【摘要】 为构建新型新城疫病毒(NDV)载体疫苗,前期我们将La Sota全长c DNA中的F基因编码区替换为基因Ⅶ型NDV的F基因编码区,并将其裂解位点突变为弱毒株的序列,构建获得了p NDFLSP-m F重组质粒。为探索p NDFLSP-m F是否能够作为载体有效地表达外源蛋白,在p NDFLSP-m F的P基因与M基因之间引入PmeⅠ酶切位点,并将EGFP基因编码区克隆至构建好的p NDFLSP-m F载体中,构建含有EGFP基因的重组质粒p NDFLSP-m F-EGFP。将p NDFLSP-m F-EGFP以及辅助质粒p CI-NP-K、p CI-P-K、p CI-LK共转染表达T7 RNA聚合酶的重组痘病毒预感染的BSR-T7/5细胞,拯救获得了重组病毒La Sota-m FEGFP。通过RT-PCR证明,被拯救的重组病毒中含有插入的外源基因EGFP。荧光观察和Western-blot分析结果表明,EGFP蛋白能在重组病毒中稳定表达。生物学特性测定结果显示,La Sota-m F-EGFP具有低毒力毒株特征,MDT为144 h,能够在鸡胚上有效地复制,EID50为1×108.75/0.1 m L。上述研究结果为研制和开发新型NDV载体疫苗奠定了基础。更多还原

【Abstract】 To generate a novel NDV vaccine vector,the coding region of La Sota F gene was replaced by the coding region of genotype VII NDV F gene.Subsequently,the cleavage site was mutated to the motif of avirulent strain to obtain the recombinant plasmid named p NDFLSP-m F.In this study,to examine whether p NDFLSP-m F can effectively express foreign protein,restriction enzyme site of Pme I was introduced into p NDFLSP-m F between P and M genes,then the coding region of enhanced green fluorescent protein was inserted into the Pme I site of p NDFLSP-m F,generating the plasmid named p NDFLSP-m F-EGFP.Then the p NDFLSP-m F-EGFP together with the helper plasmids p CI-NP-K,p CI-P-K,and p CI-L-K were cotransfected into BSR-T7/5 cells which were pre-infected with recombinant fowl poxvirus expressing T7 RNA ploymerase to obtain recombinant virus La Sota-m F-EGFP.The stable presence of inserted EGFP gene in La Sota-m F-EGFP was confirmed via RT-PCR.Fluorescence assay and Western-blotting results showed that the EGFP could be stable expressed.Biological characterization results showed that the MDT of La Sota-m F-EGFP was 144 h,indicating that La Sota-m F-EGFP was an avirulent strain.This recombinant virus could effectively replicate in chicken embryos,with a EID50 of 108.75/0.1 m L.Our results provide foundation for the development of novel NDV live vector vaccine.更多还原