猪II型圆环病毒全基因组序列分析及其ORF1和ORF2基因的克隆与表达

Genomic sequence determination of PCV-2 and expression of ORF1 and ORF2 genes in E. coli

猪II型圆环病毒(PCV＿2)的2个主要开放阅读框ORF1和ORF2分别编码病毒的复制酶Rep和结构蛋白Cap。本试验将分离株L株病毒感染PK15细胞系,提取总DNA并以其为模板,通过PCR分别扩增了全病毒基因组、完整的Rep和Cap基因,并克隆到pMD18T载体中,进行了序列测定。将Rep和Cap基因亚克隆到大肠杆菌表达载体pPROEXTMHT中,以融合蛋白的形式表达了这两种蛋白。根据文献报道,致病性的PCV＿2与无致病性的PCV1的型特异性抗原决定簇主要位于Cap基因的羧基端部分,本实验利用原核表达载体pGEX＿6p＿1融合表达了Cap基因羧基端423bp的片段。通过薄层扫描各重组菌诱导表达产物的SDS＿PAGE结果,重组质粒pPRO＿Cap和pPRO＿Rep以及pGEX＿ΔCap的外源基因表达量分别占总菌体总蛋白的15.1%、30.4%和19.6%。将表达的三种蛋白作为抗原,通过酶联免疫吸附试验分别与PCV＿2阳性猪血清和SPF猪血清进行了反应,pGEX＿ΔCap表达的蛋白(Cap蛋白的羧基端部分)抗原反应性最强,这表明截短的Cap蛋白羧基端部分将是一个良好的诊断PCV＿2感染用候选抗原。

Porcine circle virus type 2 (PCV-2) consists of two open reading frames, ORF1 and ORF2 encoding for viral replicase (Rep) and capsid protein (Cap), respectively. In the study, the entire PCV-2 DNA, ORF1 and ORF2 were amplified from PK15 cell line infected with PCV-L Strain. The PCR products were cloned into plasmid pMD18T and sequenced. The complete ORF1 and ORF2 were then subcloned into expression plasmid pPROEXTMHT and generated two recombinant plasmids, pPRO-Rep and pPRO-Cap. In addition, a fragment of 423 bp at N-terminal of ORF2 was subcloned into expression vector pGEX-6p-1 resulting in a recombinant plasmid pGEX-Δcap. SDS-PAGE analysis of bacterial harboring above three recombinant plasmids showed that the expressed proteins could represent 30.6 %(ORF1), 15.9 %(ORF2) or 19.6 %(truncated ORF2) of the total bacterial proteins, respectively. To compare the antigenic reactivity of the three recombinant proteins, PCV-2 positive sera and negative sera (from SPF pigs) were tested by ELISA. The results indicated that the truncated ORF2 nesting putative type-specific epitopes, showing much stronger reaction with PCV-2 positive sera than others. This study demonstrated that the recombinant truncated ORF2 protein might be functional as potential diagnostic antigen for PCV infection.