表达犬瘟热H基因重组伪狂犬病毒的构建及生物学特性研究

摘要

[目的]构建表达犬瘟热Onderstepoort株H蛋白的重组伪狂犬病毒,并研究其生物学特性.[方法]通过RT-PCR方法获得Onderstepoort株H基因,插入pcDNA3.1(+)建立好完整的真核细胞表达盒并将此表达盒亚克隆到转移栽体p8AA上.在此基础上再将报告基因LacZ的表达盒插入转移栽体,命名为p8AAZH.将p8AAZH与伪狂犬病毒(PRV) Bartha-K61株基因组共转染至BHK-21细胞中进行基因重组包装出毒,待细胞病变后收集病毒液.通过蓝色蚀斑筛选、PCR、电镜观察以及Western blot,筛选纯化重组病毒并鉴定目的基因的表达.同时在BHK-21细胞上测定重组病毒的生长曲线.[结果]获得了表达H蛋白的重组伪狂犬病毒,重组病毒与亲本Bartha-K61株的生长曲线、病变特征相一致.[结论]成功构建了表达犬瘟热Onderstepoort株H蛋白的重组伪狂犬病毒,H基因的插入不影响重组病毒的增殖特性,为犬瘟热活病毒载体疫苗的研制与开发打下了基础.%[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and to investigate its biological characters. [ Method] H gene of canine distemper virus ( CDV) strain Onderstepoort was produced by RT-PCR,inserted into pcD-NA3. 1( + ) vector to construct a expression cassette,which was then subcloned into transfer vector p8AA, prior to the insertion of LacZ expression cassette. The resulting new transfer vector was named as p8AAZH. Subsequently ,p8AAZH was co-transfected with the genome of pseudorabies virus (PRV) Bartha-K61 into BHK-21 cells to enable gene recombination and virus package,and the virus solution was collected as cyto-pathic effect occurring. A series of procedures including blue plaque purification, PCR identification, observation under electron microscope and Western blot were carried out to screen the recombinant pseudorabies virus and to identify the protein expression of target gene. Meanwhile, growth curve of the recombinant virus was determined in BHK-21 cells. [ Result] The H gene had been inserted into the genome of Bartha-K61 * strain,and rPRV-H was the same as Bartha-K61 in the one-step growth curve and cytopathic effect in BHK-21 cells. [ Conclusion] The recombinant pseudorabies virus was constructed ,and the insertion of H gene did not influence proliferation of recombinant virus,which laid a foundation for development of recombinant canine distemper virus vaccine.