猪繁殖与呼吸综合征病毒GS15株NSP2蛋白多位点缺失病毒的拯救与体外生长特性分析

Rescue and in vitro growth characterization of NSP2 multisite-deleted strain of porcine reproductive and respiratory syndrome virus GS15

【目的】本实验室前期在高致病性毒株PRRSV/GSWW/2015的感染性克隆上,拯救获得了非结构蛋白(non-structural protein 2,NSP2)第519-565位和第628-747位氨基酸双缺失的工程病毒(r GS15-?2)。本研究旨在在双缺失病毒的感染性克隆上,构建拯救获得NSP2三个位点缺失的工程病毒。【方法】在前期双缺失病毒感染性克隆的基础上,利用融合PCR方法分别构建缺失NSP2第323-364位和第372-433位优势抗原表位的两个3个位点缺失的重组质粒。经脂质体介导,转染Marc-145细胞拯救病毒,通过电子显微镜观察、免疫荧光实验、测定病毒滴度、绘制生长曲线等方法对缺失病毒的生长特性进行分析。【结果】成功获得拯救病毒r GS15-?3-1和r GS15-?3-2。电子显微镜下可以观察到直径大小为50-80 nm的病毒粒子;免疫荧光实验检测表明,拯救病毒与亲本病毒GS15一致,都能检测到PRRSV N蛋白表达;缺失区域RT-PCR扩增鉴定,拯救病毒传至40代缺失标记稳定存在;r GS15-△3-1与r GS15-△3-2病毒滴度分别为2.00×10^(6.0)TCID_(50)/m L和2.25×10^(5.8)TCID50/m L,与亲本病毒相比病毒滴度差异显著(P<0.05);生长曲线分析表明拯救病毒复制水平低于亲本病毒达到最高滴度的培养时间比亲本病毒延迟24 h。【结论】本研究通过对PRRSV基因2型非结构蛋白NSP2多位点缺失病毒的体外生长特性分析,为研制新型PRRSV标记疫苗奠定了基础,也为猪繁殖与呼吸综合征的防控提供新的策略。

[Objective]A genetically engineered virus strain rGS15-∆2 with deletion of the residues at the positions 519–565 and 628–747 of the non-structural protein 2(NSP2)had been rescued based on the PRRSV/GSWW/2015 infectious clone.This study aims to construct and rescue an engineered virus strain with the deletion of three sites in NSP2 based on rGS15-∆2.[Methods]Based on the infectious clones of rGS15-∆2,two recombinant plasmids with the deletion of three sites were constructed by fusion PCR.Specifically,the dominant epitope at the amino acid site 323–364 or 372–433 of NSP2 was further deleted on the basis of rGS15-∆2.The recombinant plasmids were linearized and mixed with liposome,which were transfected into Marc-145 cells for virus rescue.The growth characteristics of the engineered virus strains were analyzed by electron microscopy,immunofluorescence assay(IFA),virus titer determination,and growth curve establishment.[Results]The engineered virus strains rGS15-∆3-1 and rGS15-∆3-2 were rescued successfully.Virions with the diameter from 50 nm to 80 nm can be observed under an electron microscope.The results of IFA confirmed the expression of PRRSV N protein by the rescued virus strains and the parent strain GS15.Furthermore,the rescued viruses were cultured with Marc-145 cells for 40 passages,and the deletion regions were confirmed to be stable by RT-PCR and sequencing.The titers of rGS15-∆3-1 and rGS15-∆3-2 were 2.00×10^(6.0)TCID50/mL and 2.25×10^(5.8)TCID_(50)/mL,respectively,which had differences from that of the parent strain(P<0.05).The growth curves showed that the rescued viruses had lower replication levels than the parent strain,and they reached the peak titers 24 h later than the parent strain.[Conclusion]We characterized the growth of the viruses with the deletion of multiple sites in NSP2 of PRRSV.The findings laid a foundation for the development of novel PRRSV-labeled vaccines and provided a new strategy for the prevention and control of porcine reproductive and respiratory syndrome.