猪圆环病毒1型和2型血清抗体鉴别诊断技术的研究
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摘要
猪圆环病毒分为2个血清型,猪圆环病毒1型(Porcine circovirus type 1, PCV1)和猪圆环病毒2型(Porcine circovirus type 2, PCV2)。PCV1对猪无致病性,但是在猪群中广泛检测到其血清抗体。PCV2具有致病性,可以引起免疫抑制,从而并发多种疾病,如仔猪断奶后多系统衰竭综合征、猪皮炎肾炎综合征、繁殖和呼吸障碍等。PCV1和PCV2的ORF1同源性可以达到86%,存在血清学交叉反应,而PCV1感染常与PCV2伴发,因此PCV1和PCV2感染的鉴别诊断方法尤为重要。本研究通过建立PCV1-IPMA诊断方法,利用PCV1和PCV2重组Cap蛋白制备单克隆抗体,应用单抗建立检测PCV2中和抗体的阻断ELISA方法可以从血清学上鉴别诊断PCV1和PCV2感染。以上研究为猪圆环病毒感染的流行病学调查、疫苗免疫效果的评价、抗原表位的研究等方面提供了一定的技术基础。
本研究建立了一种免疫过氧化物酶单层细胞试验(Immunoperoxidase monolayer assay,IPMA)用于PCV1血清抗体的检测。对该方法的反应条件、特异性、敏感性及重复性等进行了系统试验,并与昆虫杆状病毒表达的PCV1-rCap蛋白建立的间接ELISA检测进行比较。结果显示,用IPMA法对已知PCV1和PCV2参考阳性血清进行试验表明,血清稀释倍数≥1:100可消除两种病毒间存在的低水平交叉反应,与其它几种猪病毒参考阳性血清无交叉反应。该方法与用昆虫杆状病毒表达的PCV1-rCap蛋白建立的间接ELISA检测符合率为92.6%。对来自黑龙江、吉林、河北、上海、辽宁等地猪场送检的257份血清样品进行检测,PCV1和PCV2血清抗体阳性检出率分别为19.1%和80.5%;PCV1血清抗体阳性猪中有95.9%为PCV2阳性,表明我国猪群已存在PCV1感染,在临床上PCV1与PCV2多以混合感染形式存在。本研究建立的PCV1-IPMA方法具有操作简便、特异和敏感等特点,可为PCV1流行病学调查及血清抗体检测提供了一种技术手段。
利用重组杆状病毒表达PCV1-rCap蛋白(rBac/PCV1-Cap),对重组蛋白表达参数、纯化工艺及免疫活性进行了研究。PCV1-rCap蛋白表达量占总蛋白的20.7%。利用包涵体纯化方法获得重组蛋白的纯度为85.1%,浓度为1.123 mg/mL。Western-blot分析证实,该重组蛋白可被PCV1特异性抗体所识别,具有良好的免疫活性。以纯化的PCV1-rCap和PCV2-rCap免疫兔制备高免血清,经过PCV1-IPMA和PCV2-IPMA检测结果表明,两种病毒Cap蛋白抗原无血清学交叉反应,它们可作为PCV1和PCV2血清学鉴别诊断的理想靶抗原。
以纯化的杆状病毒表达的重组PCV1-Cap蛋白(PCV1-rCap)和重组PCV2-Cap蛋白(PCV2-rCap)分别免疫BALB/c小鼠,利用淋巴细胞杂交瘤技术分别获得了4株稳定分泌抗PCV1-rCap蛋白和5株抗PCV2-rCap蛋白的杂交瘤细胞株,分别命名为2C10、3A8、3F7和5D1及1D2、2E8、3A10、5F2和6F10。这9株杂交瘤细胞培养上清和诱导同品系小鼠产生的腹水抗体效价分别为1:320～1:10240和1: 320000～1:5120000。抗PCV1-rCap单克隆抗体2C10和3A8亚类为IgG1, 3F7为IgM, 5D1为IgG3;以上单克隆抗体轻链均为κ型。抗PCV2-rCap单克隆抗体1D2、2E8、5F2和6F10亚类为IgG2a,仅3A10为IgG1;2E8、3A10、5F2和6F10轻链为λ型,仅1D2轻链为κ型。Western blot结果显示,抗PCV1-rCap单克隆抗体均可以与自然PCV1-Cap发生反应;抗PCV2-rCap单克隆抗体中2E8、3A10、5F2和6F10可与自然PCV2-Cap发生反应,而1D2无反应。IPMA和抗原捕获ELISA结果表明,抗PCV2-rCap单克隆抗体1D2可与PCV2发生反应,而与PCV1无交叉反应;病毒中和试验证实,1D2具有中和活性,是一株针对PCV2-Cap蛋白中和表位的单克隆抗体。本试验制备的单克隆抗体为猪圆环病毒抗原表位分析及鉴别诊断提供了技术手段。
采用淋巴细胞杂交瘤技术,获得了一株具有分泌抗PCV2中和活性的单克隆抗体,利用这株单抗建立了一种阻断ELISA方法,用于检测该病毒感染猪的血清中和抗体。该方法先用病毒多克隆抗体包被ELISA板以捕获已知病毒抗原,然后与待检猪血清抗体及辣根过氧化物酶标记的单抗进行反应,通过测定酶标单抗显示阻断ELISA检测结果。该方法与IPMA对353份试验猪血清样品进行了平行试验,两种方法检测符合率为96.0%;该方法的敏感性为97.9%,特异性为92.4%,与其它几种猪病毒参考阳性血清无交叉反应。用阻断ELISA和血清中和试验检测PCV2血清中和抗体结果表明,两种方法呈显著正相关性(r=0.9970),其敏感性优于血清中和试验。用阻断ELISA对PCV2灭活疫苗免疫与攻毒试验猪的血清样品检测结果显示,疫苗接种2 w开始能够检测到病毒中和抗体,4 w后血清抗体阳转率达100%;此外,对来自东北3个省不同地区送检的703份血清样品进行检测,抗体阳性检出率为73.4%,表明我国养猪场中PCV2感染率较高,该病毒危害日益严重。本研究建立的阻断ELISA具有操作简便、特异和敏感等特点,可为PCV2流行病学调查及血清抗体检测提供了一种有效技术手段。
