猪胸膜肺炎放线杆菌感染特异性诊断方法的建立：PCR和夹心ELISA
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摘要
猪传染性胸膜肺炎是由胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae, APP)引起的一种高度接触传染的呼吸道疾病,给集约化养猪业造成严重的经济损失,该病以出血性坏死性肺炎和纤维素性胸膜炎为特征,APP有两个生物型,15个血清型。
猪传染性胸膜肺炎最急性型引致猪的急性死亡,无临床病症慢性感染猪往往是再次爆发和流行的潜在传染源,感染猪易引发其它病原的继发感染。所以APP感染的早期诊断成为防控该病的关键。
APP致病机理非常复杂,有众多毒力因子,Apx(A. pleuropneumoniae-RTX- toxins, Apx)是最重要的毒力因子。其中apxⅣ基因只在活体内表达,体外培养则不表达该毒素,apxⅣ基因存在于所有血清型中且高度保守,具有种特异性。本研究致力于提高、完善APP感染的诊断技术,建立PCR诊断方法并开发研制夹心ELISA试剂盒。
1、建立APP生物Ⅰ型的PCR诊断方法
PCR是一种快速的病原学诊断方法,本研究据已发表的apxⅣ毒素基因序列(登录号:CS056137,位置:1543~2418)设计一对特异性引物,建立检测APP 1~12血清型的PCR方法。血清型1~12标准菌株和5株野毒株均能扩增出预期876bp片段,而副猪嗜血杆菌、巴氏杆菌、猪放线杆菌、猪霍乱沙门氏菌、奇异变形杆菌、支气管败血波氏菌、猪丹毒杆菌PCR扩增结果为阴性。可检出最低活菌数为2×102cfu/mL,最低检出DNA浓度为9pg。
2、重组蛋白rApxⅣ的表达
参照GenBank中已发表的apxⅣ序列(登录号:CS056137)设计一对特异性引物,扩增血清1型标准菌株apxⅣ基因963bp片段(位置:2518~3480),PCR产物经NdeI、NotI酶切处理后克隆到同样处理的质粒pET-22b(+)中,酶切鉴定和序列分析后将此重组质粒转化E. coli BL 21(DE3),0.1 mM IPTG诱导4h获得高效表达的重组蛋白His-ApxⅣ,SDS-PAGE、Western blot检测证实rApxⅣ分子量为35.3 KDa,以可溶状态存在,含His·Tag。融合蛋白经Novagen公司的固定化镍离子亲和层析试剂盒进一步纯化。
3、单克隆抗体的制备及双抗体夹心ELISA方法的建立
按常规方法制备单克隆抗体。以纯化的重组蛋白rApxⅣ免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,以rApxⅣ和表达宿主菌的菌体蛋白为检测抗原,进行杂交瘤细胞的筛选,三次亚克隆后挑选两株分泌稳定、为IgG的杂交瘤细胞:5B7、5C11,竞争性结合试验证明它们有不同的抗原结合表位。这两株单抗制得腹水的ELISA滴度分别为1:6.4×104,1:1.28×105,以(NH4)2SO4盐析法纯化单抗。纯化的5B7用标准生物素方法进行标记并测得标记效价为106。建立检测ApxⅣ毒素的双抗体夹心ELISA方法,方阵滴定法确定捕获抗体最佳工作浓度为4μg/mL,Biotin-5B7最佳工作浓度为0.8μg/mL,rApxⅣ最低检出量为60pg/mL。
将建立的PCR和双抗体夹心ELISA分别检测10份猪病变肺组织和血清样品,阳性6份,与细菌分离鉴定结果一致,三种鉴定方法结果相符合。表明本研究建立的PCR和双抗体夹心ELISA方法都可用于APP感染的临床诊断。
Actinobacillus pleuropneumoniae, divided into 2 biovars and 15 serotypes, is the causative agent of porcine pleuropneumonia. The disease occurs worldwide and causes important economic losses to the swine industry. The disease is characterized by a hemorrhagic necrotizing pneumonia and fibrinous pleuritis.
