基因Ⅶ型新城疫病毒样颗粒作为疫苗候选株的免疫效果

Immune effect of genotypeⅦ ND VLPs as a vaccine candidate

利用昆虫细胞/杆状病毒表达载体系统,将已构建编码新城疫病毒(NDV)M、F和HN结构基因的3种单顺反子重组杆粒共感染悬浮培养的Sf9细胞以大量制备新城疫病毒样颗粒(ND VLPs)。收集细胞培养上清经超速离心浓缩,蔗糖密度梯度纯化后,利用Western blot检测和透射电镜观察。结果显示,3种目的蛋白均正确表达且组装形成与原病毒粒子形态相似的颗粒,测定其血凝活性效价可达12log2。动物试验结果表明,与LaSota灭活油乳苗相比,将纯化后的病毒样颗粒以含25μg蛋白的剂量或联合佐剂免疫商品蛋鸡后能产生较高水平的HI抗体,且可有效减少攻毒鸡只口咽、泄殖腔途径的排毒时间,从而减少病毒在健康鸡群中的传播感染机会;表明所研制的基因Ⅶ型ND VLPs可作为疫苗候选株具有很大的防控优势。

In order to study the effect of the genotype Ⅶ Newcastle disease virus-like particles （ND VLPs） ,the monocistronic recombinant bacmids containing M,F and HN gene of Newcastle disease virulent strain was constructed by the insect cell/baculovirus expression vector system. Meanwhile,Sf9 cells were co-infected with recombinant baculovirus to prepare ND VLPs on a large scale. Then,the collected cell supernatants were purified and concentrated by ultracentrifugation and sucrose density gradient. The results of Western blot and transmission electron microscopy have shown that the three target proteins were correctly expressed and assembled into particles whose configuration is similar to that of Newcastle disease virus. The titer of hemagglutination activity could reach 121og2. In addition, the result of animal assay has shown that the commercial chickens immunized by the purified virus-like particles containing 25μg proteins or combined adju vant can produce higher HI antibodies compared with that of inactivated LaSota oil emulsion vaccine,and thus effectively reduce virus shedding from oropharyngeal, cloacal, thereby reducing the chance of virus infection in healthy flocks. Overall,the results above showed that the genotype ND VLPs has a great advantage as vaccine candidate.