基于口蹄疫病毒非结构蛋白3AB的ELISA鉴别检测方法的研究
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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)引起的偶蹄动物共患的、接触性传染病。FMDV有七种血清型、七十多种亚型,变异性强,给病毒的防治造成很大困难。它可导致国家的畜产品进出口贸易受限,造成巨大的经济损失和政治影响。因此,建立准确、快捷的区分感染活病毒(发病或注射弱毒疫苗)和注射灭活疫苗动物的诊断方法,在检测和防止FMD的发生、活畜检疫等方面都是十分迫切需要的。
以口蹄疫非结构蛋白3AB作为抗原的ELISA,适用于鉴别诊断感染和注苗动物。以3ABC基因片断为模板,经PCR扩增得到了684bp的3AB基因片断,与pET32а(+)连接后,转化宿主菌BL21(DE3) plysS,待菌体浓度为0.8OD550时,IPTG诱导表达目的蛋白。SDS-PAGE和Western Blot结果表明,3AB基因得到了正确表达,表达的重组3AB蛋白为非溶性融合蛋白,分子量约为50Kda。ELISA结果显示,重组3AB蛋白可用于猪、牛口蹄疫病毒感染与疫苗免疫抗体的鉴别诊断。
重组口蹄疫病毒非结构蛋白3AB作为检测抗原包被酶标板反应孔,以辣根过氧化物酶标记的重组蛋白A/G作为第二抗体,建立了抗非结构蛋白抗体检测的方法。结果表明,在1746份免疫猪血清中,3AB-ELISA对免疫猪血清的特异性为98.68%;在1077份健康非免疫猪血清中,3AB-ELISA对健康非免疫猪血清的特异性为97.96%;在36份人工感染猪血清中,3AB-ELISA对阳性检出率为100%。而牛特异性的各项指标分别为94.04%,97.96%和阳性检出率为100%。
进一步改进了3AB-ELISA检测方法,使用了脱脂奶粉和大肠杆菌裂解液做为该方法的阻断剂,有效降低了背景值,消除了非特异性反应。采用TMB作显色剂,对检测环境的要求比OPD的更宽,使该检测方法的实用性更好。同时,3AB-ELISA与国外同类试剂盒比较,对阳性牛血清检出率,3AB-ELISA的阳性率为91.7%,UBI试剂盒为79.2%,高出UBI试剂盒12.5个百分点。对阴性牛血清检测率,两种试剂盒几乎一致。
Foot and Mouth Disease (FMD) virus by the foot and mouth disease (FMDV) from cloven-hoofed animals were suffering from the reach of sexually transmitted diseases. There are seven FMDV serotypes, more than 70 kinds of subtypes, high variability, and to prevent the virus have caused great problems. It can lead to the import and export of animal products trade restricted, causing huge economic losses and political influence between countries. Establish an accurate and fast distinction between infected live virus vaccine and injected animals in the detection and diagnosis of FMD control the incidence of livestock quarantine areas are very urgently needed.
The ELISA of using antigen of Non-structural protein 3AB in Foot-and-mouth disease virus can be used for differentiation of infection from vaccination. The 3AB gene fragment amplified by PCR from the template of 3ABC gene fragment was inserted into pET32а(+). The recombinant plasmid pET3AB was transferred into BL21(DE3) plysS and the Target protein was induced by IPTG with 0.8OD550. The SDS-PAGE and Western Blot results showed the recombinant plasmid pET3AB was constructed successfully and the NS-3AB was expressed strictly. The NS-3AB is unsoluble .The MW of NS-3AB is about 50Kda. the ELISA result indicated that the NSP-3AB can be used as antigen to differentiation FMDV-infected from vaccinated pig and cattle.
NSP-3AB ELISA were developed using the recombinant Non-structural protein 3AB as detecting antigen. It can diagnose if the detected animal infected FMDV. Detecting antigen based on the non-structural protein 3AB was coated onto reaction microplate wells, recombined protein A/G marked with horseradish peroxide enzyme was used as the second antibody, the anti-non-structural protein antibody detecting method was established. Specific of NSP-3AB ELISA was 96.68% in 1,746 immunity swine serum; it was 97.96% in 1,077 health swine serum; its positive rate was 100% for 36 swine serum infected FMDV by man. However, the specific of bovine serum was respectively 94.04%, 97.96%, 100%.
