A reverse genetic technology platform of Porcine reproductive and respiratory syndrome virus (PRRSV) with foreign gene is established, which can be used to develop a DIVA (differentiating infected from vaccinated animals) marker vaccine. In this study, a molecular marker of immunodominant B-cell epitope (49 amino acids) of Newcastle disease virus (NDV) nucleoprotein (NP) was inserted into nonessential region of nsp2 by the method of mutant PCR, basing on the infectious clone of HuN4-F112 vaccine strain. The marker full-length cDNA clone (psk-HuN4-F112-A52+NP49) was assembled by cloning and splicing of the gene fragments. The completely assembled full-length cDNA clone was confirmed by sequence and Swa I enzyme digestion. Capped RNA was transcribed in vitro from a full-length cDNA clone of the viral genome and transfected into baby hamster kidney cells (BHK-21) by liposome to acquire the rescued virus. The rescued recombinant virus was passaged on a highly permissive subclone of African green monkey kidney epithelial cell line(MARC-145). The successfully rescued virus was tested by RT-PCR, digestion, and genome sequence. The results showed that the rescued virus could be distinguished from the parental with the mutant genetic marker (Mlu I enzyme site of genome at 14667nt) and the inserted nsp2 gene region. The results of indirect immunofluorescence assay (IFA) showed that the inserted foreign marker could be expressed by the recombinant virus and the inserted foreign gene was stable during the virus serial passage. The results of plaque assay and viral growth curve showed that the recovery vims possessed similar characters of viral growth to those of the parental virus. The results suggest that this stable infectious clone can be used as an important tool for development of novel vaccine against PRRSV.%为建立表达标记基因的重组猪繁殖与呼吸综合征病毒(PRRSV)反向遗传操作平台,本研究在PRRSV HuN4-F112疫苗株感染性分子克隆的基础上,采用突变PCR技术将新城疫病毒(NDV)NP蛋白末端优势抗原区插入PRRSV NSP2蛋白的复制非必须区,经基因片段的克隆、拼接,构建了含有外源标记基因的感染性的PRRSV cDNA克隆psk-HuN4-F112-△52+NP49.用限制性内切酶SwaⅠ将psk-HuN4-F112-△52+NP49线性化后通过细胞外转录获得病毒RNA,用脂质体法将病毒RNA转染叙利亚仓鼠肾细胞(BHK-21)包装出病毒粒子,再转接到猴胚胎肾上皮细胞(MACR-145)传代拯救病毒.对拯救的病毒进行RT-PCR扩增、酶切和序列分析鉴定.结果表明,拯救病毒含有不同于亲本病毒的分子标记(病毒基因组14667位人工产生的Mlu Ⅰ酶切位点)和插入的标记基因序列.间接免疫荧光试验表明,外源基因NP49在拯救的PRRSV中表达,且能够在PRRSV传代过程中稳定遗传.病毒生长特性比较显示,拯救病毒与亲本病毒在MARC-145细胞上具有相似的增殖特性.本研究构建了含有标记基因PRRSV的感染性克隆并获得了拯救病毒,为进一步开发新型PRRS疫苗提供了有效的反向遗传操作平台.
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