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

作　　者：赵冉[1,2] 齐甜铭 孙军峰[2] 刘怀然[2] 韩宗玺[2] 杨孝朴[1] 刘胜旺[2] ZHAO Ran QI Tian-ming SUN Jun-feng LIU Huai-ran HAN Zong-xi YANG Xiao-pu LIU Sheng-wang(College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China State Key Loboratory of Veterinary Biotechnology/Division of A vian Infectious Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences ,Harbin 150001, China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽呼吸道病研究团队,黑龙江哈尔滨150069

出　　处：《中国兽医科学》2017年第7期843-849,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31502088);黑龙江省自然科学基金项目(QC2015038);国家公益性行业(农业)科研专项(201303033)

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。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 Pme 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 10^8.75/0.1 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 

分 类 号：S852.659.5[农业科学—基础兽医学]