基于全基因组序列合成的禽副黏病毒Ⅱ型反向遗传操作系统的建立

Establishment of reverse genetic system of avian paramyxovirus typeⅡbased on the whole genome synthesis

为建立禽副黏病毒Ⅱ型(avian paramyxovirus 2,APMV-2)的反向遗传操作平台,对NCBI已经公布的12条APMV-2全基因组序列进行分析比对,选取较为保守的F8株为模板,将该毒株基因组中特有的10处核苷酸突变为其他毒株共有的一致序列,增加一段大部分毒株共有的非编码区序列,获得了1株新的APMV-2(命名为Y2株)的全基因组序列。通过人工合成和克隆的方法,构建并获得了Y2株全长cDNA克隆质粒和3个分别表达NP、P和L的辅助质粒。将4个重组质粒共转染至感染痘病毒的BHK-21细胞,成功拯救出APMV-2Y2株。生物学特性测定结果显示,Y2株的鸡胚最低致死剂量的平均致死时间(MDT)大于168h,1日龄雏鸡的脑内致病指数(ICPI)为0.09,判定为弱毒株,鸡胚尿囊液的病毒含量为10^(8.83) EID_(50)/mL,HA效价为9log_(2)。病毒增殖曲线测定结果显示,Y2株感染BHK-21细胞后60h滴度达到峰值。APMV-2Y2株反向遗传操作系统的建立为该病原的致病机理研究及新型禽用疫苗研发提供了依据。

To establish the reverse genetic system of the avian paramyxovirus type 2(APMV-2),a total of 12complete genome sequences of APMV-2published in NCBI were aligned,and the more conservative APMV-2F8strain was selected as the template.Ten nucleotide sites in the genome of F8strain were mutated into the consensus sequence shared by the other strains,then the complete genome sequence of a new APMV-2strain named Y2was obstained.Through artificial synthesis and clone technology,the plasmid containing the full-length cDNA clone of Y2strain and the expressing plasmids of NP,P and L gene were constructed and co-transfected into BHK-21cells preinoculated with MAV-T7.Then the recombinant APMV-2strain Y2was rescued and tested for its biological characterization.The results showed that the mean death time in chicken embryos(MDT)of Y2strain was higher than 168h,the intracerebral pathogenicity index in day-old chickens(ICPI)was 0.09.The Y2strain belonged to the lentogenic pathotype.The viral titer of Y2 strain propogated in chicken embryo was 10^(8.83) EID_(50)/mL,and its HA titer was 9log_(2).The results of the virus proliferation curve assay showed that the Y2strain reached a peak titer 60hafter infection with BHK-21cells.The establishment of reverse genetic system of APMV-2strain Y2provided a technical platform for the pathogenicity research of APMV-2and development of novel aivian vaccines.