDV antibodies, as detected by immunoblot and enzyme-linked immunosorbent assay tests. These antibodies did not prevent changes in the bursa and mortality when birds were challenged with a virulent IBDV strain after vaccination with the recombinant VP3. The results show that VP3 polypeptide cannot be used as a subunit vaccine against IBDV and raise questions concerning the nature of the neutralizing epitope on this structural protein.