摘要
【目的】猪圆环病毒2型(PCV2)是引起断奶仔猪多系统衰竭综合症(PMWS)的重要病原,其基因组ORF2编码的Cap蛋白为病毒的主要结构蛋白,起保护性抗原作用,对其进行抗原表位鉴定十分必要。【方法】本研究采用杆状病毒表达的重组Cap蛋白作为免疫原制备5株单克隆抗体,通过原核表达系统对ORF2基因进行了截短表达,先行对Cap蛋白抗原表位进行宽幅定位,然后利用合成多肽对Cap抗原表位做精确扫描定位。【结果】5株单抗中有4株针对同一抗原表位,坐落在Cap蛋白N末端核定位信号区,经多肽扫描证实,核心序列为26RPWLVHPRHRY36;另1株单抗(1D2)仅与重组Cap蛋白产生免疫活性反应,对4个分段截短表达的Cap蛋白无免疫活性反应,鉴于该单抗具有中和病毒活性,针对的可能是构象表位。【结论】本研究首次鉴定出位于Cap蛋白核定位信号区的一个抗原表位,为以后Cap蛋白功能及核定位机理的研究奠定基础。
[Objective] Porcine circovirus type 2 （PCV2） is an important causative agent of postweaning multisystemic syndrome in pigs and capsid protein （Cap） encoded by ORF2 as the main structural protein is involved in the host immune protective response, so it is very important to identify the antigenic epitopes of PCV2 Cap. [Method] In this study, five monoclonal antibodies （MAbs） against recombinant PCV2-ORF2 Cap expressed by baculovirus system were generated and preliminary epitopes region was localized using the five MAbs after expression of truncated fragments from ORF2 gene in Echerichia coli system. Subsequently, peptides were synthesized to further accurately identify epitopes on the Cap. [ Result ] Of the five MAbs, four MAbs were against the same core epitope （26RPWLVHPRHRy36） verified by peptides mapping, locating in nuclear localization signal region at the N terminal of Cap. Besides, another MAb （1D2） could not react with the four truncated fragments only to react with the whole recombinant Cap, and it may be a conformational epitope based on its neutralizing effect against PCV2. [Conclusion] It was concluded that an epitope located in nuclear localization signal region of Cap was firstly reported, which will provide a basis for further studies on the functions of Cap as well as on the mechanism of Cap localization into nuclear.
出处
《中国农业科学》
CAS
CSCD
北大核心
2010年第7期1480-1486,共7页
Scientia Agricultura Sinica
基金
国家自然科学基金项目(30871859)
国家科技支撑计划项目(2006BAD06A07)
中央级公益性科研院所基本科研业务费(2008-07)
兽医生物技术国家重点实验室基金(NKLVBP2008-07)
关键词
猪圆环病毒2型
CAP蛋白
抗原表位
多肽扫描
核定位信号
porcine circovirus type 2
capsid protein
antigenic epitope
peptide mapping
nuclear localization signal