Porcine circovirus (PCV) has two serotypes. One is porcine circovirus type 1 (PCV1) which is nonpathogenic. Another is porcine circovirus type 2 (PCV2) which can induce immunosuppression and is considered to be an important emerging pathogen associated with a number of di?erent syndromes and diseases in pigs such as postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex, reproductive failure, and so on. There are cross-reactivity due to 85% homology in ORF1 between PCV1 and PCV2. In addition, PCV1 and PCV2 as a mixture infection emerge in the field condition. It is important to develop differential diagnosis methods for PCVs. An immunoperoxidase monolayer assay (IPMA) and a blocking ELISA based on a monoclonal antibody (McAb) for neutralization antibody detection of PCV2 were developed and validated in this study. The two assays offer some convenient tools for epidemiology survey and vaccine evaluation. McAbs also can be used for antigen epitope analysis of PCVs.
An IPMA was developed for antibody detection of PCV1 in the study. The reaction, specificity, sensitivity, and repeatability of IPMA were optimized and evaluated, and compared with ELISA using baculovirus-expressed PCV1 recombinant capsid protein as antigen. In results, different dilution of PCV1 and PCV2 positive reference sera were assayed by the IPMA, and there was a low level antibody cross-reaction between two viruses could be eliminated by serum dilution more than 1:100. There was no cross-reaction with reference sera against other porcine viruses. In comparison, 92.6% agreement rate was obtained between IPMA and an ELISA using baculovirus-expressed PCV1 recombinant capsid protein as antigen. Total 257 serum samples from Heilongjiang, Jilin, Hebei, Shanghai, Liaoning provinces were detected using PCV1-IPMA and PCV2-IPMA. The positive rate was 19.1% and 80.5% for PCV1 and PCV2, respectively. About 95.9% of PCV1-positive samples belong to PCV2-pisitive either. These data demonstrates that these pig herds has contaminated with PCV1. PCV1 and PCV2 as a mixture infection emerge under the clinic condition. PCV1-IPMA offers a convenient tool for epidemiology survey and evaluation of antibodies of the virus.
PCV1 recombinant Cap (PCV1-rCap) protein was expressed by recombinant baculovirus (rBac/PCV1-Cap). PCV1-rCap protein expression parameters, purification and immunological activity were studied. The recombinant protein yield was about 20.7% of the total proteins after optimizing the expression. PCV1-rCap protein was purified by inclusion body purification method, the purity and density reached 85.1% and 1.123 mg/mL. The recombinant protein was identified by Western blot using the virus-specific antibodies, and had good immunoreactivity. The rabbit serum against PCV1-rCap and PCV2-rCap were produced and could be detected by IPMA. As a result, there was no cross reaction between above anti-serum. No cross reaction between the two Cap of PCV1 and PCV2 was concluded. Therefore, Cap was considered as a target protein in the studies of differential diagnosis between porcine circovirus type 1 and 2.
