非洲猪瘟病毒P54蛋白原核表达及间接ELISA检测方法建立

Prokaryotic Expression of P54 Protein of African Swine Fever Virus and Establishment of an Indirect ELISA Detection Method

【目的】建立一种具有良好特异性和敏感性的非洲猪瘟病毒(African swine fever,ASFV)抗体间接ELISA检测方法。【方法】根据ASFV P54蛋白基因序列进行大肠杆菌密码子优化,将优化后的P54序列克隆到pET-28a(+)质粒中,构建重组质粒pET28a-P54,利用大肠杆菌表达系统表达目的蛋白并用His镍柱进行纯化,经SDS-PAGE和Western blotting鉴定后,以纯化的P54蛋白作为检测抗原进行包被,建立一种间接ELISA方法,对包被条件、封闭液、封闭时间和血清孵育等反应条件进行优化后,对其临界值、特异性、灵敏性和重复性进行验证。【结果】SDS-PAGE结果显示,P54蛋白分子质量为42 ku,纯度在95%以上;Western blotting结果显示,P54蛋白与ASFV阳性血清能产生特异性反应。优化后的ELISA反应条件为:0.3125μg/mL抗原37℃包被1 h,5%脱脂奶粉37℃封闭1 h,1∶800稀释的血清37℃孵育2 h,1∶15000稀释的酶标二抗37℃孵育1 h。该ELISA检测方法仅与ASFV阳性血清产生特异性反应,与猪瘟病毒、猪细小病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型、牛病毒性腹泻病毒、猪伪狂犬病病毒和乙型脑炎病毒的阳性血清均无明显交叉反应;该方法灵敏性较高,ASFV阳性血清稀释度为1∶3200时仍有明显反应。批间重复和批内重复变异系数均<10%;与商品化试剂盒相比,其阳性符合率为95.45%,阴性符合率为96.05%,总符合率为95.83%。【结论】本研究建立了一种以ASFV P54蛋白为检测抗原的ASFV间接ELISA方法,该方法具有良好的特异性和敏感性,能运用于ASFV的血清学检测,为非洲猪瘟的快速诊断提供更多选择。

【Objective】This study was aimed to establish an indirect ELISA method for detecting antibodies against African swine fever virus(ASFV)with good specificity and sensitivity.【Method】According to the gene sequence of ASFV P54 protein,E.coli codon optimisation was carried out,and the optimised P54 sequence was cloned into the pET-28a(+)plasmid to construct the recombinant plasmid pET28a-P54,which was used to express the target proteins using the E.coli expression system.Protein purification was carried out with a His nickel column,and after identification by SDS-PAGE and Western blotting,the purified P54 protein was encapsulated as a detection antigen to establish an indirect ELISA method,which was validated for its critical value,specificity,sensitivity and reproducibility after optimisation of the reaction conditions such as encapsulation conditions,closure solution,closure time and serum incubation.【Result】The results of SDS-PAGE showed that the molecular weight of P54 protein was 42 ku,and the purity was above 95%.The results of Western blotting showed that P54 protein could react specifically with ASFV positive serum.The optimized ELISA conditions were as follows:0.3125μg/mL antigen was coated at 37℃for 1 h,5%skimmed milk powder was blocked at 37℃for 1 h,serum diluted at 1∶800 was incubated at 37℃for 2 h,and enzyme-labeled secondary antibody diluted at 1∶15000 was incubated at 37℃for 1 h.The ELISA method only reacted specifically with ASFV positive sera,and did not cross-react with positive sera of Swine fever virus,Porcine parvovirus,Porcine reproductive and respiratory syndrome virus,Porcine circovirus type 2,Bovine viral diarrhoea virus,Porcine pseudorabies virus and Encephalitis B virus.The sensitivity of the method was high,and the ASFV-positive serum was still significantly responsive when the dilution was 1∶3200.The intra-batch and inter-batch duplicates were both<10%.Compared with the commercial kits,the positive compliance rate was 95.45%,the negative compliance rate was 96.05%,and the total compliance rate was 95.83%.【Conclusion】In this study,an indirect ELISA method using ASFV P54 protein as antigen was established.The method had good specificity and sensitivity,and could be used for serological detection of ASFV,providing more options for rapid diagnosis of African swine fever.