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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。
Three insect cells / baculovirus expression vector system were used to co-infect Sf9 cells cultured in suspension with three monocistronic recombinant baculovirus plasmids encoding the M, F and HN structural genes of Newcastle disease virus (NDV) to produce Newcastle disease Virus-like particles (ND VLPs). The collected cell culture supernatant was concentrated by ultracentrifugation and purified by sucrose density gradient. Western blot and transmission electron microscopy were used to observe the cell culture supernatants. The results showed that the three kinds of target proteins were correctly expressed and assembled to form granules with the same virion morphology. The titer of hemagglutination activity could reach 12 log 2. Animal experiments showed that compared with LaSota inactivated oil emulsion, the purified virus-like particles can produce higher HI antibody after being immunized with commercial 25μg protein or commercial adjuvant, and can effectively reduce Toxicity of chickens with orogenic and cloacal pathogenic factors in chickens was reduced, and the chances of transmission of virus in healthy chickens were reduced. It indicated that the NDV type Ⅶ developed could be used as candidate vaccine candidate for prevention and control.