小反刍兽疫病毒Nigeria 75/1株病毒样颗粒的构建及其免疫原性

Constructing virus-like particles of peste des petits ruminants virus Nigeria 75/1 strain and its immunogenicity in mice

为了构建具有天然构象的小反刍兽疫病毒Nigeria 75/1株病毒样颗粒,本试验扩增了Nigeria 75/1株M、F和H基因,并分别克隆至双启动子载体pFastBac^(TM) Dual中,构建含有双目的基因的重组供体质粒pFastBac^(TM) Dual-2M、pFastBac^(TM) Dual-2F和pFastBac^(TM) Dual-2H;测序正确后转化至DH10Bac^(TM) 感受态细胞,同源重组获得穿梭质粒rBacmid-2M、rBacmid-2F和rBacmid-2H;将其分别转染昆虫细胞Sf9获得重组杆状病毒rpFB-2M、rpFB-2F和rpFB-2H。以鼠抗M、H蛋白的主要抗原表位区多克隆抗体与绵羊抗PPRV阳性血清对重组杆状病毒感染细胞进行间接免疫荧光(IFA)鉴定,可见特异性荧光;以3种重组杆状病毒共感染昆虫细胞Sf9的方式组装病毒样颗粒,放大培养后进行病毒样颗粒纯化。Western blot检测纯化后样品可见相对分子质量为38 000,59 000,68 000左右的条带,表明基质膜蛋白与2种囊膜糖蛋白成功组装出病毒样颗粒,且免疫小鼠可诱导产生保护性中和抗体。本试验为后续小反刍兽疫病毒(PPRV)病毒样颗粒疫苗的进一步研发奠定了基础。

In order to build virus-like particles with a natural conformation of peste des petits ruminants virus （PPRV） vaccine strain Nigeria 75/1,M,F and H genes fragment were amplified by RT-PCR and then cloned into pFastBacrMDual vector containing double promoter,to build recombinant donor plasmid pFastBacXMDual-2M,pFastBacXVDual-2F and pFastBacTMDual-2H witch contain double purpose genes. The shuttle plasmid rBacmid-2M, rBacmid-2F and rBacmid-2H were ex- tracted after donor plasmids were transformed into DH10BacTM cells for homologous recombination, then transfected to Sf9 cells to obtain recombinant baculovirus rpFB-2M, rpFB-2F and rpFB-2H. The expression of membrane protein （M）,fusion protein （rF） and hemagglutinin protein （rH） were identified by indirect immunofluoreseence assay（IFA）. It also showed that the positive sera could recognize non denatured full length M,F and tt protein by IFA. Virus-like patti cles were constructed with three recombinant baculovirus co-infecting Sf9 cells and then were puri- fied after enlarge cultivation. The purified sample was identified by Western blot, showing 38 000, 59 000 and 68 000 protein hands,indicating that virus-like particles were constructed successfully with matrix membrane protein and two capsule membrane glycoprotein. Protective neutralizing an tibody can be induced in mice after VLPs were immuned to mice for three times,this study laid a solid foundation for subsequent PPRV virus like particles vaccine research and development.