牛口蹄疫病毒VP2-ELISA和3B表位串联肽间接ELISA试剂盒的研制
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摘要
口蹄疫(Foot and Mouth Disease, FMD)是由口蹄疫病毒(Foot-and-mouth disease virus, FMDV)引起的以感染偶蹄动物为主的急性、热性、高度传染性疫病。该病的发生和流行严重危害畜牧业的健康持续发展,造成巨大的经济损失,该病被世界动物卫生组织列为必须通报的多种动物共患传染病之一。从2005年起国内不断发生牛口蹄疫,目前面临着Asia 1型口蹄疫的持续危害、O型FMDV的复发感染、新型毒株A型的侵袭风险。因此,国内口蹄疫的防控任务艰巨。我国防控FMD主要采取疫苗免疫和抗体监测为主的综合防控政策,通过接种疫苗提高畜群整体抗体水平来预防口蹄疫的感染和传播。因此牛口蹄疫感染的快速诊断、免疫效果评价、自然感染和疫苗免疫的鉴别诊断成为防控口蹄疫的关键技术。
本研究通过pPROEXTM HTb表达载体在大肠杆菌DH5α中成功表达了FMDV的vp2基因,获得大小为35ku的融合蛋白,Western blot证实目的蛋白可与FMDV 5种血清型的牛阳性血清发生特异性反应。以此纯化蛋白为抗原建立了牛FMDV VP2蛋白间接ELISA方法。特异性试验表明,该抗原不与常见的7种牛病阳性血清发生交叉反应。检测非免疫无口蹄疫地区牛阴性血清特异性为100%;检测感染血清敏感性为97.3%;与4种商品化试剂盒比较检测O-Asia 1二价灭活苗免疫牛血清364份,符合率分别为69.0%、95.0%、90.4%和86.8%。试验结果表明建立的ELISA方法可用于牛口蹄疫感染和免疫抗体检测。
本研究利用实验室融合表达的3B表位串联肽(8BF)为检测抗原建立的间接ELISA为基础,优化组装了一种特异、敏感的间接ELISA试剂盒。通过检测大量未免疫、免疫健康牛血清和感染牛血清,确定了判定标准:S/P≥0.3,阳性;0.2≤S/P<0.3,可疑;S/P<0.2,阴性。8BF-ELISA试剂盒检测NSP抗体持续期试验表明,自然感染牛3B表位肽感染后10个月仍为阳性。与3种商品化试剂盒比较检测101份临床样本,和Ceditest? FMDV-NS ELISA、3ABC-I-ELISA符合率高达95%;与3ABC-I-ELISA进一步比较检测528份不同背景的牛血清,总符合率为94.5%(499/528)。大规模检测不同来源的6个牛场共2026份临床牛血清,部分牛群全部阴性,大多数牛群含有阳性牛,阳性率从3%到17%不等,个别感染牛群阳性率高达72.2%。8BF-ELISA试剂盒方便、安全、实用,特异性和敏感性高,用于鉴别诊断牛FMD自然感染和疫苗免疫,为FMD综合防控提供了技术支持。
Foot-and-mouth disease (FMD) caused by foot-and-mout disease virus (FMDV) is a highly contagious and acutely infected disease of cattle, pigs, sheep, goats and wild ruminant species. It has an economically devastating impact on affected countries, where it creates great losses to productivity and considerable economic losses to the husbandry industry. Whenever the disease has happened; it is must reported to the OIE. Since the Asia 1 FMDV first reported in 2005 in china, there are several outbreaks with the Asia 1, O and A serotypes. There is a complex policy to control the FMD mainly by the preventive vaccination in china, where the most animals are vaccinated 2-4 times every year. So the accurate and in-time diagnosis and routine surveillance become very important for the disease control. Thus, diagnostic methods play an important role for FMD control.
The complete gene encoding the structural protein VP2 of FMDV was subcloned into expression vector pPROEXTM HTb and expressed in DH5αcells. Purified VP2 protein reacted positively with serotype-specific cattle sera of 5 serotypes of FMDV (O, A, C, SAT 2 and Asia 1) by western blot. An indirect ELISA (VP2-ELISA) was developed using purified protein to detect FMDV antibodies in cattles. The protein had no cross-reaction with the positive sera of other bovine diseases. The specificity was 100% to detect the negative cattle serum from Canada where was free of FMD without vaccination. The sensitivity was 97.3% to detect infected sera. Comparison with four commercial kit showed a coincidence rate of 69.0%、95.0%、90.4% and 86.8% against vaccinated cattle serum, respectively. Thus the VP2-ELISA can be used to detect the infected and vaccinated cattle serum.
To differentiate antibodies induced by FMDV infection from those induced by vaccination, an indirect ELISA was established then the kit was developed, using purified tandem epitopes of 3B protein as antigen. A large number of sera from naive, vaccinated and infected cattle were examined for determination of the cut-off value of the method. The cut-off value was established as follows: positive, S/P≥0.3; suspicious, 0.2≤value<0.3; negative, S/P <0.2. Seroreactivity to 8BF in some infected cattle was maintained through 10 months. The rate of agreement with Ceditest? FMDV-NS ELISA and the 3ABC-I-ELISA was up of 95% by detection of 101 field cattle serum. The performance of this assay was further validated by 3ABC-I-ELISA kits. The coincident rate was 94.5% (499/528), respectively. The number of 2026 cattle sera derived from with different situations (na?ve, unknown, immune and infectious status) were detected. The prevalence of 8BF antibodies reached 72.2% in some diseased cattle herds. The described 8BF-ELISA is safe, cheap and also easy to perform in large scale of serological surveys. The high specificity and sensitivity makes this test a good tool for better distinguishing between infected and vaccinated cattle.
