基于F和HN蛋白的基因Ⅶ型新城疫病毒嵌合疫苗的构建及免疫效力评估

Construction and Immune Efficacy Evaluation of a GenotypeⅦNewcastle Disease Virus Chimeric Vaccine Based on F and HN Proteins

【目的】试验旨在构建一种基因Ⅶ型新城疫病毒(Newcastle disease virus,NDV)嵌合疫苗,并对其免疫效力进行评估。【方法】利用反向遗传学技术,以含有禽偏禽腮腺炎病毒2型(Avian metaavulavirus-2,AMAV-2)Y2株基因组的重组质粒pT7-Y2为模板,将Y2株的F和HN蛋白的胞外区替换为基因Ⅶ型NDV HB0901株的F和HN蛋白的胞外区,将HB0901株的F蛋白裂解位点突变为LaSota弱毒株的F蛋白裂解位点,构建嵌合重组病毒rY2-FHNR株。对rY2-FHNR株的增殖特性、致病力及遗传稳定性等生物学特性进行检测,并通过接种2周龄SPF鸡评估rY2-FHNR株的免疫原性及其免疫血清与Y2株的交叉反应性,利用NDV NP蛋白的间接ELISA方法对免疫血清进行检测,验证其鉴别诊断效果。【结果】试验成功获得了嵌合重组病毒rY2-FHNR株,生物学特性检测结果显示,rY2-FHNR株在鸡胚中的增殖滴度和致病性符合弱毒特征,其鸡胚传代的遗传稳定性良好。rY2-FHNR株可诱导机体产生针对NDV的抗体,且免疫血清不与rY2株抗原发生交叉反应。通过NDV NP ELISA抗体检测方法可以实现区分疫苗免疫与野毒感染。【结论】本试验研发了一种基因Ⅶ型NDV嵌合候选疫苗,为基因Ⅶ型NDV的监测、防控和净化提供了技术支撑。

【Objective】This study was aimed to construct a genotypeⅦNewcastle disease virus(NDV)chimeric vaccine,and access its immune efficacy.【Method】The F and HN protein ectodomains of recombinant plasmid pT7-Y2 of the Avian metaavulavirus-2(AMAV-2)Y2 strain were replaced with the corresponding F and HN protein ectodomains of genotypeⅦNDV HB0901 strain by utilizing reverse genetic technology.Then F protein cleavage site of HB0901 strain was transformed into that of lentogenic LaSota strain,and the chimeric virus was recovered and named rY2-FHNR.The growth characteristics,pathogenicity and genetic stability of rY2-FHNR strain were evaluated,then 2-week-old SPF chickens were immunized with rY2-FHNR to determine the immunogenicity and cross-reactivity between immune serum and Y2 strain.The immune serum was identified by indirect NDV NP ELISA to verify its diagnosis effect.【Result】Chimeric recombinant virus strain rY2-FHNR was constructed successfully.The biological characteristics results showed that the proliferation titer and pathogenicity in chicken embryos of rY2-FHNR strain were avirulent,while the chimeric virus was genetic stable when serially passaged in chicken embryos.Immunization with rY2-FHNR stimulated antibodies against NDV,and the immune serum had no cross-reactivity with rY2 strain.Through indirect NDV NP ELISA could distinguish infected animals from vaccinated animals.【Conclusion】This study developed a genotypeⅦNDV chimeric vaccine candidate,which could be used as chimeric vaccine,provided technological support for the monitoring,prevention and eradication of genotypeⅦNDV.