表达呼吸道合胞病毒F蛋白的新城疫重组病毒的构建及其免疫原性

Construction and immunogenicity of recombinant Newcastle disease virus expressing respiratory syncytial virus F protein

目的利用新城疫病毒(Newcastle disease virus,NDV)反向遗传系统构建表达呼吸道合胞病毒(respiratory syncytial virus,RSV)F蛋白的重组病毒,并以BALB/c小鼠为动物模型,评价其免疫原性。方法在已建立的NDV反向遗传系统的基础上,将RSV F基因插入NDV全长cDNA克隆中,构建并拯救获得表达RSV F蛋白的重组病毒rLS/RSV-F。采用间接免疫荧光(indirect immunoinfluscent assay,IFA)法鉴定RSV F蛋白的表达;以HA、病毒半数鸡胚感染量(50%infective dose,EID50)、半数组织感染量(50%tissue infectious dose,TCID50)及生长曲线等生物学指标对其生物学特性进行鉴定;将rLS/RSV-F滴鼻免疫BALB/c小鼠,于免疫后第49天经眼眶后静脉丛采血,分离血清,ELISA法检测IgG抗体效价,中和试验检测中和抗体效价。结果成功拯救表达RSV F蛋白的NDV重组病毒rLS/RSV-F,具有与亲本病毒相似的感染能力及生长动力学特性;rLS/RSV-F可刺激小鼠机体产生较高水平的针对RSV F蛋白的特异抗体及RSV中和抗体,与亲本病毒对照组相比,差异均有统计学意义(P <0. 05)。结论成功拯救了表达RSV F蛋白的NDV重组病毒,并通过动物实验证明重组病毒rLS/RSV-F具有较好的免疫原性,为RSV疫苗的研发提供了新思路。

Objective To construct a recombinant virus expressing respiratory syncytial virus(RSV)F protein by reverse genetic system of Newcastle disease virus(NDV)and evaluate its immunogenicity in BALB/c mice. Methods On the basis of successful establishment of reverse genetic system of NDV in the early stage,F gene of RSV was inserted into the full-length cDNA plasmid of NDV to construct and rescue the recombinant virus expressing RSV F protein. The expression of RSV F protein was confirmed by indirect immunofluorescence assay(IFA). The biological characteristics of recombinant virus were identified with HA,50% infective dose(EID50),50% tissue infectious dose(TCID50)and growth curve.BALB/c mice were immunized intranasally with the constructed recombinant virus rLS/RSV-F,of which the serum samples were collected on day 49 after immunization,and determined for IgG by ELISA,and for neutralizing antibody titer by neutralization test. Results Recombinant virus rLS/RSV-F expressing RSV F protein was successfully rescued,of which the infectious ability and dynamic growth characteristics were similar to its parental virus. The recombinant virus stimulated high levels of specific antibody against RSV F protein and RSV neutralizing antibody in mice. The antibody levels showed significant difference with those stimulated by the parental virus as control(P < 0. 05). Conclusion The recombinant NDV expressing RSV F protein was successfully rescued,which showed good immunogenicity as proved by animal experiment. It provided a new idea for the development of RSV vaccine.