NADC30-like猪繁殖与呼吸综合征病毒样颗粒的制备与鉴定

Preparation and verification of NADC30-like porcine reproductive and respiratory syndrome virus-like particles

摘要:

【背景】猪繁殖和呼吸综合征病毒(porcine reproductive and respiratory syndrome virus，PRRSV)可以造成怀孕母猪的繁殖障碍及仔猪的呼吸系统疾病，近年来，NADC30-like谱系PRRSV已成为国内的优势流行毒株。【目的】研制针对NADC30-like谱系PRRSV的病毒样颗粒(virus-like particle，VLP)疫苗。【方法】将PRRSV NADC30-like毒株编码GP5蛋白开放阅读框5(open reading frame 5，ORF5)、ORF6(编码M蛋白)分别连接至pFastBac^TMDual载体P₁₀和P_H启动子下游多克隆位点，获得穿梭质粒pFB-30-ORF5及pFB-30-ORF6，酶切鉴定后，将ORF6基因插入到穿梭质粒pFB-30-ORF5 P_H启动子下游，构建穿梭质粒pFB-30-ORF5-OPF6。将上述3种穿梭质粒分别转化大肠杆菌DH10Bac感受态细胞，通过蓝白斑筛选及PCR鉴定重组杆粒。再将获得的重组杆粒转染至SF9昆虫细胞，发现细胞病变后收获病毒液，继续盲传3代，在透射电镜下观察是否有病毒样颗粒。用第3代病毒液感染SF9细胞后，分别用GP5蛋白、His-tag、Flag-tag单克隆抗体作为一抗，通过免疫电镜、间接免疫荧光(indirect immunofluorescence assay，IFA)、Western blotting鉴定重组蛋白。【结果】成功构建了3种穿梭质粒pFB-30-ORF5、pFB-30-ORF6和pFB-30-ORF5-OPF6，酶切鉴定正确。通过蓝白斑筛选及PCR验证后获得重组杆粒，分别命名为Bacmind-30-ORF5、Bacmind-30-ORF6和Bacmind-30-ORF5-ORF6。重组杆粒感染SF9细胞120h时出现明显的细胞病变，收获病毒液后，在透射电子显微镜可观察到大小为50nm左右呈现球形结构的VLPs。免疫电镜可以观察到胶体金颗粒结合在VLPs周围；IFA结果显示实验组均出现了明显绿色的特异性荧光灶；Western blotting结果表明，3种VLPs均出现特异性条带，并与预期大小一致。【结论】制备了3种NADC30-like谱系PRRSV的病毒样颗粒，为针对PRRSV新谱系流行株疫苗的研发奠定了基础。

Abstract:

[Background] Porcine reproductive and respiratory syndrome virus (PRRSV) can cause reproductive disorders in pregnant sows and respiratory diseases in piglets. In recent years, NADC30-like strains of PRRSV have become dominant in China. [Objective] To develop a virus-like particle (VLP) vaccine against NADC30-like strains of PRRSV. [Methods] We linked the encoding GP5 protein open reading frame 5 (ORF5) and ORF6 (encoding M protein) genes of NADC30-like strain to the downstream polyclonal sites of P₁₀ and P_H promoters in pFastBac^TM dual vector to obtain the shuttle plasmids pFB-30-ORF5 and pFB-30-ORF6, respectively. After identification by restriction endonuclease digestion, the ORF6 gene was inserted into the downstream region of P_H promoter in the shuttle plasmid pFB-30-ORF5 to construct the shuttle plasmid pFB-30-ORF5-OPF6. The above three shuttle plasmids were respectively transformed into DH10Bac competent Escherichia coli cells, and the recombinant plasmids were identified by blue-white screening and PCR. The recombinant plasmids were then transfected into SF9 insect cells. After the appearance of cytopathic changes, the virus was harvested and passed on blindly for three generations. Whether there were virus-like particles was observed under a transmission electron microscope. After the SF9 cells were infected with the third-generation virus, the GP5, His-tag, and Flag-tag antibodies were used as the primary antibodies for the recombinant protein identification by indirect immunofluorescence assay (IFA) and Western blotting. [Results] Three shuttle plasmids pFB-30-ORF5, pFB-30-ORF6, and pFB-30-ORF5-OPF6 were successfully constructed and identified by restriction endonuclease digestion. After blue-white screening and PCR verification, the recombinant rod particles were obtained and named Bacmind-30-ORF5, Bacmind-30-ORF6, and Bacmind-30-ORF5-ORF6, respectively. Obvious cytopathic effect was observed in SF9 cells 120 h post infection with recombinant rod particles. After harvesting of the virus suspension, the spherical VLPs with the diameter of about 50 nm were observed under a transmission electron microscope. The binding of colloidal gold particles around VLPs was observed via immunoelectron microscopy, and the results of immunofluorescence assay showed obvious green specific fluorescent foci in the experimental group. Western blotting showed that all the three kinds of VLPs generated specific bands with the expected size. [Conclusion] Three kinds of virus-like particles of NADC30-like PRRSV were prepared, which laid a foundation for the research and development of vaccines against the new prevalent strains of PRRSV.