表达大熊猫源犬瘟热病毒H蛋白复制缺陷型重组腺病毒的构建及鉴定  

Construction and Identification of Replication-Deficient Recombinant Adenovirus Expressing H Protein of Canine Distemper Virus Isolated from Giant Panda

作　　者：陈廷炜 孟宪踊 王铁成 高玉伟 冯娜 夏咸柱[1,2] CHEN Tingwei;MENG Xianyong;WANG Tiecheng;GAO Yuwei;FENG Na;XIA Xianzhu(College of Animal Science and Technology,Jilin Agricultural University,Changchun,130118,China;Military Veterinary Institute of the Academy of Military Medical Sciences,Key Laboratory of Zoonosis Prevention and Control of Jilin Province,Changchun,130122,China)

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]军事医学研究院,军事兽医研究所,吉林省人兽共患病预防与控制重点实验室,长春130122

出　　处：《野生动物学报》2021年第3期747-754,共8页CHINESE JOURNAL OF WILDLIFE

基　　金：国家重点研发计划项目(2017YFD0501704)。

摘　　要：为构建能够表达大熊猫源犬瘟热病毒(CDV)分离株giant panda/SX/2014 H蛋白的复制缺陷型重组腺病毒,利用反转录聚合酶链反应(RT-PCR)扩增株giant panda/SX/2014 H基因,通过TOPO连接克隆至入门载体pENTR/D-TOPO,得到重组入门质粒pENTR/D-TOPO-CDV-H,然后与腺病毒骨架载体pAd/CMV/V5-DEST进行LR重组,获得重组质粒pAd-CDV-H;重组质粒用PacⅠ酶切线性化后转染293A细胞,获得重组腺病毒rAd-CDV-H,连续传代后对获得的重组腺病毒分别进行病毒滴定、免疫荧光和Western blot鉴定。经免疫荧光方法及Western blot鉴定结果表明,重组腺病毒rAd-CDV-H感染293A细胞、Vero细胞后均可以成功表达H蛋白,获得的重组腺病毒的滴度高达1010IFU/mL。成功获得能够表达大熊猫源CDV H蛋白的重组腺病毒rAd-CDV-H,在进一步证明其安全有效后,可为大熊猫犬瘟热免疫预防提供候选疫苗株。The aim of this study was to construct a defective recombinant adenovirus that could express the H protein of giant panda/SX/2014,a canine distemper virus(CDV)isolated from giant panda.The H gene of giant panda/SX/2014 was amplified by reverse transcription polymerase chain reaction(RT-PCR),and cloned into the entry vector pENTR/D-TOPO through TOPO ligation to obtain the recombinant entry plasmid pENTR/D-TOPO-CDV-H,then LR recombination with the adenovirus backbone vector pAd/CMV/V5-DEST was conducted to obtain the recombinant plasmid pAd-CDV-H.The recombinant plasmid was linearly digested by PacⅠenzyme and transfected into 293A cells to obtain the recombinant adenovirus rAd-CDV-H.After successive passage,the obtained recombinant adenovirus was identified by viral titration,immunofluorescence and Western blot,respectively.The results of immunofluorescence and Western blot analysis showed that the recombinant adenovirus rAd-CDV-H successfully expressed H protein after infecting 293A cells and Vero cells,and the titer of the obtained recombinant adenovirus was as high as 1010 IFU/mL.After further proving its safety and effectiveness,the rAd-CDV-H constructed in this study could be a candidate vaccine strain for the immunoprevention of giant panda canine distemper.

关 键 词：犬瘟热病毒 H基因 复制缺陷型重组腺病毒 大熊猫源 

分 类 号：S852.65[农业科学—基础兽医学]