PRRSV-PCV2重组病毒的构建及PRRSV转录调控机制研究

Construction of recombinant porcine reproductive and respiratory syndrome virus expressing porcine circovirus type Ⅱ(PRRSV-PCV2) antigen and study of PRRSV transcription mechanisms with this novel viral vector

目的本研究旨在探讨猪繁殖与呼吸综合征病毒(PRRSV)感染性cDNA克隆作为猪圆环病毒(PCV2)的主要免疫原开放阅读框架2(ORF2)表达载体的可行性,以及利用重组病毒对PRRSV复制转录过程进行解剖。方法以北美株PRRSV感染性克隆pAPRRS为平台进行反向遗传操作,分别在pAPRRS的ORF1和ORF2间,ORF5和ORF6间,ORF6和ORF7间插入PCV2ORF2,且对拯救病毒vPCV进行了病毒学及分子生物学鉴定。结果vPCV的sg mRNA2.1利用了PRRSV mRNA2的转录调控序列(TRS),形成由PRRSVGP2和PCV2衣壳蛋白组成的sgm RNA2.1。外源基因的插入同时也导致重组病毒启用新的TRS而产生3条新亚基因组,其中sgm RNA2.2和sgm RNA2.3采用PCV2上的序列来取代PRRSVRNA2本身的TRS;另一条sgm RNA2.4则为非经典型亚基因组,其TRS在PRRSV相应的AUG下游。结论2株PRRSV-PCV2重组病毒可以稳定传代,为进一步研发PRRSV遗传标疫苗奠定了基础;插入片段上一些类似TRS序列的引入及插入片段(718bp)过长导致PRRSV ORF2TRS本身和其两翼序列的RNA结构变化是引起重组病毒遗传不稳定性的最主要原因;PRRSV可以利用TRS样外源序列作为转录启动子,这为进一步解剖PRRSV复制转录过程奠定了基础。

Objective The goal of the cun＇ent study is to develop infectious porcine reproductive and respiratory syndrome virus （PRRSV） cDNA clones as expression vectors for porcine circovirus type Ⅱ （PCV2） ORF2 antigens and to use this new vector to dissect the replication and transcription abilities of PRRSV.Methods ORF2 from PCV2 was inserted into our previously established infectious clone of PRRSV,designated pAPRRS,at three locations： 1 ） between ORFlb and ORF2a; 2） between ORF5 and ORF6; and 3） between ORF6 and ORFT.The three full length mutant clones were then transfected into MARC-145 cells, resulting in three recombinant viruses.The genetic instability of recombinant virus was further examined via PRRSV＇ s mRNA2-spocific RT-PCR sequence analysis of sgmRNA2, which enabled us to determine the origin of the sequence at the junction site. Results Ore- results demonstrated that the sgmRNA2 of this recombinant PRRSV- PCV2 has one TRS derived from the PRRSV ORF2 TRS, and two TRS segments were derived from the sequence of PCV2. One TRS was derived from the PRRSV ORF2 noncanonical TRS located at the downstream of PRRSV OPF2 initiation cordon. Conclusion Two recombinant PRRSV- PCV viruses are stable for more than ten passages, which makes them useful as marker vaccines. The most common reason for genetic instability of the PRRSV-PCV recombinant virus is an insertion of a sequence that resembles TRS and a subsequent change of the RNA structure for PRRSV ORF2 TRS and its flanking sequence. PRRSV can use a PCV2 sequence as its own TRS, which is useful for dissecting the mechanism behind PRRSV replication and transcription.