中国兽医科学2017，Vol.47Issue(7)：843-849,7.DOI:10.16656/j.issn.1673-4696.2017.07.007
表达EGFP蛋白新型重组新城疫病毒疫苗载体的构建
Development of a novel recombinant Newcastle disease virus vector expressing green fluorescent protein
摘要
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 pNDFLSP-mF.In this study,to examine whether pNDFLSP-mF can effectively express foreign protein,restriction enzyme site of P me Ⅰ was introduced into pNDFLSP-mF between P and M genes,then the coding region of enhanced green fluorescent protein was inserted into the Pme Ⅰ site of pNDFLSP-mF,generating the plasmid named pNDFLSP-mF-EGFP.Then the pNDFLSP-mF-EGFP together with the helper plasmids pCI-NP-K,pCI-P-K,and pCI-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-mF-EGFP.The stable presence of inserted EGFP gene in La Sota-mF-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-mF-EGFP was 144 h,indicating that La Sota-mF-EGFP was an avirulent strain.This recombinant virus could effectively replicate in chicken embryos,with a EID50 of 108.75/0.1 mL.Our results provide foundation for the development of novel NDV live vector vaccine.关键词
新城疫病毒/基因Ⅶ型/绿色荧光蛋白Key words
Newcastle disease virus/genotype type Ⅶ/EGFP分类
农业科技引用本文复制引用
赵冉,齐甜铭,孙军峰,刘怀然,韩宗玺,杨孝朴,刘胜旺..表达EGFP蛋白新型重组新城疫病毒疫苗载体的构建[J].中国兽医科学,2017,47(7):843-849,7.基金项目
国家自然科学基金项目(31502088) （31502088）
黑龙江省自然科学基金项目(QC2015038) （QC2015038）
国家公益性行业(农业)科研专项(201303033) （农业）
