犬细小病毒颗粒疫苗的研制与免疫效果评价

Preparation and immunogenicity of virus-like particles of canine parvovirus

犬细小病毒性肠炎是由犬细小病毒(CPV)引起的一种高度传染性疾病,目前最为有效的防控方法为疫苗接种。本研究将CPV VP2基因克隆于pQB3载体中,将鉴定正确的pQB3-VP2重组质粒与qBac-ⅢG杆粒共转染至sf9昆虫细胞中,拯救获得表达CPV VP2蛋白和绿色荧光蛋白的重组杆状病毒rBac-VP2。rBac-VP2感染sf9细胞可出现明显的细胞病变,在荧光显微镜下可见明显的绿色荧光,间接免疫荧光鉴定显示rBac-VP2感染的sf9细胞可见明显的红色荧光。对细胞表达的VP2蛋白进行Western-blot鉴定,结果显示在约65 ku处可见明显的目标蛋白条带,电镜下可见与天然细小病毒形态大小相似的粒子,表明rBac-VP2感染昆虫细胞后可组装形成病毒样颗粒(VLPs),收获的VLPs血凝效价可达1∶210。将CPV VLPs免疫小鼠后诱导机体产生血凝抑制抗体,效价达1∶27,与宠必威幼犬保商业化疫苗产生的免疫效果相当。本研究为犬细小病毒新型基因工程疫苗的研制奠定了基础。

Canine parvovirus enteritis is a highly infectious disease caused by canine parvovirus(canine parvovirus,CPV).At present,vaccine immunization remains the most effective means for disease control.In this study,the CPV VP2 gene was cloned into the pQB3 vector,the pQB3-VP2 recombinant plas-mid and qBac-IG bacmid were co-transfected into sf9 insect cells to rescue the recombinant bac-ulovirus rBac-VP2,which can induce the cytopathic effect to sf9 insect cells and express CPV VP2 pro-tein with green fluorescent protein,the red fluorescence of the recombinant baculovirus rBac-VP2 was also observed by Indirect immunofluorescence assay.VP2 protein was identified clearly at about 65 ku band by Western-blot.The particles with similar morphology and size like parvovirus were observed un-der electron microscope,indicating that rBac-VP2 could assemble into virus-like particles(VLPs)after in-fecting sf9 insect cells,and the hemagglutination titer of VLPs was 1:21o.The CPV VLPs can induce the titer of 1:27 hemagglutination inhibition antibody after immunizing mice,which was equivalent to the commercial vaccine of CPV.This study laid a foundation for the development of new genetic engineering vaccineof CPV.