Development of a competitive enzyme-linked immunosorbent assay for detection of turkey coronavirus antibodies

A competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of turkey coronavirus (TCV) antibodies. The cELISA utilized a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus)-expressed TCV nucleocapsid (N) protein and biotin-labeled TCV N protein-specific monoclonal antibody. Sensitivity and specificity of the cELISA for detection of TCV antibodies were determined by comparison with the indirect fluorescent antibody test (IFAT) with 1269 reference, experimentally derived, and field-origin sera. Sera with discordant cELISA and IFAT results were further evaluated by western immunoblot analyses. The cELISA detected antibodies specific for TCV and infectious bronchitis virus, a closely related coronavirus, but did not detect antibodies specific for other avian viruses. A high degree of concordance was observed between the cELISA and IFAT; sensitivity and specificity of the cELISA relative to IFAT were 92.9% and 96.2%, respectively. Western immunoblot analyses provided additional evidence of cELISA specificity. The findings indicate that the cELISA is a rapid, sensitive, and specific serologic test for detection of TCV antibodies in turkeys.     

鸡传染性支气管炎病毒GX-YL5株S蛋白在昆虫杆状病毒表达系统的表达及其免疫原性

为了对鸡传染性支气管炎病毒(avian infectious bronchitis virus,IBV)广西优势血清型代表株GX-YL5的S蛋白进行真核表达并研究其免疫原性,设计GX-YL5毒株S基因特异引物,扩增出目的片段后,构建重组表达载体pFastBacTM/HBM-TOPO-S,转化DH10Bac细胞获得重组杆...     

犬冠状病毒N蛋白的原核表达及其多克隆抗体的制备

本研究旨在克隆犬冠状病毒（canine coronavirus,CCV）N基因,体外表达N蛋白,并制备抗该蛋白质的多克隆抗体,用于CCV的诊断及其抗原的检测。参考GenBank中CCV的N基因序列（登录号：KY063618.2）,选择CCV流行毒株的N基因,通过对该基因密码子进行优化和基因合成,最后选择一段有效基因构建重组表达质粒pET-B2M-N,将成功构建的重组质粒转化大肠杆菌DH5α感受态细胞,挑取阳性克隆提取质粒,转化大肠杆菌BL21（DE3）感受态细胞,通过0.5 mmol/L IPTG 30 ℃进行诱导表达。结果表明,优化诱导条件后成功表达出大小约为49 ku的重组蛋白。将重组蛋白与弗氏佐剂按一定比例混合,每隔2周免疫G767、G768两只日本大耳白兔数次,用间接ELISA检测G768抗体效价可达1∶512 000,选用G768抗体进行抗体纯化,纯化后浓度可达10 mg/mL,用间接ELISA、Western blotting和间接免疫荧光试验对N蛋白纯化后制备的兔多克隆抗体进行检测分析,表明表达的重组N蛋白免疫原性良好,制备的多克隆抗体具有良好的反应原性。本研究为犬冠状病毒抗原抗体检测以及靶标CCV诊断试剂盒的建立奠定了一定的基础。     

Detection of turkey enteric coronavirus by enzyme-linked immunosorbent assay and differentiation from other coronaviruses

A double-antibody ELISA for the detection of coronaviruses in intestinal contents from turkey poults with diarrhea was developed. Antibodies were raised in rabbits and guinea pigs against a Minnesota isolate of turkey enteric coronavirus (TCV) propagated in embryonating turkey eggs and were purified by density-gradient centrifugation. The specificity of antisera was confirmed by hemagglutination-inhibition and immunoelectron microscopy. Absorption of anti-TCV hyperimmune sera with egg extracts or egg ovalbumin and the use of different dilution and blocking buffers influenced the sensitivity and specificity of the ELISA. Reciprocal cross-reactivity was detected among turkey, chicken, bovine, and murine coronaviruses. Antisera to the transmissible gastroenteritis virus of swine, the rabbit enteric coronavirus, or the human coronavirus strain 299E failed to react with TCV. The TCV cross-reacted only moderately with the avian infectious bronchitis virus and the hemagglutinating encephalomyelitis virus of swine. Investigations with samples from 47 commercial turkey flocks in Quebec with episodes of transmissible enteritis revealed that the ELISA was more sensitive than was electron microscopy for detection of TCV.     

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.     

4型禽腺病毒抗体间接hexon-ELISA检测方法的建立

以4型禽腺病毒(FAdV-4)的DNA为模板,扩增其六邻体(hexon)部分基因并进行重组表达,以重组hexon蛋白为包被抗原,优化ELISA检测条件,建立了FAdV-4的ELISA抗体检测方法。该方法对FAdV-4阳性血清检测为阳性,对于其他鸡常见病毒,如禽流感病毒5、7、9型,新城疫病毒以及鸡传染性支气管炎病毒阳性血清检测均为阴性;与PCR方法的阳性符合率为83.3%。试验表明,建立的以重组hexon蛋白为包被抗原检测FAdV-4血清抗体的ELISA方法可以用于检测FAdV-4的感染及相关流行病学调查。     