BALB/c mice were immunized using the PCV1-rCap and PCV2-rCap prepared by baculovirus expression system. Nine McAbs, named as 2C10, 3A8, 3F7, 5D1 and 1D2, 2E8, 3A10, 5F2, 6F10 to PCV1-rCap and PCV2-rCap were prepared by lymphocyte hybridoma technique. Nine McAbs supernatant and ascites titers were 1:320～1:10240 and 1: 320000～1:5120000. The McAbs of PCV1 subclass belonged to IgG1 for 2C10 and 3A8, IgM for 3F7, IgG3 for 5D1, and all of McAbs light chains belonged toκchain. The McAbs of PCV2 subclass belonged to IgG2a for 1D2, 2E8, 5F2 and 6F10; and only IgG1 for 3A10. The McAb light chains belonged toλchain for 2E8, 3A10, 5F2 and 6F10, and onlyκchain for 1D2. Western blot analysis showed that all of McAbs to PCV1 were able to react with native PCV1-Cap. McAbs of 2E8, 3A10, 5F2 and 6F10 were able to react with native PCV2-Cap, but not for 1D2. However, the McAb of 1D2 was able react with PCV2 by IPMA and antigen capture ELISA, and it could be differentiated with PCV1 at the same condition. The McAb of 1D2 was confirmed to have the neutralizing ability to PCV2 by neutralization test. These McAbs which to the PCVs-rCap were obtained in the study would be applied in the differential diagnostic method and antigen epitope analysis of the virus.
A blocking ELISA was developed for detecting neutralization antibodies against PCV2 based on a McAb to the virus, which was prepared by lymphocyte hybridoma technique. The virus antigens were captured by PCV2 specific positive sera as coating antigen and reacted with the sera antibodies and peroxidase from horseradish (HRP)-labeled with the McAb, respectively. The results were developed by detection of the HRP-labeled McAb. In comparison with IPMA, there was 96.0% coincidence between the blocking ELISA and IPMA based on detection of 353 serum samples. The sensitivity and specificity of the blocking ELISA were 97.9% and 92.4% and showed no cross-reactivity with the reference sera of other swine viruses. The results of detection PCV2 neutralization antibodies in serum by blocking ELISA and serum neutralization test(SNT) show that the two methods were significant positive correlation (r=0.9970). The blocking ELISA was more sensitive than SNT. Neutralization antibodies could be detected from the vaccinated swine sera with PCV2 inactivated vaccine after two weeks, 100% positive after four weeks. Test of 703 serum samples collected from the provinces of Heilongjiang, Jilin, Liaoning showed 73.4% positive rates. These data demonstrate that pig herds in China have been contaminated heavily with PCV2. Thus, the blocking ELISA offers a convenient tool for epidemiology survey and evaluation of neutralization antibodies of PCV2.
