高致病性猪繁殖与呼吸综合征病毒HuN4传代致弱株HuN4-F30感染性克隆的构建及拯救病毒致病性分析

Recused and identification of the porcine reproductive and respiratory syndrome virus from the infectious cDNA clone of the attenuated strain HuN4-F30

为构建高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)Hu N4株传代致弱株Hu N4-F30感染性克隆,本研究利用高保真DNA聚合酶,分6个片段进行病毒全基因组序列扩增,并通过点突变在其基因组10 808位引入Mlu I作为分子标记。通过重叠延伸PCR在5'端上游引入CMV启动子,3'端引入丁型肝炎病毒(HDV)核酶基因和牛生长素多聚腺苷酸化信号(BGH)。将各片段依次连接克隆于改造的p Belo BAC11载体中,构建含有全长HP-PRRSV致弱株c DNA感染性克隆p Belo BAC11-Hu N4-F30,并将全长质粒直接转染Marc-145细胞拯救出病毒。通过核酸鉴定与测序、分子标记鉴定、间接免疫荧光试验和动物致病性试验等进行鉴定。结果表明,拯救的病毒能够通过Mlu I酶切与亲本鉴别,拯救病毒的间接免疫荧光与亲本病毒一致,拯救病毒表现出低致病性。HP-PRRSV Hu N4-F30感染性克隆的构建与病毒的拯救,为进一步研究HP-PRRSV传代致弱机制奠定了基础。

To construct the infectious cDNA clone of the attenuated HuN4-F30 of the highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） HuN4 strain, six cDNA fragments covered the whole genome of the HuN4-F30 were amplified by PCR, and a Mlu I restriction endonuclease site as genetic marker was introduced into the genome by a point mutation of G 10.808 A. In addition, the CMV promoter was introduced to the 5＇ end of the viral genome and the hepatitis delta virus ribozyme （HDV） and the bovine growth hormone polyadenylation sequence were incorporated to the 3＇ end of the viral genome. Using the appropriate ligation strategy, the whole genome cDNA were inserted into a modified pBeloBACll vector to construct the recombinant plasmid pBeloBAC11-HuN4-F30, which was transfected into Marc-145 cells to generate the virus. The result showed that the virus with Mlu I site in genome was rescued from the transfected Marc-145 cells, which was able to be distinguished from the parental virus by the Mlu I digestion. However, the rescued virus and the parental virus showed similar green fluorescence by indirect immunofluorescence. The rescued virus showed no significant pathogenicity in piglets. The construction of infectious cDNA clone of HP-PRRSV HuN4-F30 strain provided a useful tool for the further study of the attenuated mechanism of HP-PRRSV.