口蹄疫病毒2C3AB基因的原核表达及ELISA方法的建立

口蹄疫病毒(FMDV)的2C蛋白中存在对自然感染和灭活疫苗免疫动物具有鉴别诊断意义的抗原表位。为建立一种敏感的血清学鉴别诊断方法,本研究截取FMDV 2C蛋白的C端B细胞表位较为富集的区域与全长3AB基因组合,经原核表达获得分子量约为44 ku的目的蛋白。Western blot分析表明,表达产物2C3AB与FMDV感染动物的阳性血清呈特异性反应。以纯化的目的蛋白作为包被抗原建立间接ELISA方法,敏感性检测表明该方法比3ABC-ELISA具有更高的敏感性;同时检测猪圆环病毒、猪繁殖和呼吸障碍综合征病毒、猪瘟病毒标准阳性血清均无交叉反应。批内和批间重复性试验显示,OD450nm值的变异系数小于9%。采用该方法检测不同背景的临床样品,并与3ABC-ELISA及进口试剂盒比较,总符合率分别为98.5%和90%。结果表明,该ELISA检测方法具有更高的敏感性和良好的特异性、重复性。     

重组马传染性贫血病病毒核心蛋白抗原在 A GID 和ELISA中应用的评价

用昆虫杆状病毒表达系统获得的马传染性贫血病病毒（ Ｅ Ｉ Ａ Ｖ） 核心蛋白（ Ｇａｇ） 和 Ｐ２６ 蛋白, 作为免疫琼脂双扩散（ Ａ Ｇ Ｉ Ｄ） 和酶联免疫吸附试验（ Ｅ Ｌ Ｉ Ｓ Ａ） 抗原。对７６ 份已知马传贫非特异性血清进行检查, 同时与市售 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 试剂盒作比较。证明, 用表达蛋白作抗原的 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 检测结果均为阴性反应, 而用市售 Ａ Ｇ Ｉ Ｄ 试剂盒检查有５４ 份马血清出现非特异性反应, 市售 Ｅ Ｌ Ｉ Ｓ Ａ 试剂盒检查也出现了非特异性反应, Ｏ Ｄ 值比表达抗原 Ｅ Ｌ Ｉ Ｓ Ａ 高３５ 倍。初步证明在 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 法中, 表达抗原优于常规马传贫病毒抗原。     

H5N1和H9N2亚型禽流感病毒型特异性抗原捕捉ELISA检测方法的建立

采用禽流感病毒多克隆抗体及型特异性单克隆抗体，研究建立了型特异性抗原捕捉ELISA检测方法，用于检测H5N1和H9N2亚型禽流感病毒。优化了反应条件，确定了包被抗体、检测抗体及酶结合物的最佳工作浓度，对该方法的敏感性、特异性、重复性及稳定性进行了分析，并与病毒分离鉴定的结果进行了比较。结果表明，该方法敏感、特异，具有良好的重复性和稳定性，可用于临床样品及鸡胚、细胞培养物中禽流感病毒的检测。     

基因Ⅶ型新城疫病毒F蛋白在昆虫细胞中的表达及免疫效力研究

本试验旨在利用杆状病毒表达系统对基因Ⅶ型新城疫病毒（NDV）F基因进行表达研究。RT-PCR扩增F基因,将其克隆到pFastBac HT A载体中,阳性重组质粒转化DH10Bac感受态细胞,PCR鉴定获得阳性克隆,碱裂解法提取阳性质粒,转染Sf9昆虫细胞,获得含F基因的重组杆状病毒质粒,重组病毒感染Sf9细胞72 h后,进行SDS-PAGE电泳、间接免疫荧光和Western blotting检测。免疫SPF鸡,间接ELISA测定抗体滴度,攻毒保护试验检测重组F蛋白保护性。结果显示,F蛋白在昆虫细胞中能够特异性表达,该蛋白诱导了高滴度的NDV特异性抗体,具有良好的免疫原性;重组F蛋白免疫组攻击保护率达到90%,明显高于阴性对照组。本研究结果为NDV新型亚单位疫苗的研究奠定了基础。     

猪囊尾蚴小热休克蛋白的免疫原性研究

利用反转录聚合酶链式反应（RT—PCR），从猪囊尾蚴虫体中扩增出小热休克蛋白（small Heat shock protein，sHSP）基因，克隆至原核表达载体pGEX-4T-1，在大肠杆菌BL21中以GST融合蛋白的形式表达。SDS-PAGE分析证明，表达产物为64Ku的融合蛋白，经凝胶扫描分析，表达量约占菌体可溶性蛋白的30％。免疫学分析（Western blot，ELISA）结果表明，sHSP能与猪囊尾蚴血清反应。将表达产物点电泳后切胶回收免疫小鼠，进行Western blot和ELISA检测，其抗血清能与猪囊虫粗抗原及纯化的sHSP重组融合蛋白产物发生免疫学反应。该研究为丰富新的诊断试剂和免疫用重组抗原具有重要意义，同时，预测糖基化、磷酸化作用在sHSP实现生物学功能方面也具有广泛的意义。     