Further improve the 3AB-ELISA detection method, using skim milk and E. coli lysate as the method of blocking agent, effectively reducing the background value and eliminate non-specific response. TMB used for the color of the testing environment than the requirements of the OPD wider to enable the detection of practicality better.The result of Comparison between 3AB-ELISA and similar foreign kit, the detection rate of positive bovine serum, 3AB-ELISA the positive rate was 91.7%, UBI kit was 79.2%, higher than the UBI kit 12.5 percentage points. On the negative bovine serum testing, they are almost unanimous.
以口蹄疫非结构蛋白3AB作为抗原的ELISA,适用于鉴别诊断感染和注苗动物。以3ABC基因片断为模板,经PCR扩增得到了684bp的3AB基因片断,与pET32а(+)连接后,转化宿主菌BL21(DE3) plysS,待菌体浓度为0.8OD550时,IPTG诱导表达目的蛋白。SDS-PAGE和Western Blot结果表明,3AB基因得到了正确表达,表达的重组3AB蛋白为非溶性融合蛋白,分子量约为50Kda。ELISA结果显示,重组3AB蛋白可用于猪、牛口蹄疫病毒感染与疫苗免疫抗体的鉴别诊断。
重组口蹄疫病毒非结构蛋白3AB作为检测抗原包被酶标板反应孔,以辣根过氧化物酶标记的重组蛋白A/G作为第二抗体,建立了抗非结构蛋白抗体检测的方法。结果表明,在1746份免疫猪血清中,3AB-ELISA对免疫猪血清的特异性为98.68%;在1077份健康非免疫猪血清中,3AB-ELISA对健康非免疫猪血清的特异性为97.96%;在36份人工感染猪血清中,3AB-ELISA对阳性检出率为100%。而牛特异性的各项指标分别为94.04%,97.96%和阳性检出率为100%。
进一步改进了3AB-ELISA检测方法,使用了脱脂奶粉和大肠杆菌裂解液做为该方法的阻断剂,有效降低了背景值,消除了非特异性反应。采用TMB作显色剂,对检测环境的要求比OPD的更宽,使该检测方法的实用性更好。同时,3AB-ELISA与国外同类试剂盒比较,对阳性牛血清检出率,3AB-ELISA的阳性率为91.7%,UBI试剂盒为79.2%,高出UBI试剂盒12.5个百分点。对阴性牛血清检测率,两种试剂盒几乎一致。
Foot and Mouth Disease (FMD) virus by the foot and mouth disease (FMDV) from cloven-hoofed animals were suffering from the reach of sexually transmitted diseases. There are seven FMDV serotypes, more than 70 kinds of subtypes, high variability, and to prevent the virus have caused great problems. It can lead to the import and export of animal products trade restricted, causing huge economic losses and political influence between countries. Establish an accurate and fast distinction between infected live virus vaccine and injected animals in the detection and diagnosis of FMD control the incidence of livestock quarantine areas are very urgently needed.
The ELISA of using antigen of Non-structural protein 3AB in Foot-and-mouth disease virus can be used for differentiation of infection from vaccination. The 3AB gene fragment amplified by PCR from the template of 3ABC gene fragment was inserted into pET32а(+). The recombinant plasmid pET3AB was transferred into BL21(DE3) plysS and the Target protein was induced by IPTG with 0.8OD550. The SDS-PAGE and Western Blot results showed the recombinant plasmid pET3AB was constructed successfully and the NS-3AB was expressed strictly. The NS-3AB is unsoluble .The MW of NS-3AB is about 50Kda. the ELISA result indicated that the NSP-3AB can be used as antigen to differentiation FMDV-infected from vaccinated pig and cattle.
NSP-3AB ELISA were developed using the recombinant Non-structural protein 3AB as detecting antigen. It can diagnose if the detected animal infected FMDV. Detecting antigen based on the non-structural protein 3AB was coated onto reaction microplate wells, recombined protein A/G marked with horseradish peroxide enzyme was used as the second antibody, the anti-non-structural protein antibody detecting method was established. Specific of NSP-3AB ELISA was 96.68% in 1,746 immunity swine serum; it was 97.96% in 1,077 health swine serum; its positive rate was 100% for 36 swine serum infected FMDV by man. However, the specific of bovine serum was respectively 94.04%, 97.96%, 100%.
Further improve the 3AB-ELISA detection method, using skim milk and E. coli lysate as the method of blocking agent, effectively reducing the background value and eliminate non-specific response. TMB used for the color of the testing environment than the requirements of the OPD wider to enable the detection of practicality better.The result of Comparison between 3AB-ELISA and similar foreign kit, the detection rate of positive bovine serum, 3AB-ELISA the positive rate was 91.7%, UBI kit was 79.2%, higher than the UBI kit 12.5 percentage points. On the negative bovine serum testing, they are almost unanimous.
