Preparation of monoclonal antibody against N protein of porcine epidemic diarrhea virus and establishment of indirect immuno-fluorescence assay
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摘要:目的
制备猪流行性腹泻病毒(PEDV) N蛋白单克隆抗体，并建立检测PEDV的间接免疫荧光试验方法。
方法以重组表达的PEDV N蛋白为免疫原，免疫8周龄雌性BALB/c小鼠，分离高抗体效价小鼠的脾细胞，与SP2/0细胞融合。筛选分泌抗PEDV N蛋白单克隆抗体的杂交瘤细胞株。在已经感染PEDV的Vero细胞中，以抗PEDV N蛋白的单克隆抗体为一抗，FITC−羊抗鼠IgG为二抗，建立PEDV的间接免疫荧光检测方法。
结果制备的杂交瘤细胞株可以稳定分泌抗PEDV N蛋白抗体，细胞上清液的ELISA抗体效价在1∶3 200以上，而诱导的小鼠腹水抗体效价在1∶1 000 000以上。将单克隆抗体应用在间接免疫荧光试验时，最适条件为−20 ℃ 80%（φ）丙酮溶液中固定30 min；一抗用PBS缓冲液按体积比1∶1 000稀释，4 ℃条件下过夜孵育；二抗用PBS缓冲液按体积比1∶100稀释，37 ℃条件下孵育1 h。以建立的间接免疫荧光试验方法检测细胞中的猪传染性胃肠炎病毒(TGEV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病毒(PPRV)、猪肠道α冠状病毒（PEAV）、猪轮状病毒（PoRV）和PEDV，只有PEDV显示阳性，其他病毒均为阴性。
结论制备了抗PEDV N蛋白单克隆抗体，以该抗体为一抗建立检测PEDV的间接免疫荧光试验方法具有良好的特异性，为PEDV的实验室检测及PEDV在培养细胞中的定位和动态分布提供了有效的手段。
Abstract:ObjectiveTo prepare monoclonal antibodies against porcine epidemic diarrhea virus (PEDV) N protein, and develop an indirect immuno-fluorescence assay method used for detecting PEDV.
MethodThe expressed recombinantly PEDV N protein was used as an immunogen and 8-week-old female BALB/c mice were immunized. Then their spleen cells with high antibody titer were isolated and fused with SP2/0 cells. The hybridoma cell lines secreting monoclonal antibodies against PEDV N protein were screened. In Vero cells infected with PEDV, monoclonal antibody of anti-PEDV N protein was used as the primary antibody and FITC-goat-anti-mouse IgG was used as the secondary antibody to develop indirect immuno-fluorescence assay method used for detecting PEDV.
ResultThe prepared hybridoma cell lines could stably secrete anti-PEDV N protein antibodies, ELISA antibody titer in cell supernatant was above 1∶3 200, and in mouse ascites above 1∶1 000 000. While monoclonal antibodies were applied in established indirect immuno-fluorescence assay, the optimal conditions were that cells were fixed with 80% (φ) acetone at −20 ℃ for 30 min; The primary antibody was diluted 1 000 times by PBS buffer solution and incubated at 4 ℃ overnight; The secondary antibody was diluted 100 times by PBS buffer solution and incubated at 37 ℃ for 1 h. Transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine reproductive virus (PRV), porcine enteric α corone virus (PEAV), porcine rotavirus (PoRV) and PEDV were detected by established indirect immuno-fluorescence assay method, only PEDV showed positive, all the else viruses showed negative.
ConclusionAn anti-PEDV N protein monoclonal antibody is prepared, and the indirect immuno-fluorescence assay method used for detecting PEDV is established with high specificity. It provides an effective method for laboratory detection of PEDV and for localization and dynamic distribution of PEDV in infected cells.
