表达禽网状内皮组织增生病病毒gp90蛋白的重组马立克氏病病毒的构建及生物学特性分析  

Construction and characterization of a recombinant Marek's disease virus expressing gp90 protein of reticuloendotheliosis virus

作　　者：许曾焜 李凯[1] 刘永振[1] 高立[1] 刘长军[1] 张艳萍[1] 高玉龙[1] 祁小乐[1] 崔红玉[1] 王笑梅[1] XU Zeng-kun;LI Kai;LIU Yong-zhen;GAO Li;LIU Chang-jun;ZHANG Yan-ping;GAO Yu-long;QI Xiao-le;CUI Hong-yu;WANG Xiao-mei(Avian Immunosuppressive Diseases Division,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]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/禽免疫抑制病研究创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2020年第9期867-871,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省青年科学基金(QC2016042);国家重点研发计划(2016YFE0203200)。

摘　　要：禽网状内皮组织增生病(RE)是禽类一种重要免疫抑制性疾病,目前尚无有效的商品化疫苗预防该病。为研制表达RE病毒(REV)gp90蛋白的重组鸡马立克氏病病毒(MDV),本研究构建了含有REV保护性抗原基因gp90的pCAGGS-gp90表达质粒,将gp90表达框架克隆入pENTR1载体,获得重组质粒pENTR1-gp90。通过pENTR1-gp90与p814-5US2KanccdB重组粘粒的LR反应,将gp90表达框架插入p814-5粘粒中MDV 814疫苗株基因组US2基因内,构建重组粘粒p814-5-gp90。将该重组粘粒转染鸡胚成纤维细胞(CEF),获得重组MDV r814-gp90株。将该重组病毒在CEF中连续传至20代后对第5、10、15、20代病毒经PCR扩增并测序鉴定;利用间接免疫荧光试验(IFA)和western blot检测重组MDV感染细胞中gp90蛋白的表达;绘制重组MDV的生长曲线,分析其体外复制特性。结果显示,正确构建了含有gp90表达框架的重组粘粒p814-5-gp90。将该重组粘粒转染CEF后能够引起典型的蚀斑病变,且蚀斑形态大小与亲本病毒814株无明显差异。PCR及测序鉴定结果显示,重组病毒r814-gp90基因组中正确插入gp90基因表达框架,且其能够在该重组病毒中稳定存在。IFA及western blot检测结果表明,r814-gp90株能够在感染的CEF中表达gp90蛋白。体外生长曲线显示,r814-gp90株在CEF中的复制能力与亲本病毒无明显差异。以上结果表明,本研究正确构建了表达REV保护性抗原gp90基因的重组MDV,为研制RE重组MDV活载体疫苗奠定了基础,对RE以及REV与MDV混合感染的防控具有重要意义。Reticuloendotheliosis(RE)is an important immunosuppressive disease in poultry.However,there is no effective commercial vaccine to control and prevent the disease currently.To develop a recombinant Marek's disease virus(MDV)vector vaccine against reticuloendotheliosis virus(REV),the pCAGGS-gp90 expression plasmid containing the REV protective antigen gp90 gene was constructed,and this expression cassette of gp90 was cloned into pENTR1 vector to construct pENTR1-gp90 recombinant plasmid.Then through the LR reaction between pENTR1-gp90 and p814-5US2KanccdB recombinant fosmid,the gp90 expression cassette was inserted into the US2 gene of the MDV 814 vaccine strain in the p814-5 fosmid to construct the p814-5-gp90 recombinant fosmid.The recombinant fosmids were transfected into the chicken fibroblast(CEF)to rescue the recombinant virus r814-gp90.After the recombinant virus was continuously passaged to 20 generations in CEF,the 5th,10th,15th,and 20th generation viruses were identified by PCR and sequenced.The gp90 expression of the recombinant virus was detected by immunofluorescence assay(IFA)and western blot.The replication ability of the recombinant virus was analyzed by the replication kinetics studies.The results showed that the recombinant fosmid p814-5-gp90 containing the gp90 expression cassette was correctly constructed.The recombinant virus r814-gp90 can cause typical cytopathic effect on CEF,and the plaque size was not significantly different from that of the parental virus 814 strain.The PCR and sequencing results showed that the gp90 gene expression cassette was correctly inserted into the recombinant virus r814-gp90 genome,and it can exist stably in the recombinant virus.The results of IFA and western blot indicated that r814-gp90 could express gp90 protein in the infected CEF.The replication kinetics study in vitro showed that the replication ability of r814-gp90 in CEF is not significant difference from that of the parent virus.The above results showed that a recombinant MDV expressing the REV protectiv

关 键 词：禽网状内皮组织增生病 gp90蛋白 马立克氏病病毒 重组病毒 

分 类 号：S852.65[农业科学—基础兽医学]