| 摘要: |
| 【目的】制备猪δ冠状病毒（porcine deltacoronavirus,PDCoV）N蛋白及其多克隆抗体，建立检测PDCoV N蛋白抗体的间接ELISA方法。【方法】从PDCoV CH-01毒株中扩增N全基因，克隆至原核表达载体pET-28a中，构建重组表达质粒pET-28a-PDCoV-N，转入BL21（DE3），用IPTG诱导表达重组N蛋白，并对该蛋白的反应原性进行鉴定。诱导表达的N蛋白经镍柱纯化后免疫新西兰大白兔制备多克隆抗体。同时用镍柱纯化后的N蛋白作为包被抗原，建立PDCoV的间接ELISA检测方法，对该方法的特异性、重复性和稳定性进行检测，并利用该方法对河南部分地区猪场送检的165份临床猪血清样本进行PDCoV抗体检测。【结果】成功克隆了1 026 bp的N基因，构建了重组表达载体pET-28a-PDCoV-N，经诱导表达后获得了重组N蛋白，其相对分子质量约42.9 ku，Western blot分析表明所表达的蛋白可与抗PDCoV全病毒猪阳性血清发生反应。制备的PDCoV N蛋白多克隆抗体经Western blot检测表明，该抗体具有特异性；间接免疫荧光试验测定该多克隆抗体在ST细胞上的最佳使用稀释度为1∶200。以N蛋白作为包被抗原建立了间接ELISA方法，确定最佳的反应条件如下：抗原2 μg/mL在4 ℃过夜包被，10 g/L BSA在37 ℃下封闭2 h，血清（1∶80稀释）37 ℃孵育1 h，酶标二抗(1∶6 000稀释)37 ℃孵育1 h，TMB在37 ℃下显色15 min；待检血清OD450值≥0.272判定为阳性。特异性试验结果显示，该包被抗原与其他猪常见病毒的阳性血清均无交叉反应，批内和批间重复试验变异系数均小于5%。用间接ELISA法对来自河南不同地区的165份血清样本进行检测，结果表明各地样本PDCoV抗体阳性率平均为53.94%。【结论】成功表达了PDCoV N蛋白，制备了其多克隆抗体，建立了PDCoV间接ELISA检测方法，该方法具有良好的特异性、重复性和稳定性，可用于临床猪血清中PDCoV抗体的检测。 |
| 关键词: 猪δ冠状病毒 N蛋白 原核表达 多克隆抗体 血清学检测 |
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| 基金项目:国家重点研发计划项目（2016YFD0500102）；国家自然科学基金项目（31772773）；河南省科技开放合作项目（15210-6000047） |
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| Prokaryotic expression of N protein and establishment of an indirect ELISA for antibody detection of porcine deltacoronaviru |
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YAN Ruijie,ZHANG Yunfei,LIU Lingling,et al
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| Abstract: |
| 【Objective】This study prepared N protein and polyclonal antibody of porcine deltacoronavirus (PDCoV),and developed an indirect ELISA to detect PDCoV antibody of clinical pig serum samples from pig farms.【Method】The N gene was amplified from the PDCoV CH-01 strain and cloned into pET-28a vector.The recombinant protein was expressed in E. coli BL21 by induction with IPTG.The recombinant N protein purified from the nickel column was emulsified with Freund’s adjuvant,and the polyclonal antibody of anti-PDCoV-N protein was prepared by immunizing rabbits.An indirect ELISA method for detection of PDCoV antibody was established using N protein.After the specificity,repeatability and stability were detected,it was used for clinical detection of 165 clinical swine serum samples from pig farms in Henan.【Result】The N gene was cloned successfully,and the recombinant expression vector pET-28a-PDCoV-N was constructed.After induced expression,N protein was obtained and the protein molecular mass was approximately 42.9 ku.The recombinant N protein can react specifically with PDCoV positive serum.The specificity of polyclonal antibody was detected by Western blot.The titers of polyclonal antibodies were 200 by indirect immunofluorescence test in ST cell.This protein was used to establish an indirect ELISA method,the optimal antigen coating concentration was 2 μg/mL at 4 ℃ overnight and blocked at 37 ℃ for 2 h with 10 g/L BSA.The optimal serum dilution was 1∶80 and incubated at 37 ℃ for 1 h.The optimal dilution of the secondary antibody was 1∶6 000 and incubated at 37 ℃ for 1 h,and TMB color at 37 ℃ for 15 min.The serum was determined to be positive when the OD450 value was ≥0.272.The specific test results showed that there were no cross reactions of the coated antigen with the positive sera of other porcine viruses.Intra and inter assay variation coefficients were less than 5%.The ELISA method was used to detect 165 clinical swine serum samples and the positive rate of PDCoV antibody average was 53.94%.【Conclusion】This study expressed the PDCoV N protein,prepared rabbit polyclonal antibody and established the indirect ELISA method.The method had good specificity,reproducibility and stability,and can be used for the detection of PDCoV antibody in clinic. |
| Key words: porcine deltacoronavirus N protein prokaryotic expression polyclonal antibody serological detection |
