猪2型圆环病毒原核表达产物的免疫原性测定

目的检测猪2型圆环病毒(PCV2)全基因组原核表达产物的免疫原性。方法根据PCV2JXL株序列(GenBank登录号AY491310),设计合成引物,利用多聚酶链式反应(PCR)从含有PCV2接种猪肾细胞PK15中扩增了Cap蛋白的氨基端片段(737-421nt),克隆入上游带有谷光苷肽-S-转移酶的原核表达载体pGEX-6p-1中,获得重组质粒pGEX-PCV737-421。PCV2Cap蛋白羧基端(426-37nt)和Rep蛋白已在过去的研究中分别融合表达。利用IPTG对3种重组大肠杆菌BL21进行诱导,进行聚丙烯酰胺凝胶电泳分析(SDS-PAGE)以及对PCV2阳性猪多抗血清的蛋白免疫印迹试验,在此基础上,将3种SDS-PAGE分离粗纯的重组表达产物碾碎后分别免疫BALB/c小鼠,用获得的抗鼠血清与PCV2病毒感染PK15细胞做间接免疫荧光抗体试验(IFA)。结果PCV2Cap蛋白的氨基端片段能在大肠杆菌中表达,且Cap蛋白羧基端和Rep蛋白的原核表达产物都能在免疫印迹试验中被PCV2阳性猪血清检测出特异性条带,重组蛋白免疫鼠血清可在IFA试验中观察到特异性的荧光分布,即检测到培养细胞中的病毒抗原。结论PCV2全基因组都可以用原核系统高效表达,且表达产物具有免疫原性。

Detection of Immunogenicity of Bacteria-Expressed PCV2 Genome

Objective To find out whether porcine circovirus type 2 (PCV_2) total genome could be expressed by E.coli and whether the recombinant proteins still be biologically active. MethodsThe N-end fragment of PCV2 ORF2 gene of 737-421nt was PCR amplified, cloned and fusion expressed with the expression vector pGEX-6p-1 in E. coli, resulting in the recombinant clone of pGEX-PCV737-421. Both the C-end fragment of the cap gene and the rep gene of PCV2 virus were expressed by E. coli previously. Then SDS-PAGE assay of the three recombinant proteins was performed and Western-blotting against PCV2-specific antiserum was carried out in turn. Finally both the recombinant proteins of the Cap gene were inoculated into SPF BALB/c mice to prepare anti-mouse polyserum in order to find out if the serum could be used to identify the virus antigens propagated in PK15 cell line through indirect fluorescence assay (IFA) experiment. ResultBoth Western-blotting and IFA assays could identify the expressed protein bands and virus antigens specifically. ConclusionThe immunogen assay demonstrated that E.coli expressed recombiant proteins of PCV2 total genome have immunoreactivity.