牛病毒性腹泻病毒1型病毒样颗粒的制备及其对豚鼠的免疫原性分析

Preparation of bovine viral diarrhea disease virus 1 virus-like particles and evaluation of its immunogenicity in a guinea pig model

摘要:

为了获得预防牛病毒性腹泻病毒1型(bovine viral diarrhea virus 1，BVDV-1) 感染的病毒样颗粒，扩增C-E^rns-E1-E2编码区段并克隆至pFastBacDaul载体，转化大肠杆菌DH10Bac感受态细胞与Bacmid重组获得Bacmid-BVDV-1，转染至Sf9细胞，获得重组杆状病毒Baculo-BVDV-1。利用免疫荧光试验、免疫印记、电镜观察确定目的蛋白表达和病毒样颗粒的组装。制备病毒样颗粒灭活抗原并辅以Montanide ISA-201佐剂免疫豚鼠，分别用中和抗体滴度测定和淋巴细胞增殖试验分析体液免疫和细胞免疫应答，然后用10⁶ TCID₅₀ BVDV-1型AV69株攻毒，评价病毒样颗粒的免疫保护效果。结果表明，构建的重组杆状病毒Baculo-BVDV-1感染Sf9细胞可高效表达BVDV结构蛋白并形成病毒样颗粒。病毒样颗粒免疫诱导豚鼠产生较高滴度(1︰144) 的中和抗体并激活细胞免疫，降低了BVDV感染豚鼠血液中的病毒RNA水平。文中以昆虫细胞表达系统制备BVDV病毒样颗粒并评价其免疫原性，为进一步研制BVD病毒样颗粒疫苗奠定了基础。

关键词:牛病毒性腹泻病毒;杆状病毒;病毒样颗粒;免疫原性

Abstract:

In order to obtain virus-like particles (VLPs) for prevention of bovine viral diarrhea virus 1 (BVDV-1), the C-E^rns-E1-E2 region was cloned into a pFastBacDaul vector for generating the recombinant Bacmid-BVDV-1 in DH10Bac Escherichia coli. The recombinant baculovirus Baculo-BVDV-1 was produced by transfecting the Sf9 cells with Bacmid-BVDV-1. The expressed protein and the assembled VLPs were determined by immunofluorescence, Western blotting and electron microscopy. Guinea pigs were immunized with inactivated VLPs coupled with the Montanide ISA-201 adjuvant. The immunogenicity of VLPs was evaluated by monitoring the humoral immune response with neutralizing antibody titer determination, as well as by analyzing the cell-mediated immune response with lymphocyte proliferation assay. The protective efficacy of VLPs was evaluated by challenging with 10⁶ TCID₅₀ virulent BVDV-1 strain AV69. The results showed that the recombinant Baculo-BVDV-1 efficiently expressed BVDV structural protein and form VLPs in infected Sf9 cells. The immunization of guinea pigs with VLPs resulted in a high titer (1:144) of neutralizing antibody, indicating an activated cellular immunity. Significantly lower viral RNA in the blood of the post-challenged immunized guinea pigs was observed. The successful preparation of BVDV VLPs with insect cell expression system and the observation of the associated immunogenicity may facilitate further development of a VLPs-based vaccine against BVD.

Key words:bovine viral diarrhea virus;baculovirus;virus-like particles;immunogenicity