The disease course may be acute and fatal, and the infected animals sometimes died within a few hours. However, in many herds, pigs may be subclinically infected without presenting clinical signs. And it has been proposed that subclinically infected pigs are the main cause of A. pleuropneumoniae dissemination. Concomitant infections with other pathogens of the respiratory tract may also aid the development of pleuropneumonia. Early detection of infected herds is essential for control of the disease.
The pathogenicity of A. pleuropneumoniae is considered to be multifactorial. A number of virulence factors have been identified. It is now clear that Apx toxins (A. pleuropneumoniae-RTX-toxins) play an important role in the pathogenesis of porcine pleuropneumonia. Only ApxⅣis expressed by all serotypes of A. pleuropneumoniae after infection in pigs in vivo, but not under in vitro conditions. The apxⅣgene, which is highly conserved in different A. pleuropneumoniae serotypes, was shown to be species-specific.
This paper developed a PCR test and sandwich ELISA for detection of A. pleuropneumoniae.
ⅠTo develop a PCR test for detection of A. pleuropneumoniae biovar I strains.
PCR techniques that amplify well-defined sequences have been described as valuable tools for the rapid and affordable detection of pathogen. A PCR assay was developed using primers specific to apxⅣof A. pleuropneumoniae (Accession number: CS056137, Sit: 1543~2418). Test on reference strains of A. pleuropneumoniae serotypes 1~12 and 5 wild types A. pleuropneumoniae showed that a 876 bp fragment was specifically amplified, but not from other bacterial species such as H. parasuis, P. multocida, S. suis, S. choleraesuis, P. miraillis, B. bronchiseptica, E. rhusiopathiae. With this test, it was possible to detect as low as 2×102 cfu/mL and 9 pg of DNA.
ⅡExpression and purification of rApxⅣ
The apxⅣgene (Accession number: CS056137, Sit: 2518~3480) was amplified by PCR using the pair of primers and the template from A. pleuropneumoniae serotype 1 genomic DNA. The PCR product with the restriction enzyme sites at each end (NdeI and NotI) were digested and then cloned into the expression vector pET-22b(+) to construct recombinant plasmid, which was confirmed by the means of combination with restriction endonuclease analysis and sequencing, and then the recombinant plasmid was transformed to E. coli BL21(DE3). After IPTG inducing for 4 hours, the recombinant BL21 (DE3) with recombinant plasmid expressed the soluble ApxⅣprotein (rApxⅣ) with size of 35.5kD by SDS-PAGE and the recombinant protein was confimed by Western blotting analysis with anti-his monoclonal antibody. The fusion proteins rApxⅣwere purified by Ni-TED (tris-carboxymethyl ethylene diamine) immobilized metal iron affinity chromatography (IMAC).
ⅢDevelopment of monoclonal antibodies and double monoclonal antibody-mediated sandwich ELISA against ApxⅣ
Monoclonal antibodies (mAb) are used extensively in diagnosis of disease. They are typically made by fusing myeloma cells with the spleen cells. The hybridoma cell lines, which secreting mAb against rApxⅣprotein were obtained by indirect ELISA using the coated antigens of rApxⅣand E. coli BL 21(DE3) protein as a negative control. Limiting dilution method was applied to subclone positive hybridoma cells three times and two positive clones, named 5B7 and 5C11, were obtained from hybridoma cell lines. The ascites ELISA titers of 5B7 and 5C11 were 1:6.4×104 and1:1.28×105 respectivly. They were typed to be IgG and had different recognized epitopes, which were analysed by competitive binding ELISA. The mAbs were purificated using ammonium sulfate precipitation, and then the purified mAb 5B7 was labled with biotin with ELISA titers of 106. A double mAb-mediated sandwich ELISA was developed using mAb 5C11 as capture antibody and biotinylated mAb 5B7 as detection antibody. In a checker-board analysis, the optimal concentration of the capture mAb 5C11 was 4μg/mL, and the biotinylated mAb 5B7 was 0.8μg/mL. With this system, it was possible to detect rApxⅣconcentration as low as 60pg/mL.
Furthermore, clinical samples with sera and necrotic lung lesions were tested by both methods of sandwich ELISA and PCR, the results showed that 6 samples were tested positive, which were confirmed by isolation and identification of A. pleuropneumoniae. And the result from the both assays was 100%correlation rate.
