5'端非病毒核苷酸对猪繁殖与呼吸综合征病毒感染性克隆拯救的影响

为研究猪繁殖与呼吸综合征病毒(PRRSV)5'端非病毒核苷酸对感染性克隆拯救的影响,优化PRRSV反向遗传操作平台,本试验构建了含有CMV启动子的PRRSVJXA1-R株全长克隆质粒,在CMV启动子的下游引入锤头核酶序列,并在PRRSV基因组的5'端上游和锤头核酶下游之间引入非病毒核苷酸,分别构建成5'端含有非病毒核苷酸GG和GCTAGC的PRRSV全长感染性cDNA克隆Pgg-PRRSV和PNhe-PRRSV。新构建的两株克隆质粒直接转染BHK-21细胞拯救,48h后用间接免疫荧光检测病毒M蛋白,结果表明两株克隆均可拯救成活,测定Pgg-PRRSV和PNhe-PRRSV转染上清的病毒毒价分别为3.0和1.25TCID₅₀/mL,拯救病毒在Marc-145细胞上传3代后均稳定在6.5TCID₅₀/mL,病毒滴度无明显差异。本研究成功构建了两株5'端上游含有非病毒核苷酸的PRRSV基因组全长感染性cDNA克隆,表明PRRSV的5'端上游含有两个非病毒核苷酸GG拯救效率较高,且5'端的病毒外源核苷酸对拯救的影响仅存在于拯救的最初阶段。

Effect of 5' Terminal Non Viral Nucleotide on the Rescue of PRRSV Infectious Clone

To determine the effect of non viral nucleotide on the rescue of infectious PRRSV and optimize the reverse genetics system of PRRSV, two full-length PRRSV plasmids were constructed containing the CMV promoter, and inserted ribozyme sequences in the downstream of CMV promoter in this study.Two non viral nucleotide sequences GG and GCTAGC were inserted into a position between the ribozyme sequence and the 5' UTR of the PRRSV genome, respectively, resulting two full-length PRRSV infectious cDNA clones, Pgg-PRRSV and PNhe-PRRSV.The plasmids were transfected into BHK-21 cells.The M protein of PRRSV was detected by indirect immunofluorescence 48 h after transfection.Two infectious viruses, Pgg-PRRSV and PNhe-PRRSV, had been successfully generated.The titers of the two viruses were determined to be 3.0 and 1.25 TCID₅₀/mL, respectively.And the titer of the two rescued viruses reached a peak as high as 6.5 TCID₅₀/mL after passaged for 3 generations in Marc-145 cells.The results demonstrated that the presence of the two non viral nucleotides (GG) could enhance the rescue in a higher level.Nevertheless, the effect of the two nucleotide sequences was indistinguishable when the viruses passaged for 3 generations.Together, our result indicated that the additional nucleotide sequence was effective only in the initial stage of rescue.