本研究建立了一种免疫过氧化物酶单层细胞试验(Immunoperoxidase monolayer assay,IPMA)用于PCV1血清抗体的检测。对该方法的反应条件、特异性、敏感性及重复性等进行了系统试验,并与昆虫杆状病毒表达的PCV1-rCap蛋白建立的间接ELISA检测进行比较。结果显示,用IPMA法对已知PCV1和PCV2参考阳性血清进行试验表明,血清稀释倍数≥1:100可消除两种病毒间存在的低水平交叉反应,与其它几种猪病毒参考阳性血清无交叉反应。该方法与用昆虫杆状病毒表达的PCV1-rCap蛋白建立的间接ELISA检测符合率为92.6%。对来自黑龙江、吉林、河北、上海、辽宁等地猪场送检的257份血清样品进行检测,PCV1和PCV2血清抗体阳性检出率分别为19.1%和80.5%;PCV1血清抗体阳性猪中有95.9%为PCV2阳性,表明我国猪群已存在PCV1感染,在临床上PCV1与PCV2多以混合感染形式存在。本研究建立的PCV1-IPMA方法具有操作简便、特异和敏感等特点,可为PCV1流行病学调查及血清抗体检测提供了一种技术手段。
利用重组杆状病毒表达PCV1-rCap蛋白(rBac/PCV1-Cap),对重组蛋白表达参数、纯化工艺及免疫活性进行了研究。PCV1-rCap蛋白表达量占总蛋白的20.7%。利用包涵体纯化方法获得重组蛋白的纯度为85.1%,浓度为1.123 mg/mL。Western-blot分析证实,该重组蛋白可被PCV1特异性抗体所识别,具有良好的免疫活性。以纯化的PCV1-rCap和PCV2-rCap免疫兔制备高免血清,经过PCV1-IPMA和PCV2-IPMA检测结果表明,两种病毒Cap蛋白抗原无血清学交叉反应,它们可作为PCV1和PCV2血清学鉴别诊断的理想靶抗原。
以纯化的杆状病毒表达的重组PCV1-Cap蛋白(PCV1-rCap)和重组PCV2-Cap蛋白(PCV2-rCap)分别免疫BALB/c小鼠,利用淋巴细胞杂交瘤技术分别获得了4株稳定分泌抗PCV1-rCap蛋白和5株抗PCV2-rCap蛋白的杂交瘤细胞株,分别命名为2C10、3A8、3F7和5D1及1D2、2E8、3A10、5F2和6F10。这9株杂交瘤细胞培养上清和诱导同品系小鼠产生的腹水抗体效价分别为1:320～1:10240和1: 320000～1:5120000。抗PCV1-rCap单克隆抗体2C10和3A8亚类为IgG1, 3F7为IgM, 5D1为IgG3;以上单克隆抗体轻链均为κ型。抗PCV2-rCap单克隆抗体1D2、2E8、5F2和6F10亚类为IgG2a,仅3A10为IgG1;2E8、3A10、5F2和6F10轻链为λ型,仅1D2轻链为κ型。Western blot结果显示,抗PCV1-rCap单克隆抗体均可以与自然PCV1-Cap发生反应;抗PCV2-rCap单克隆抗体中2E8、3A10、5F2和6F10可与自然PCV2-Cap发生反应,而1D2无反应。IPMA和抗原捕获ELISA结果表明,抗PCV2-rCap单克隆抗体1D2可与PCV2发生反应,而与PCV1无交叉反应;病毒中和试验证实,1D2具有中和活性,是一株针对PCV2-Cap蛋白中和表位的单克隆抗体。本试验制备的单克隆抗体为猪圆环病毒抗原表位分析及鉴别诊断提供了技术手段。
采用淋巴细胞杂交瘤技术,获得了一株具有分泌抗PCV2中和活性的单克隆抗体,利用这株单抗建立了一种阻断ELISA方法,用于检测该病毒感染猪的血清中和抗体。该方法先用病毒多克隆抗体包被ELISA板以捕获已知病毒抗原,然后与待检猪血清抗体及辣根过氧化物酶标记的单抗进行反应,通过测定酶标单抗显示阻断ELISA检测结果。该方法与IPMA对353份试验猪血清样品进行了平行试验,两种方法检测符合率为96.0%;该方法的敏感性为97.9%,特异性为92.4%,与其它几种猪病毒参考阳性血清无交叉反应。用阻断ELISA和血清中和试验检测PCV2血清中和抗体结果表明,两种方法呈显著正相关性(r=0.9970),其敏感性优于血清中和试验。用阻断ELISA对PCV2灭活疫苗免疫与攻毒试验猪的血清样品检测结果显示,疫苗接种2 w开始能够检测到病毒中和抗体,4 w后血清抗体阳转率达100%;此外,对来自东北3个省不同地区送检的703份血清样品进行检测,抗体阳性检出率为73.4%,表明我国养猪场中PCV2感染率较高,该病毒危害日益严重。本研究建立的阻断ELISA具有操作简便、特异和敏感等特点,可为PCV2流行病学调查及血清抗体检测提供了一种有效技术手段。
Porcine circovirus (PCV) has two serotypes. One is porcine circovirus type 1 (PCV1) which is nonpathogenic. Another is porcine circovirus type 2 (PCV2) which can induce immunosuppression and is considered to be an important emerging pathogen associated with a number of di?erent syndromes and diseases in pigs such as postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex, reproductive failure, and so on. There are cross-reactivity due to 85% homology in ORF1 between PCV1 and PCV2. In addition, PCV1 and PCV2 as a mixture infection emerge in the field condition. It is important to develop differential diagnosis methods for PCVs. An immunoperoxidase monolayer assay (IPMA) and a blocking ELISA based on a monoclonal antibody (McAb) for neutralization antibody detection of PCV2 were developed and validated in this study. The two assays offer some convenient tools for epidemiology survey and vaccine evaluation. McAbs also can be used for antigen epitope analysis of PCVs.
