表达猪流行性腹泻病毒变异株S基因重组伪狂犬病病毒的构建与鉴定  

Construction and Characterization of A Recombinant Pseudorabies Virus Expressing S Gene of A Porcine Epidemic Diarrhea Virus Variant

作　　者：张传健[1,2] 郭仕琦 郭容利 刘晓琳[1,2,3] 陈赛赛 王志胜 曾容愚[5] 咼荣兵 王继春 ZHANG Chuanjian;GUO Shiqi;GUO Rongli;LIU Xiaolin;CHEN Saisai;WANG Zhisheng;ZENG Rongyu;WAI Rongbing;WANG Jichun(Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;TECON Pharmaceutical(Suzhou)Co.,Ltd,Suzhou 215000,China;Animal Husbandry and Veterinary Station,Longchi Street,Liuhe District,Nanjing City,Nanjing 211500,China)

机构地区：[1]江苏省农业科学院动物免疫工程研究所,南京210014 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009 [3]南京农业大学动物医学院,南京210095 [4]江苏省农业科学院兽医研究所,南京210014 [5]天康制药(苏州)有限公司,苏州215000 [6]南京市六合区龙池街道畜牧兽医站,南京211500

出　　处：《中国动物传染病学报》2023年第2期51-58,共8页Chinese Journal of Animal Infectious Diseases

基　　金：江苏省自然科学基金(BK20181243)。

摘　　要：为研制预防猪流行性腹泻病毒(PEDV)变异株感染的重组伪狂犬病病毒(PRV)活载体疫苗,本研究应用PRV变异株的细菌人工染色体和同源重组技术,将PEDV流行株的S基因表达盒插入PRV基因组UL40与UL41之间的非编码区,构建重组病毒rPRV-S(UL40-41),该毒株还缺失了TK和gE基因。生长动力学显示该毒株在猪睾丸(ST)细胞的增殖效率与亲本毒株相似,表明S基因表达盒的插入对病毒生长性能无显著影响。重组病毒在ST细胞传至15代,PCR和测序鉴定S基因未发生碱基的丢失和变异,间接免疫荧光显示重组病毒感染鸡胚成纤维细胞后可稳定表达S蛋白。应用该毒株接种断奶仔猪,产生低水平的针对PEDV的抗体。该研究为PRV活载体疫苗的构建奠定了基础。To develop a recombinant Pseudorabies virus(PRV)vaccine against a Porcine epidemic diarrhea virus(PEDV)variant,this study constructed a recombinant PRV with TK and gE deletion(rPRV-S(UL40-41))expressing S gene of PEDV variant in which S expression cassette was inserted in the UL40-41(noncoding region)through bacterial artificial chromosome of AH02LA and homologous recombination.Growth kinetics experiments showed that proliferation efficiency of rPRV-S(UL40-41)in swine testis(ST)cells was similar to that of the parental strain,indicating that the inserted S expression cassette had no effect on virus growth performance.The recombinant virus was passaged 15 times on ST cells,and the correct sequences of the inserted S expression cassette without base deletions and variation were confirmed by PCR and sequencing.Indirect immunofl uorescence assay revealed that S protein was stably expressed in chicken embryo cells infected with rPRV-S(UL40-41).Weaned piglets inoculated with rPRV-S(UL40-41)induced a low level of anti-PEDV antibodies.This study laid a foundation for the construction of PRV live vector vaccine.

关 键 词：伪狂犬病病毒 猪流行性腹泻病毒 重组病毒 S基因 细菌人工染色体 

分 类 号：S852.65[农业科学—基础兽医学]