引文
[1] 谢庆阁,赵启祖,江鹏斐等.口蹄疫研究进展.中国农业科学.1999,32(6),93-100.
[2] 卢永干.用口蹄疫非结构蛋白鉴别康复与注射疫苗动物的研究及应用.中国兽医科技.2000,30(10),18-20.
[3] 江鹏斐,刘在新,赵启祖等.口蹄疫病毒2C基因的克隆与表达.畜牧兽医学报.2001,32(3),231-234.
[4] 潘文波,徐海聂,廖秀云等.口蹄疫合成肽酶联免疫吸附试验试剂盒在口蹄疫抗体检测中的应用.中国动物检疫,2001,18(12),41-43.
[5] 尤永进,朱彩珠,葛艳等.重组口蹄疫病毒非结构蛋白 3ABC 基因的克隆与表达.中国病毒学,2003,18(2),155-158.
[6] 尤永进,朱彩珠,葛艳等.重组口蹄疫病毒非结构蛋白 3ABC 基因的诱导表达及表达蛋白的纯化.中国预防兽医学报.2002,24(Suppl),175-177.
[7] 朱彩珠,张强,常惠芸等.NSP-ELISA 鉴别 FMDV 感染与免疫.中国兽医科技.2003,33(8),3-6.
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[10] 邱德新,陈焕春,何启盖等. 间接 ELISA 检测猪伪狂犬病血清抗体[J]. 中国兽医学报 2002,22(2),149~152.
[11] 王宏伟,孙明,田克恭等.口蹄疫病毒 3ABC、3AB、3BC 基因克隆、表达及其抗体消长规律的研究.中国农业科学.2004,37(7),1065-1070.
[12] Lane D 编著,沈关心,龚非力译 . 抗体技术实验指南 . 北京科学出版社.2002,165-177.
[13] 阮力,钱平,何启盖等.口蹄疫病毒非结构蛋白 3B 基因的克隆表达及 3B-ELISA鉴别诊断方法的初步建立.中国兽医学报.2005,5,68-69.
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[15] 杨汉春.动物免疫学.中国农业大学出版社.1996,305-306.
[16] 杨柳 李康然.欧洲使用疫苗接种控制口蹄疫.广西畜牧兽医.2003,6,86-87.
[17] 谢庆阁.口蹄疫.中国农业出版社.2004,174-181.
[18] Doel TR, T Collen. Qualitative assessment of 146S paticles of FMDV in preparations destined for vaccines. J Biol Stand, 1982, 10,69-81.
[19] Mackay DKJ, Forsyth MA, Davies PR, etc. Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant,non-structural proteins in ELISA. Vaccine, 1998 16,446-459.
[20] Geering WA. Food and Agricultural Organization of the United Nations(FAO).The disease and their Diagnosis,FAO,Rome,Italy, 1984,43-51.
[21] Roeder PL, Le Blanc Smith PM. The detection and typing of footand-mouth disease virus by enzyme-linked immunosorbent assay,a sensitive,rapid and reliable technique for primary diagnosis, Res Vet Sci.1987, 43,225-232.
[22] De Diego M, Brocchi E, Mackay D, etc. The non-structural polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in ELISA to differentatiate infected from vaccinated cattle, Arch Virol, 1997 ,142,2021-2033.
[23] Silberstein E,Kaplan G,Taboga O et al. Foot and mouth disease virus-infected but not vaccinated cattle develop antibodies against recombinant 3 AB1 nonstructural protein.Arch Virol,1997,142,795-805.
[24] Shen F,Chen PD, Walfield AM, etc. Differentiation of convalescent animals from those vaccinated against foot and mouth disease by a peptide ELISA. Vaccine, 1999,17,3039-3049.
[25] Meyer RF,Babcock GD, Newman JFE etc. Baculovirus expressed 2C of foot and mouth disease virus has the potential for differentiating conva-lescent from vaccinated animals. Virol Methods, 1997,65,33-43.
[26] Doel TR,Williams L, Bamett PV, etc. Emergency vaccination against foot-and-mouth disease rate of development of immunity and its implications foe the carrier state, Vaccine,1994,12,592-600
[27] Barnett PV,Pullen L, Willians L etc. International bank for foot-and-mouth disease vaccine,assenssment of Montanide ISA 25 and ISA 206,two commercially available oil adjuvants.Vaccine,1996,9,75-88.
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[2] 卢永干.用口蹄疫非结构蛋白鉴别康复与注射疫苗动物的研究及应用.中国兽医科技.2000,30(10),18-20.