Based on the results that apxⅣis specific to the species A. pleuropneumoniae, we make the conclution that the PCR test and double mAbs-mediated sandwich ELISA array may be valuable methods for highly sensitive detection of A. pleuropneumoniae.
猪传染性胸膜肺炎最急性型引致猪的急性死亡,无临床病症慢性感染猪往往是再次爆发和流行的潜在传染源,感染猪易引发其它病原的继发感染。所以APP感染的早期诊断成为防控该病的关键。
APP致病机理非常复杂,有众多毒力因子,Apx(A. pleuropneumoniae-RTX- toxins, Apx)是最重要的毒力因子。其中apxⅣ基因只在活体内表达,体外培养则不表达该毒素,apxⅣ基因存在于所有血清型中且高度保守,具有种特异性。本研究致力于提高、完善APP感染的诊断技术,建立PCR诊断方法并开发研制夹心ELISA试剂盒。
1、建立APP生物Ⅰ型的PCR诊断方法
PCR是一种快速的病原学诊断方法,本研究据已发表的apxⅣ毒素基因序列(登录号:CS056137,位置:1543~2418)设计一对特异性引物,建立检测APP 1~12血清型的PCR方法。血清型1~12标准菌株和5株野毒株均能扩增出预期876bp片段,而副猪嗜血杆菌、巴氏杆菌、猪放线杆菌、猪霍乱沙门氏菌、奇异变形杆菌、支气管败血波氏菌、猪丹毒杆菌PCR扩增结果为阴性。可检出最低活菌数为2×102cfu/mL,最低检出DNA浓度为9pg。
2、重组蛋白rApxⅣ的表达
参照GenBank中已发表的apxⅣ序列(登录号:CS056137)设计一对特异性引物,扩增血清1型标准菌株apxⅣ基因963bp片段(位置:2518~3480),PCR产物经NdeI、NotI酶切处理后克隆到同样处理的质粒pET-22b(+)中,酶切鉴定和序列分析后将此重组质粒转化E. coli BL 21(DE3),0.1 mM IPTG诱导4h获得高效表达的重组蛋白His-ApxⅣ,SDS-PAGE、Western blot检测证实rApxⅣ分子量为35.3 KDa,以可溶状态存在,含His·Tag。融合蛋白经Novagen公司的固定化镍离子亲和层析试剂盒进一步纯化。
3、单克隆抗体的制备及双抗体夹心ELISA方法的建立
按常规方法制备单克隆抗体。以纯化的重组蛋白rApxⅣ免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,以rApxⅣ和表达宿主菌的菌体蛋白为检测抗原,进行杂交瘤细胞的筛选,三次亚克隆后挑选两株分泌稳定、为IgG的杂交瘤细胞:5B7、5C11,竞争性结合试验证明它们有不同的抗原结合表位。这两株单抗制得腹水的ELISA滴度分别为1:6.4×104,1:1.28×105,以(NH4)2SO4盐析法纯化单抗。纯化的5B7用标准生物素方法进行标记并测得标记效价为106。建立检测ApxⅣ毒素的双抗体夹心ELISA方法,方阵滴定法确定捕获抗体最佳工作浓度为4μg/mL,Biotin-5B7最佳工作浓度为0.8μg/mL,rApxⅣ最低检出量为60pg/mL。
将建立的PCR和双抗体夹心ELISA分别检测10份猪病变肺组织和血清样品,阳性6份,与细菌分离鉴定结果一致,三种鉴定方法结果相符合。表明本研究建立的PCR和双抗体夹心ELISA方法都可用于APP感染的临床诊断。
Actinobacillus pleuropneumoniae, divided into 2 biovars and 15 serotypes, is the causative agent of porcine pleuropneumonia. The disease occurs worldwide and causes important economic losses to the swine industry. The disease is characterized by a hemorrhagic necrotizing pneumonia and fibrinous pleuritis.
The disease course may be acute and fatal, and the infected animals sometimes died within a few hours. However, in many herds, pigs may be subclinically infected without presenting clinical signs. And it has been proposed that subclinically infected pigs are the main cause of A. pleuropneumoniae dissemination. Concomitant infections with other pathogens of the respiratory tract may also aid the development of pleuropneumonia. Early detection of infected herds is essential for control of the disease.
