CAJ | 학술논문

[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载体p8AA上。在此基础上再将报告基因LacZ的表达盒插入转移载体,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Westernblot,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基础。
[목적]구건표체견온열Onderstepoort주H단백적중조위광견병독,병연구기생물학특성。[방법]통과RT-PCR방법획득Onderstepoort주H기인,삽입pcDNA3.1（＋）건립호완정적진핵세포표체합병장차표체합아극륭도전이재체p8AA상。재차기출상재장보고기인LacZ적표체합삽입전이재체,명명위p8AAZH。장p8AAZH여위광견병독（PRV）Bartha-K61주기인조공전염지BHK-21세포중진행기인중조포장출독,대세포병변후수집병독액。통과람색식반사선、PCR、전경관찰이급Westernblot,사선순화중조병독병감정목적기인적표체。동시재BHK-21세포상측정중조병독적생장곡선。[결과]획득료표체H단백적중조위광견병독,중조병독여친본Bartha-K61주적생장곡선、병변특정상일치。[결론]성공구건료표체견온열Onderstepoort주H단백적중조위광견병독,H기인적삽입불영향중조병독적증식특성,위견온열활병독재체역묘적연제여개발타하기출。
[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstepoort was produced by RT-PCR,inserted into pcDNA3.1（＋）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 cytopathic 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 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.