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图 1 N基因的PCR扩增
M：DL 2000 DNA marker；1：N基因的PCR产物；2：阴性对照
Figure 1. PCR amplification of N gene
M: DNA marker; 1: PCR products of N gene; 2: Negative control
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图 2 PET-N表达产物的SDS-PAGE
Mr： 相对分子质量；M：蛋白marker；1：空载体宿主菌诱导产物；2、5：未诱导的含PET-N宿主菌菌株；3、4：pET-N诱导产物
Figure 2. SDS-PAGE of expressed product PET-N
Mr: Relative molecular mass; M: Protein marker; 1: Induction products of empty vector host bacteria; 2, 5: Uninduced host bacteria strain with PET-N; 3, 4: pET-N induced products
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图 3 纯化N蛋白的Western blot检测
Mr： 相对分子质量；M：蛋白预染marker；1：纯化N蛋白
Figure 3. Western blot detection of purified N protein
Mr: Relative molecular mass; M: Protein pre-staining marker; 1: Purified N protein
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图 4 经不同浓度咪唑洗脱的纯化产物的SDS-PAGE
Mr：相对分子质量；M：蛋白 marker；1：300 mmol·L−1咪唑洗脱的蛋白液；2：500 mmol·L−1咪唑洗脱的蛋白液；3、4：400 mmol·L−1咪唑洗脱的蛋白液
Figure 4. SDS-PAGE of purified product eluted by different concentrations of imidazole
Mr: Relative molecular mass; M: Protein marker; 1: 300 mmol·L−1 imidazole eluted protein solution; 2: 500 mmol·L−1 imidazole eluted protein solution; 3, 4: 400 mmol·L−1 imidazole eluted protein solution
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图 5 重组PEDV N蛋白免疫小鼠血清中的抗体效价
1~6：免疫小鼠；7：阴性小鼠
Figure 5. Antibody titer in serum of mice immunized with recombinant PEDV N protein
1−6: Immunized mice; 7: Negative mice
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图 8 单克隆抗体与PEDV N蛋白反应的Western blot鉴定
Mr：相对分子质量；M：蛋白marker；1：小鼠腹水
Figure 8. Western blot identification of monoclonal antibodies reacting with PEDV N protein
Mr: Relative molecular mass; M: Protein marker; 1: Ascites in mice
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图 9 不同稀释度N蛋白单克隆抗体的间接免疫荧光试验结果
a、b、c、d、e的稀释比例分别为 1∶100、1∶200、1∶500、1∶1 000、1∶2 000；f：阴性血清；g：空白对照
Figure 9. Indirect immuno-fluorescenece assay results of N protein monoclonal antibodies at different dilution ratios
The dilution ratios of a，b，c，d and e are 1∶100, 1∶200, 1∶500, 1∶1 000 and 1∶2 000 respectively；f：Negative serum；g：Blank control
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图 10 不同稀释度FITC-羊抗鼠IgG的间接免疫荧光试验结果
a、b、c、d的稀释比例分别为 1∶100、1∶200、1∶500、1∶800；e：自发光样品
Figure 10. Indirect immuno-fluorescenece assay results of FITC–goa–anti–mouse IgG at different dilution ratios
The dilution ratios of a，b，c and d are 1∶100, 1∶200, 1∶500 and 1∶800；e: Self-illuminated sample
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图 11 N蛋白单克隆抗体孵育时间的确定
a：4 ℃条件下过夜孵育；b：37 ℃条件下孵育2 h
Figure 11. Determination of incubation time of N protein monoclonal antibody
a: Incubation at 4 ℃ overnight; b: Incubation at 37 ℃ for 2 h
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图 12 间接免疫荧光特异性试验结果
a：猪瘟病毒；b：猪繁殖与呼吸综合征病毒；c：猪伪狂犬病毒；d：猪传染性胃肠炎病毒；e：猪肠道α冠状病毒；f：猪轮状病毒；g：阳性对照；h：阴性对照
Figure 12. Specific test results of indirect immuno-fluorescenece assay
a: Classical swine fever virus; b: Porcine reproductive and respiratory syndrome virus; c: Porcine pseudorabies virus; d: Transmissible gastroenteritis virus; e: Porcine enteric α corone virus; f: Porcine rotavirus; g: Positive control; h: Negative control
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图 13 间接免疫荧光试验重复性试验结果
a, b：同批次 Vero 细胞；c：与a、b不同批次 Vero 细胞
Figure 13. Repeatability test results of indirect immuno-fluorescenece assay
a, b: The same batch Vero cells; c: Different batch Vero cells with a and b
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图 14 间接免疫荧光试验敏感性试验结果
a：PEDV病毒液用不完全培养基DMEM按1∶10稀释；b：PEDV病毒液用不完全培养基DMEM按1∶100 000稀释；c：空白对照
Figure 14. Sensitivity test results of indirect immuno-fluorescenece assay
a：PEDV virus solution is diluted by incomplete medium DMEM at 1∶10；b：PEDV virus solution is diluted by incomplete medium DMEM at 1∶100 000；c：Blank control
表 1 间接ELISA法检测不同稀释比例病毒的抗体特异性
Table 1 Detection of antibody specificity of viruses with different dilution ratios by indirect ELISA
病毒名称1)
Virus nameD450 nm 1∶1 1∶100 1∶1 000 1∶3 000 1∶5 000 1∶10 000 1∶50 000 1∶100 000 TGEV 0.022 0.019 0.026 0.021 0.022 0.018 0.024 0.026 PRV 0.020 0.020 0.023 0.023 0.026 0.022 0.023 0.021 CSFV 0.018 0.017 0.019 0.020 0.017 0.021 0.020 0.016 PRRSV 0.018 0.020 0.023 0.021 0.017 0.020 0.022 0.018 PEDV 0.489 0.474 0.492 0.491 0.479 0.472 0.468 0.492 1)TGEV、PPRV、CSFV、PRRSV、PEDV 分别代表猪传染性胃肠炎病毒、猪伪狂犬病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪流行性腹泻病毒
1) TGEV, PPRV, CSFV, PRRSV and PEDV N indicate transmissible gastroenteritis virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus
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