The pathogenicity of A. pleuropneumoniae is considered to be multifactorial. A number of virulence factors have been identified. It is now clear that Apx toxins (A. pleuropneumoniae-RTX-toxins) play an important role in the pathogenesis of porcine pleuropneumonia. Only ApxⅣis expressed by all serotypes of A. pleuropneumoniae after infection in pigs in vivo, but not under in vitro conditions. The apxⅣgene, which is highly conserved in different A. pleuropneumoniae serotypes, was shown to be species-specific.
This paper developed a PCR test and sandwich ELISA for detection of A. pleuropneumoniae.
ⅠTo develop a PCR test for detection of A. pleuropneumoniae biovar I strains.
PCR techniques that amplify well-defined sequences have been described as valuable tools for the rapid and affordable detection of pathogen. A PCR assay was developed using primers specific to apxⅣof A. pleuropneumoniae (Accession number: CS056137, Sit: 1543~2418). Test on reference strains of A. pleuropneumoniae serotypes 1~12 and 5 wild types A. pleuropneumoniae showed that a 876 bp fragment was specifically amplified, but not from other bacterial species such as H. parasuis, P. multocida, S. suis, S. choleraesuis, P. miraillis, B. bronchiseptica, E. rhusiopathiae. With this test, it was possible to detect as low as 2×102 cfu/mL and 9 pg of DNA.
ⅡExpression and purification of rApxⅣ
The apxⅣgene (Accession number: CS056137, Sit: 2518~3480) was amplified by PCR using the pair of primers and the template from A. pleuropneumoniae serotype 1 genomic DNA. The PCR product with the restriction enzyme sites at each end (NdeI and NotI) were digested and then cloned into the expression vector pET-22b(+) to construct recombinant plasmid, which was confirmed by the means of combination with restriction endonuclease analysis and sequencing, and then the recombinant plasmid was transformed to E. coli BL21(DE3). After IPTG inducing for 4 hours, the recombinant BL21 (DE3) with recombinant plasmid expressed the soluble ApxⅣprotein (rApxⅣ) with size of 35.5kD by SDS-PAGE and the recombinant protein was confimed by Western blotting analysis with anti-his monoclonal antibody. The fusion proteins rApxⅣwere purified by Ni-TED (tris-carboxymethyl ethylene diamine) immobilized metal iron affinity chromatography (IMAC).
ⅢDevelopment of monoclonal antibodies and double monoclonal antibody-mediated sandwich ELISA against ApxⅣ
Monoclonal antibodies (mAb) are used extensively in diagnosis of disease. They are typically made by fusing myeloma cells with the spleen cells. The hybridoma cell lines, which secreting mAb against rApxⅣprotein were obtained by indirect ELISA using the coated antigens of rApxⅣand E. coli BL 21(DE3) protein as a negative control. Limiting dilution method was applied to subclone positive hybridoma cells three times and two positive clones, named 5B7 and 5C11, were obtained from hybridoma cell lines. The ascites ELISA titers of 5B7 and 5C11 were 1:6.4×104 and1:1.28×105 respectivly. They were typed to be IgG and had different recognized epitopes, which were analysed by competitive binding ELISA. The mAbs were purificated using ammonium sulfate precipitation, and then the purified mAb 5B7 was labled with biotin with ELISA titers of 106. A double mAb-mediated sandwich ELISA was developed using mAb 5C11 as capture antibody and biotinylated mAb 5B7 as detection antibody. In a checker-board analysis, the optimal concentration of the capture mAb 5C11 was 4μg/mL, and the biotinylated mAb 5B7 was 0.8μg/mL. With this system, it was possible to detect rApxⅣconcentration as low as 60pg/mL.
Furthermore, clinical samples with sera and necrotic lung lesions were tested by both methods of sandwich ELISA and PCR, the results showed that 6 samples were tested positive, which were confirmed by isolation and identification of A. pleuropneumoniae. And the result from the both assays was 100%correlation rate.
Based on the results that apxⅣis specific to the species A. pleuropneumoniae, we make the conclution that the PCR test and double mAbs-mediated sandwich ELISA array may be valuable methods for highly sensitive detection of A. pleuropneumoniae.
引文
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