表达基因Ⅶ型新城疫病毒F蛋白重组LaSota疫苗株的构建

Construction of a recombinant vaccine strain expressing genotype Ⅶ Newcastle disease virus fusion protein

基因Ⅶ型新城疫病毒(NDV)为我国NDV的主要流行病毒株,而经典疫苗株LaSota不能对鸡群起到有效的免疫保护。为研发针对NDV基因Ⅶ型流行病毒株的疫苗,本研究采用反向遗传操作技术,将LaSota的F基因编码区(CDS)替换为基因Ⅶ型流行病毒株CK/CH/LHLJ/1/06株的F基因CDS,并将替换后的F蛋白的裂解位点突变为LaSota的裂解位点,构建重组质粒pNDFLsp-mF。将pNDFLsp-mF与表达NP、P和L蛋白的辅助质粒共转染BSR-T7/5细胞,拯救获得表达基因Ⅶ型NDV F蛋白的重组病毒(rLaSota-mF)。生长曲线结果显示,与LaSota相似,rLaSota-mF能够在鸡胚中有效复制,病毒滴度EID50为109.375/0.1 mL。生物学特性测定结果显示rLaSota-mF的MDT为144.6 h,表明其为低毒力病毒株。将拯救获得的病毒连续传15代后所替换的基因及突变的位点能够在r LaSota-mF中稳定存在。本研究为研究与开发针对NDV不同基因型流行病毒株的疫苗奠定基础。

Genotype VII Newcastle disease virus （NDV） has been identified as the predominant NDV genotype in China, and the classical LaSota vaccine strain is unable to provide effective immune protection against genotype VII NDV. To develop vaccines against prevalent strains, the F gene ofNDV LaSota strain was replaced by that of genotype VII strain CK/CH/FHLJ/1/06, and the motif of F protein cleavage site was mutated to that of LaSota to construct the recombinant plasmid of pNDFLsp-mF, which was transfected into BSR-T7/5 cells together with helper plasmids （encoding NP, P, and L proteins） to rescue the recombinant NDV LaSota （rLaSota-mF） expressing the F protein of genotype VII NDV. The results of multicycle growth kinetics showed that rLaSota-mF was similar to the parental virus and replicate effectively in chicken eggs with the maximum titer of 10^9.375/0.1 mL. The MDT value of rLaSota-mF was 144.6 h, indicating rLaSota-mF was an avirulent strain. Moreover, after serial passaged 15 times, the replaced gene and mutated sites were stably integrated in rLaSota-mF. This study would facilitate the development of the novel vaccines against different genotype of prevalent NDVs.