重组猪瘟病毒C株E2蛋白的猪繁殖与呼吸综合征病毒的构建及鉴定

CONSTRUCTION AND ANALYSIS OF RECOMBINANT PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS EXPRESSING E2 PROTEIN OF CLASSICAL SWINE FEVER VIRUS C STRAIN

本研究在猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)弱毒株(Hu N4-F112)感染性克隆的基础上,在其基因组ORF1b和ORF2之间插入(classical swine fever,CSF)疫苗毒C株的E2基因,并在E2基因3'端下游插入PRRSV的转录调控序列6(transcription regulatory sequence 6,TRS6),构建成重组全长感染性克隆(p A-C-E2)。用SwaI线性化该质粒,并体外转录后,将RNA转染MARC-145细胞并传代1次,即可拯救出重组病毒vA-C-E2,在至少20代的传代过程中能够保持遗传稳定性。重组病毒与亲本病毒vHu N4-F112在病毒滴度、空斑形态、蛋白表达等方面没有明显差异;多步生长曲线结果显示,该重组病毒与亲本毒具有相似的生长特性。间接免疫荧光(indirect immunofl uorescence,IFA)结果显示,重组病毒不仅能够表达PRRSV N蛋白,也可以表达猪瘟病毒E2蛋白。本研究结果为研制CSF和PRRS新型基因重组疫苗奠定了坚实物质基础。

Based on type 2 Porcine reproductive and respiratory syndrome virus（PRRSV） full-length infectious c DNA clone p Hu N4-F112, using SOE PCR, the E2 gene of Classical swine fever virus（CSFV） C strain was inserted between ORF1 b and ORF2. An extra transcription regulatory sequence 6（TRS6） was inserted behind the exogenous gene in order to make E2 gene expression as an unique sg m RNA expression, then the recombinant p A-C-E2 were constructed. After Swa I linearization, in vitro transcription, synthetic RNA was transfected into MARC-145 cells, and viable virus v A-C-E2 could be rescued. v A-C-E2 was conducted for genetic stability identification, and the result showed that recombinant virus could maintain genetically stable in at least 20 times cell passage processes. Indistinguishable virologic characteristics of v A-C-E2 were verified compared with the parental virus v Hu N4-F112 in viral propagation, plaque morphology, protein expression. Multi-step growth curve showed that the peak titer and the propagation tendency were similar. The results of IFA shown that recombinant virus could not only produce PRRSV N protein, but also the E2 protein of CSFV. The recombinant v A-C-E2 would become an important tool for further PRRSV research and the bivalence vaccine innovation and development afterwards.