本研究通过pPROEXTM HTb表达载体在大肠杆菌DH5α中成功表达了FMDV的vp2基因,获得大小为35ku的融合蛋白,Western blot证实目的蛋白可与FMDV 5种血清型的牛阳性血清发生特异性反应。以此纯化蛋白为抗原建立了牛FMDV VP2蛋白间接ELISA方法。特异性试验表明,该抗原不与常见的7种牛病阳性血清发生交叉反应。检测非免疫无口蹄疫地区牛阴性血清特异性为100%;检测感染血清敏感性为97.3%;与4种商品化试剂盒比较检测O-Asia 1二价灭活苗免疫牛血清364份,符合率分别为69.0%、95.0%、90.4%和86.8%。试验结果表明建立的ELISA方法可用于牛口蹄疫感染和免疫抗体检测。
本研究利用实验室融合表达的3B表位串联肽(8BF)为检测抗原建立的间接ELISA为基础,优化组装了一种特异、敏感的间接ELISA试剂盒。通过检测大量未免疫、免疫健康牛血清和感染牛血清,确定了判定标准:S/P≥0.3,阳性;0.2≤S/P<0.3,可疑;S/P<0.2,阴性。8BF-ELISA试剂盒检测NSP抗体持续期试验表明,自然感染牛3B表位肽感染后10个月仍为阳性。与3种商品化试剂盒比较检测101份临床样本,和Ceditest? FMDV-NS ELISA、3ABC-I-ELISA符合率高达95%;与3ABC-I-ELISA进一步比较检测528份不同背景的牛血清,总符合率为94.5%(499/528)。大规模检测不同来源的6个牛场共2026份临床牛血清,部分牛群全部阴性,大多数牛群含有阳性牛,阳性率从3%到17%不等,个别感染牛群阳性率高达72.2%。8BF-ELISA试剂盒方便、安全、实用,特异性和敏感性高,用于鉴别诊断牛FMD自然感染和疫苗免疫,为FMD综合防控提供了技术支持。
Foot-and-mouth disease (FMD) caused by foot-and-mout disease virus (FMDV) is a highly contagious and acutely infected disease of cattle, pigs, sheep, goats and wild ruminant species. It has an economically devastating impact on affected countries, where it creates great losses to productivity and considerable economic losses to the husbandry industry. Whenever the disease has happened; it is must reported to the OIE. Since the Asia 1 FMDV first reported in 2005 in china, there are several outbreaks with the Asia 1, O and A serotypes. There is a complex policy to control the FMD mainly by the preventive vaccination in china, where the most animals are vaccinated 2-4 times every year. So the accurate and in-time diagnosis and routine surveillance become very important for the disease control. Thus, diagnostic methods play an important role for FMD control.
The complete gene encoding the structural protein VP2 of FMDV was subcloned into expression vector pPROEXTM HTb and expressed in DH5αcells. Purified VP2 protein reacted positively with serotype-specific cattle sera of 5 serotypes of FMDV (O, A, C, SAT 2 and Asia 1) by western blot. An indirect ELISA (VP2-ELISA) was developed using purified protein to detect FMDV antibodies in cattles. The protein had no cross-reaction with the positive sera of other bovine diseases. The specificity was 100% to detect the negative cattle serum from Canada where was free of FMD without vaccination. The sensitivity was 97.3% to detect infected sera. Comparison with four commercial kit showed a coincidence rate of 69.0%、95.0%、90.4% and 86.8% against vaccinated cattle serum, respectively. Thus the VP2-ELISA can be used to detect the infected and vaccinated cattle serum.
To differentiate antibodies induced by FMDV infection from those induced by vaccination, an indirect ELISA was established then the kit was developed, using purified tandem epitopes of 3B protein as antigen. A large number of sera from naive, vaccinated and infected cattle were examined for determination of the cut-off value of the method. The cut-off value was established as follows: positive, S/P≥0.3; suspicious, 0.2≤value<0.3; negative, S/P <0.2. Seroreactivity to 8BF in some infected cattle was maintained through 10 months. The rate of agreement with Ceditest? FMDV-NS ELISA and the 3ABC-I-ELISA was up of 95% by detection of 101 field cattle serum. The performance of this assay was further validated by 3ABC-I-ELISA kits. The coincident rate was 94.5% (499/528), respectively. The number of 2026 cattle sera derived from with different situations (na?ve, unknown, immune and infectious status) were detected. The prevalence of 8BF antibodies reached 72.2% in some diseased cattle herds. The described 8BF-ELISA is safe, cheap and also easy to perform in large scale of serological surveys. The high specificity and sensitivity makes this test a good tool for better distinguishing between infected and vaccinated cattle.
引文
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