禽流感病毒核蛋白基因在重组杆状病毒中的表达

利用ＲＴ－ＰＣＲ方法成功地扩增了我国禽流感病毒分离株Ａ／Ｘｉｎｇｊｉａｎｇ／１／９６（Ｈ１４Ｎ５）的核蛋白（ＮＰ）基因,其限制性内切酶图谱和核苷酸序列与鸭源的标准Ｈ１４Ｎ５毒株几乎完全一致,与其它毒株则有较大差异,说明该鸡源分离株与鸭源毒株有非常近的亲缘关系。将ＮＰ基因定向克隆到杆状病毒转移载体ｐＶＬ１３９３中,再与杆状病毒线性ＤＮＡ（ＢＡＣ－Ｎ－ＢｌｕｅＤＮＡ）共转染于Ｓｆ９昆虫细胞中,经过三次蚀斑筛选,获得重组病毒ｒＢ２。用其细胞表达产物裂解后作ＳＤＳ－ＰＡＧＥ蛋白电泳、Ｗｅｓｔｅｒｎ－ｂｌｏｔ和ｄｏｔ－ＥＬＩＳＡ,结果表明ＮＰ基因在杆状病毒系统中获得了表达。同时用表达产物作琼脂扩散试验,结果表明表达产物与现行标准禽流感琼扩抗原具有相同的生物学活性。     

H5N1亚型禽流感病毒NP基因的克隆与表达

根据已知H5N1亚型禽流感病毒NP基因序列设计、合成PCR克隆引物。自H5N1阳性病料中提取总RNA,反转录后采用高可信度DNA聚合酶,经PCR扩增NP基因,采用Invitrogen定向表达系统进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组NP蛋白,分子质量约66.0ku。采用单克隆抗体和阳性血清经ELISA、免疫印迹分析重组蛋白的免疫反应性。结果表明:所获得的重组NP蛋白在ELISA分析中均能与阳性血清和单克隆抗体发生特异性结合,但在免疫印迹分析中仅与阳性血清发生特异性结合。     

Immunofluorescent studies on localization of secretory immunoglobulins in the intestines of turkeys recovered from turkey coronaviral enteritis

The present study concerns the production of specific secretory antibodies in turkeys orally inoculated with turkey coronavirus (TCV). Tissue localization of secretory antibodies in the intestines of recovered birds 4 and 5 months later was demonstrated by immunofluorescence technique. Intestinal sections were stained by sandwich method, using concentrated TCV antigen and anti-TCV conjugate. It was suggested that local synthesis of antibody might be responsible for life-long immunity seen in these birds after recovery from infection.     

Characterization of the sigmaB-encoding genes of muscovy duck reovirus: sigmaC-sigmaB-ELISA for antibodies against duck reovirus in ducks

The sigmaB/sigmaC-encoding genes of muscovy duck reovirus (DRV) S12 strain were cloned, sequenced, and expressed in Escherichia coli. The sigmaC-encoding gene of DRV showed only 21-22% identity to that of avian reovirus (ARV) at both nucleotide and amino acid level. The sigmaB-encoding gene of DRV comprised 1163bp with one open reading frame (ORF). The ORF comprised 1104bp and encoded 367 amino acids with a predicted molecular mass of 40.44 kDa. A zinc-binding motif and a basic amino acid motif were found within the predicted amino acid sequence of sigmaB. The identities between the S12 and ARV were 59.3-64.0% and 60.9-62.5%, respectively, at the nucleotide and deduced amino acid levels. Phylogenetic analysis of the sigmaB-encoding gene sequence indicated that S12 separated as a distinct virus relative to other avian strains. The expressed sigmaB/sigmaC fusion proteins in E. coli could be detected, approximately 45 and 50kDa, respectively, by duck anti-reovirus polyclonal serum. In addition, an ELISA (sigmaB-sigmaC-ELISA) using the expressed sigmaB-sigmaC proteins as coating antigen for detection of antibodies to DRV in ducks was developed. In comparison with the virus neutralization test and agar gel immuno-diffusion test (AGID), the sigmaB-sigmaC-ELISA showed perfect specificity and sensitivity. The sigmaB-sigmaC-ELISA did not react with the antisera to other duck pathogens, implying that these two proteins were specific in recognition of DRV antibodies. Taken together, the results demonstrated that sigmaB-sigmaC-ELISA was a sensitive and accurate method for detecting antibodies to DRV.     

Detection of antibodies to ovine lentivirus using recombinant capsid and transmembrane proteins

The coding sequences of the capsid protein p25 and transmembrane protein of Maedi-Visna virus were amplified using polymerase chain reaction and cloned into the plasmid expression vector pRSET-B. Both DNA constructs expressed proteins tagged with polyhistidine. The recombinant proteins were purified using Ni-NTA agarose and used in immunoblot to detect antibodies against Maedi-Visna virus. A total of 260 ovine serum specimens was analysed. The total number of p25-positive sera was 111 (42.7%). Higher sensitivity was achieved with rTM antigen, which detected antibodies in 118 (45.4%) sera. The combination of both recombinant proteins as antigens resulted in higher sensitivity of serological detection compared to whole virus antigen.