鹅源副黏病毒致弱株的“拯救”及分子生物学鉴定

Attenuation of GPMV NA-1 strain by reverse genetics technique

以本课题组前期构建的鹅源副黏病毒NA-1株的全长感染性克隆为模板设计2对引物,用overlap PCR方法将其F蛋白裂解位点处的112、115和117位碱性氨基酸定点突变成为具有典型弱毒株特征的非碱性氨基酸。随后将突变后的F基因序列替换全长感染性克隆的对应序列,构建突变后的克隆质粒FL-cDNA-F′。将改造后的感染性克隆质粒与pCI-NP、pCI-P和pCI-L 3个辅助表达质粒共转染VT7细胞系,成功拯救出了致弱的鹅源副黏病毒。经过分子生物学鉴定,该毒株F基因序列具有典型的弱毒株特征。救获病毒的鸡胚最小致死剂量平均死亡时间(MDT)为96h,表明救获的病毒毒力已被成功致弱,可以作为当前流行的鹅副黏病毒病的一个较为理想的疫苗候选株。

Based on the reverse genetics system established for goose paramyxovirus（GPMV） NA-1 strain,we recombined the full-length plasmid which was generated by converting the multi-basic amino acid sequence of the F0 protein cleavage region to the non-basic amino acid sequence characteristic of non-virulent NDV strain.The full-length plasmid FL-cDNA-F′ with three helper plasmids（pCI-NP,pCI-P and pCI-L） were then co-transfected into VT7 cells expressing T7 RNA polymerase.After inoculation of the transfected cell culture supernatant into specific-pathogen-free（SPF） embryonated eggs,the recombinant infectious virus was generated.The typical morphology of the rescued GPMV was detected in the electronic microscope.The mean death time（MDT） of the rescued virus was 96 h,indicating that was highly attenuated.This attenuated genotype VⅡd NDV could be a novel vaccine candidate in controlling the current epidemic of ND in geese of China.