鸭瘟病毒gG基因缺失株的构建及其免疫保护效果研究  

Construction of duck enteritis virus gG-deficient strain and study on its immune protection effect

作　　者：相生瑞 陈柳[2] 霍苏馨 朱寅初 华炯钢[2] 张存[2] 云涛[2] 倪征[2] 丁方舟 宋厚辉 叶伟成[2] XIANG Sheng-rui;CHEN Liu;HUO Su-xin;ZHU Yin-chu;HUA Jiong-gang;ZHANG Cun;YUN Tao;NI Zheng;DING Fang-zhou;SONG Hou-hui;YE Wei-cheng(College of Animal Science and Technology&College of Veterinary Medicine,Zhejiang A&f University,Hangzhou 311300,China;Institute of Animal Husbandry and Veterinary Medicine,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]浙江农林大学动物科技学院/动物医学院,浙江杭州311300 [2]浙江省农业科学院畜牧兽医研究所,浙江杭州310021 [3]南京农业大学动物医学院,江苏南京210095

出　　处：《中国预防兽医学报》2024年第9期946-953,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：浙江省基础公益研究项目(LGN22C010005);浙江省农业科学院国际合作项目(2023)。

摘　　要：鸭瘟病毒(DEV)是引起鸭高致死率的重要病原。为探究gG基因对DEV疫苗株免疫保护效果的影响,本研究在本实验室已构建的DEV疫苗株细菌人工染色体感染性克隆pDEV-EF1的基础上,通过“Red E/T两步重组”技术,构建DEV gG基因缺失的感染性克隆pDEV-ΔgG,对其进行酶切、PCR及测序鉴定。将p DEV-EF1和pDEV-ΔgG分别转染CEF细胞,待两组细胞均出现荧光后,收获细胞培养物反复冻融3次后,连续传至20代,每隔5代采用PCR及测序鉴定,分析p DEV-ΔgG的遗传稳定性,并采用western blot进一步鉴定,结果显示,正确构建gG基因缺失株,且能在CEF上稳定传代,命名为rDEV-ΔgG。将rDEV-ΔgG和亲本病毒rDEV-EF1接种CEF,测定体外生长曲线和测定蚀斑形成能力。结果显示,rDEV-ΔgG和rDEV-EF1的病毒滴度在24 h~72 h呈上升趋势,均在72 h时达到高峰,且二者的病毒滴度无显著差异;rDEV-ΔgG感染CEF后形成的蚀斑较亲本病毒rDEV-EF1产生的蚀斑面积减小27.37%。将r DEV-ΔgG以不同剂量免疫30日龄麻鸭2周后,进行DEV攻毒试验,观察各组鸭的临床症状,统计各组麻鸭的存活率,攻毒后1 d到9 d采集各组鸭肛拭子,并在攻毒后2 d、4 d、6 d和8 d各组分别随机剖杀3只麻鸭,取其各脏器组织,经荧光定量PCR分别检测各组麻鸭各脏器病毒载量及排毒情况。攻毒试验结果显示,1.0×10^(5)TCID_(50)和1.0×10^(4)TCID_(50)rDEV-ΔgG免疫的麻鸭在强毒攻击下的保护率较相同剂量rDEV-EF1免疫的麻鸭均下降17%。各脏器病毒载量结果显示,在攻毒后2 d麻鸭胸腺的病毒载量最高,其次是脾脏,在攻毒后4 d,麻鸭食道、泄殖腔、胸腺病毒载量较高。泄殖腔排毒结果显示,攻毒后1 d~4 d泄殖腔排毒量持续升高,并攻毒后4 d各组麻鸭的排毒量达到峰值。本研究比较了gG基因缺失病毒rDEV-ΔgG和亲本病毒rDEV-EF1的生物学特性和免疫保护效果差异,为明确gG在DEV基因组中的功能提供研究基础。Duck enteritis virus (DEV) is an important pathogen that causes high mortality in duck flocks.In order to explore the effect of g G gene on the immune protection of DEV vaccine strain,based on the bacterial artificial chromosomal infectious clone p DEV-EF1 constructed in our laboratory,a DEV g G gene-deficient infectious clone p DEV-Δg G was constructed by"Red E/T two-step recombination"technology,and the genetic stability test was carried out by enzyme digestion,PCR till 20 passages.The results showed that the g G gene-deficient strain was successfully constructed and could be stably propagated on CEF,and was named r DEV-Δg G.r DEV-Δg G and the parental virus r DEV-EF1 were inoculated into CEF,and the in vitro growth curve and plaque formation ability were measured.The results showed that the viral titers of r DEV-ΔgG and r DEV-EF1 increased from 24 hours to 72 hours,and reached a peak at 72 hours,with no significant difference in viral titers.The plaques formed by r DEV-?gG infection on CEF were 27.37%smaller than those produced by the parent virus r DEV-EF1.After immunizing the 30-day-old hemp ducks with different doses for 2 weeks,a DEV challenge protection test was carried out to observe their clinical symptoms,and the survival rate of the ducks was calaulated.Anal swabs of each group were collected from 1 day to 9 days after challenge,and 3 hemp ducks were randomly dissected in each group on the 2 days,4 days,6 days and 8 days after challenge.Their tissues and organs were taken to detect the detoxification and viral load of each group of hemp ducks by fluorescence quantitative PCR.The results of the challenge protection experiment showed that the protection rate of hemp ducks immunized with 1.0×10^(5)TCID_(50)and 1.0×10^(4)TCID_(50)r DEV-Δg G under strong venomous attack was 17%lower than that of hemp ducks immunized with the same dose of r DEV-EF1.The results of viral load in each organ showed that the viral load of the thymus was the highest on the 2nd day after challenge,followed by the spleen.The

关 键 词：鸭瘟病毒 GG 免疫保护 

分 类 号：S852.65[农业科学—基础兽医学]