猪圆环病毒2型（PCV2）核衣壳蛋白免疫原性的鉴定和应用及其多抗在PCV2感染诊断中的应用

Characterization and potential use of truncated PCV2 capsid protein and its polyclonal antibody for diagnosis of PCV2 infections

摘要:

猪圆环病毒2型ORF2编码与病毒毒力相关的结构蛋白――核衣壳蛋白（Cap），该蛋白可以用于PCV2感染的血清学调查，但不同区域的PCV2分离株的ORF2特别是其抗原表位序列存在一定的突变。本研究将PCV2浙江分离株ORF2的主要抗原表位以及PCV1 ORF2进行了原核表达，将分别纯化的融合蛋白Cap2s 和Cap1s免疫SPF兔后制备多抗，并进一步分析了纯化蛋白的免疫原性和多抗的特性。Western blot结果表明无论Cap2s 和Cap1s均能与两个多抗发生交叉反应，而PCV2或PCV1阳性猪血清只能分别特异性地识别Cap2s 和Cap1s。IFA结果则证明两个多抗对于天然Cap蛋白无交叉反应性。利用Cap2s作为包被抗原对13个猪场的259份血清样品的PCV2抗体进行ELISA检测，平均阳性率为80.69％（209/259），而各猪场的阳性率差异较大（48.28%-100%）。以上结果表明Cap2s可作为一个型特异性抗原用于浙江省本地猪场猪群血清中PCV2抗体的监控，而其多抗也可用于免疫组化对PCV2感染进行有效诊断。

Abstract:

As porcine circovirus 2 (PCV2) ORF2 encodes the major structural protein (capsid) that is closely related to the pathogenesis, the capsid (Cap) protein could be used as a target antigen for serological analysis. The immunoactivities of the truncated capsid proteins containing immunogenic epitopes of PCV2 (Cap2s) or PCV1 (Cap1s) expressed in Escherichia coli were described, as well as the characteristic of their polyclonal antibodies in diagnosis of PCV2 infection. Western blot analysis revealed that both Cap2s and Cap1s gave strong signals on nitrocellulose membranes to their corresponding polyclonal antibody. Furthermore, either PCV2-positive sera from PMWS cases or PCV1-positive swine sera could only recognize its relative polyclonal antibody. There was also no cross-reactivity between the two polyclonal antibodies when reacted with natural Cap proteins of viral particles on cells by immunofluorescence assay (IFA). Thus, an ELISA was then developed using PCV2 Cap as coating antigen to evaluate the sero-prevalence of PCV2 infection in pigs. The PCV2-positive rate ranged from 48.28% to 100% among different herds (n=13) with an average of 80.69% (209/259). These results indicate that Cap2s was type-specific and could be used as a discriminative antigen for monitoring PCV2 antibody in serum. The polyclonal antibodies were also useful for differential identification of PCV1 and PCV2 infection by immunohistochemistry.