[3] 江鹏斐,刘在新,赵启祖等.口蹄疫病毒2C基因的克隆与表达.畜牧兽医学报.2001,32(3),231-234.
[4] 潘文波,徐海聂,廖秀云等.口蹄疫合成肽酶联免疫吸附试验试剂盒在口蹄疫抗体检测中的应用.中国动物检疫,2001,18(12),41-43.
[5] 尤永进,朱彩珠,葛艳等.重组口蹄疫病毒非结构蛋白 3ABC 基因的克隆与表达.中国病毒学,2003,18(2),155-158.
[6] 尤永进,朱彩珠,葛艳等.重组口蹄疫病毒非结构蛋白 3ABC 基因的诱导表达及表达蛋白的纯化.中国预防兽医学报.2002,24(Suppl),175-177.
[7] 朱彩珠,张强,常惠芸等.NSP-ELISA 鉴别 FMDV 感染与免疫.中国兽医科技.2003,33(8),3-6.
[8] 张立昌,刘尚高. 口蹄疫免疫学研究进展[J]. 中国兽医科技 1995,25(11),21~22
[9] 江鹏斐,赵启俨,谢庆阁.口蹄疫研究进展[J].中国农业科学,1999,32 ( 6 ), 93 ~ 100.
[10] 邱德新,陈焕春,何启盖等. 间接 ELISA 检测猪伪狂犬病血清抗体[J]. 中国兽医学报 2002,22(2),149~152.
[11] 王宏伟,孙明,田克恭等.口蹄疫病毒 3ABC、3AB、3BC 基因克隆、表达及其抗体消长规律的研究.中国农业科学.2004,37(7),1065-1070.
[12] Lane D 编著,沈关心,龚非力译 . 抗体技术实验指南 . 北京科学出版社.2002,165-177.
[13] 阮力,钱平,何启盖等.口蹄疫病毒非结构蛋白 3B 基因的克隆表达及 3B-ELISA鉴别诊断方法的初步建立.中国兽医学报.2005,5,68-69.
[14] 杜念兴.兽医免疫学.上海科学技术出版社.1989,208-209.
[15] 杨汉春.动物免疫学.中国农业大学出版社.1996,305-306.
[16] 杨柳 李康然.欧洲使用疫苗接种控制口蹄疫.广西畜牧兽医.2003,6,86-87.
[17] 谢庆阁.口蹄疫.中国农业出版社.2004,174-181.
[18] Doel TR, T Collen. Qualitative assessment of 146S paticles of FMDV in preparations destined for vaccines. J Biol Stand, 1982, 10,69-81.
[19] Mackay DKJ, Forsyth MA, Davies PR, etc. Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant,non-structural proteins in ELISA. Vaccine, 1998 16,446-459.
[20] Geering WA. Food and Agricultural Organization of the United Nations(FAO).The disease and their Diagnosis,FAO,Rome,Italy, 1984,43-51.
[21] Roeder PL, Le Blanc Smith PM. The detection and typing of footand-mouth disease virus by enzyme-linked immunosorbent assay,a sensitive,rapid and reliable technique for primary diagnosis, Res Vet Sci.1987, 43,225-232.
[22] De Diego M, Brocchi E, Mackay D, etc. The non-structural polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in ELISA to differentatiate infected from vaccinated cattle, Arch Virol, 1997 ,142,2021-2033.
[23] Silberstein E,Kaplan G,Taboga O et al. Foot and mouth disease virus-infected but not vaccinated cattle develop antibodies against recombinant 3 AB1 nonstructural protein.Arch Virol,1997,142,795-805.
[24] Shen F,Chen PD, Walfield AM, etc. Differentiation of convalescent animals from those vaccinated against foot and mouth disease by a peptide ELISA. Vaccine, 1999,17,3039-3049.
[25] Meyer RF,Babcock GD, Newman JFE etc. Baculovirus expressed 2C of foot and mouth disease virus has the potential for differentiating conva-lescent from vaccinated animals. Virol Methods, 1997,65,33-43.
[26] Doel TR,Williams L, Bamett PV, etc. Emergency vaccination against foot-and-mouth disease rate of development of immunity and its implications foe the carrier state, Vaccine,1994,12,592-600
[27] Barnett PV,Pullen L, Willians L etc. International bank for foot-and-mouth disease vaccine,assenssment of Montanide ISA 25 and ISA 206,two commercially available oil adjuvants.Vaccine,1996,9,75-88.
[28] Doel TR.International bank for foot-and-mouth disease vaccine,stability studies with virus cocentrates and vaccines prepared from them.Vaccine,1990,8,473-478.
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