An IPMA was developed for antibody detection of PCV1 in the study. The reaction, specificity, sensitivity, and repeatability of IPMA were optimized and evaluated, and compared with ELISA using baculovirus-expressed PCV1 recombinant capsid protein as antigen. In results, different dilution of PCV1 and PCV2 positive reference sera were assayed by the IPMA, and there was a low level antibody cross-reaction between two viruses could be eliminated by serum dilution more than 1:100. There was no cross-reaction with reference sera against other porcine viruses. In comparison, 92.6% agreement rate was obtained between IPMA and an ELISA using baculovirus-expressed PCV1 recombinant capsid protein as antigen. Total 257 serum samples from Heilongjiang, Jilin, Hebei, Shanghai, Liaoning provinces were detected using PCV1-IPMA and PCV2-IPMA. The positive rate was 19.1% and 80.5% for PCV1 and PCV2, respectively. About 95.9% of PCV1-positive samples belong to PCV2-pisitive either. These data demonstrates that these pig herds has contaminated with PCV1. PCV1 and PCV2 as a mixture infection emerge under the clinic condition. PCV1-IPMA offers a convenient tool for epidemiology survey and evaluation of antibodies of the virus.
PCV1 recombinant Cap (PCV1-rCap) protein was expressed by recombinant baculovirus (rBac/PCV1-Cap). PCV1-rCap protein expression parameters, purification and immunological activity were studied. The recombinant protein yield was about 20.7% of the total proteins after optimizing the expression. PCV1-rCap protein was purified by inclusion body purification method, the purity and density reached 85.1% and 1.123 mg/mL. The recombinant protein was identified by Western blot using the virus-specific antibodies, and had good immunoreactivity. The rabbit serum against PCV1-rCap and PCV2-rCap were produced and could be detected by IPMA. As a result, there was no cross reaction between above anti-serum. No cross reaction between the two Cap of PCV1 and PCV2 was concluded. Therefore, Cap was considered as a target protein in the studies of differential diagnosis between porcine circovirus type 1 and 2.
BALB/c mice were immunized using the PCV1-rCap and PCV2-rCap prepared by baculovirus expression system. Nine McAbs, named as 2C10, 3A8, 3F7, 5D1 and 1D2, 2E8, 3A10, 5F2, 6F10 to PCV1-rCap and PCV2-rCap were prepared by lymphocyte hybridoma technique. Nine McAbs supernatant and ascites titers were 1:320～1:10240 and 1: 320000～1:5120000. The McAbs of PCV1 subclass belonged to IgG1 for 2C10 and 3A8, IgM for 3F7, IgG3 for 5D1, and all of McAbs light chains belonged toκchain. The McAbs of PCV2 subclass belonged to IgG2a for 1D2, 2E8, 5F2 and 6F10; and only IgG1 for 3A10. The McAb light chains belonged toλchain for 2E8, 3A10, 5F2 and 6F10, and onlyκchain for 1D2. Western blot analysis showed that all of McAbs to PCV1 were able to react with native PCV1-Cap. McAbs of 2E8, 3A10, 5F2 and 6F10 were able to react with native PCV2-Cap, but not for 1D2. However, the McAb of 1D2 was able react with PCV2 by IPMA and antigen capture ELISA, and it could be differentiated with PCV1 at the same condition. The McAb of 1D2 was confirmed to have the neutralizing ability to PCV2 by neutralization test. These McAbs which to the PCVs-rCap were obtained in the study would be applied in the differential diagnostic method and antigen epitope analysis of the virus.
A blocking ELISA was developed for detecting neutralization antibodies against PCV2 based on a McAb to the virus, which was prepared by lymphocyte hybridoma technique. The virus antigens were captured by PCV2 specific positive sera as coating antigen and reacted with the sera antibodies and peroxidase from horseradish (HRP)-labeled with the McAb, respectively. The results were developed by detection of the HRP-labeled McAb. In comparison with IPMA, there was 96.0% coincidence between the blocking ELISA and IPMA based on detection of 353 serum samples. The sensitivity and specificity of the blocking ELISA were 97.9% and 92.4% and showed no cross-reactivity with the reference sera of other swine viruses. The results of detection PCV2 neutralization antibodies in serum by blocking ELISA and serum neutralization test(SNT) show that the two methods were significant positive correlation (r=0.9970). The blocking ELISA was more sensitive than SNT. Neutralization antibodies could be detected from the vaccinated swine sera with PCV2 inactivated vaccine after two weeks, 100% positive after four weeks. Test of 703 serum samples collected from the provinces of Heilongjiang, Jilin, Liaoning showed 73.4% positive rates. These data demonstrate that pig herds in China have been contaminated heavily with PCV2. Thus, the blocking ELISA offers a convenient tool for epidemiology survey and evaluation of neutralization antibodies of PCV2.
引文
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