非洲猪瘟病毒全长与截短p72蛋白的抗原性比较

Comparison of Antigenicity between Recombinant p72 and Truncated p72 Proteins of African Swine Fever Virus

比较非洲猪瘟病毒(African swine fever virus,ASFV)全长p72与截短p72蛋白(p72s)的抗原性,为ASFV特异性抗体检测技术研究提供试验依据。应用大肠杆菌表达系统表达p72和p72s蛋白,应用Western blot和液相色谱-质谱联用(LC-MS/MS)技术鉴定表达的蛋白,分析蛋白的抗原表位;用纯化的重组全长和截短p72蛋白分别免疫小鼠,比较其免疫原性;通过ELISA和Western blot,评价重组p72和p72s蛋白与ASFV阳性血清的免疫反应性。结果:重组p72和p72s蛋白均以包涵体形式表达,相对分子质量分别约为76和42 ku,与预期大小相符;表达蛋白的酶解肽段对p72和p72s蛋白推测氨基酸序列的覆盖度分别为89.3%和88.39%,两者分别包含27和17个抗原表位;用p72与p72s蛋白免疫小鼠3次后,血清特异性抗体效价分别为1∶200~1∶25600和1∶12800~1∶409600;基于重组p72与p72s蛋白的ELISA从108份猪血清中分别检出78份(72.2%)和85份(78.7%)阳性血清,两者的检测结果具有很好的一致性(kappa=0.826,P=0.016);经Western blot检测的10份ASFV抗体阳性血清中有7份与p72蛋白反应,全部与p72s蛋白反应。截短p72蛋白比全长p72蛋白的抗原性好,更适合用于ASFV抗体检测。

The antigenicity of the p72 protein of African swine fever virus(ASFV)was compared with that of a truncated p72 protein(p72s)to provide experimental basis for ASFV specific antibody detection.The p72 and p72s proteins were expressed by Escherichia coli expression system,and the expressed protein was identified by Western blotting and Liquid chromatography mass spectrometer(LC-MS/MS),and the epitopes of the protein were analyzed.Mice were immunized with the purified intact p72 protein or p72s protein,respectively,and the immunogenicity of p72 and p72s proteins was compared.The immunoreactivity of the p72 and p72s proteins with ASFV-positive serum was evaluated by ELISA and Western blotting.Both recombinant p72 and p72s proteins were expressed as inclusion bodies with molecular weights of 76 and 42 kDa,respectively,which were consistent with the expected molecular weight.The coverage of predicted amino acid sequences of p72 and p72s by the hydrolysate peptide of the expression proteins was 89.3%and 88.39%,respectively,which included 27 and 17 epitopes.After mice were immunized with p72 or p72s for three times,the serum specific antibody titers were in the range of 1∶200 to 1∶25600 and 1∶12800 to 1∶409600,respectively.Among 108 swine sera,78(72.2%)and 85(78.7%)positive sera were detected by ELISA based on p72 and p72s proteins with a good consistency(kappa=0.826,P=0.016).Seven of the 10 ASFV positive sera detected by Western blotting reacted with p72 protein,and all of them reacted with the p72s protein.The truncated p72 protein has better antigenicity than the intact p72 protein,and it is more suitable for ASFV antibody detection.