鸡毒支原体TM-1蛋白嵌合新城疫病毒样颗粒的构建及鉴定

Construction and identification of chimeric Newcastle disease virus-like particles of Mycoplasma gallisepticum TM-1protein

鸡毒支原体(Mycoplasma gallisepticum,MG)感染引起的禽类的慢性呼吸道病(chronicrespiratory disease,CRD)与新城疫(Newcastle disease,ND)是严重危害禽类的两种重要疾病,可呈并发或继发感染,给养禽业造成巨大的经济损失。因此,研制安全有效、免疫程序简便的ND-CRD二联疫苗显得极为重要。本研究将MG国际参考株MG R(low)株的保护性蛋白TM-1采取以替换新城疫病毒(Newcastle disease virus,NDV)NDV-HN胞外域的方式与我国NDV流行株(基因Ⅶ型)的HN、F蛋白共同嵌合至病毒样颗粒(VLPs)表面,使其表面展示3种结构蛋白MG-TM-1、NDV-HN和NDV-F。经鉴定,间接免疫荧光显示该cVLPs各组分蛋白均正确表达;Western blot结果显示,该cVLPs可与NDV M、NDV F、NDV HN、MG TM-1的特异性阳性血清发生反应;透射电镜观察可见大小约为100nm的粒子,表面展示纤突结构,呈现典型的病毒形态,与野生型NDV形态相似,最终成功构建嵌合型病毒样颗粒MG-ND cVLPs。本试验成功制备的MG-ND cVLPs为研制新型ND与CRD二联疫苗提供候选疫苗,并为当前NDV与MG共感染的防控提供新策略。

Avian chronic respiratory disease(chronic respiratory disease,CRD)caused by Mycoplasma gallisepticum(Mycoplasma gallisepticum,MG)infection and Newcastle disease(ND)are two serious poultry disease.The two diseases can be concurrent or secondary infection,causing huge economic losses to the poultry industry.Therefore,it is extremely important to develop a Newcastle disease-poultry chronic respiratory disease dual vaccine that is safe,effective and simple in immunization procedures.In this study,the protective protein TM-1of the international reference strain of Mycoplasma gallisepticum MG R(low)strain was chimeric with the HN and F proteins of the epidemic strain of Newcastle disease virus(geneⅦ)by replacing the extracellular domain of NDV-HN.Therefore,three structural proteins,MG-TM-1,NDV-HN,NDV-F were displayed on the surface of VLPs.The results of indirect immunofluorescence showed that each component protein of VLPs was correctly expressed.The results of Western blot showed that the cVLPs could react positively with the positive sera of NDV M,NDV F,NDV HN,and MG TM-1.The results of transmission electron microscopy showed that the particles were about 100nm in size and had a fibrous surface structure with a typical viral morphology,similar to the previously reported Newcastle disease virus-like particles,indicating that the chimeric virus-like particles MGND cVLPs were successfully constructed.The MG-ND cVLPs successfully prepared in this experiment provides candidate vaccines for the development of a new combination vaccine of poultry ND and CRD,and provides a new strategy for the prevention and control of the current Newcastle disease virus and Mycoplasmagallisepticum co-infection.