鹿流行性出血热病毒核心样颗粒的制备与鉴定  

Identification and synthesis of core-like particles of epizootic hemorrhagic disease virus

作　　者：黄超华 花群俊 吴江 曹琛福 林彦星 史卫军 林永涛 曾少灵 杨俊兴 花群义 HUANG Chao-hua;HUA Qun-jun;WU Jiang;CAO Chen-fu;LIN Yan-xing;SHI Wei-jun;LIN Yong-tao;ZENG Shao-ling;YANG Jun-xing;HUA Qun-yi(Animcd and Plant Inspection and Quarantine Technology Center,Shenzhen Customs District P.R.China,Shenzhen 518045,China;Wuding Center for Artimal Disease Control and Prevention,Wuding 651601,China)

机构地区：[1]深圳海关动植物检验检疫技术中心,广东深圳518045 [2]武定县动物疫病预防控制中心,云南武定651601

出　　处：《中国兽医科学》2021年第8期1023-1028,共6页Chinese Veterinary Science

基　　金：“十三五”国家重点研发项目(2017YFD0502304,2017YFD0501805-02)。

摘　　要：为制备鹿流行性出血热病毒(EHDV)的核心样颗粒(CLPs),本研究将鹿流行性出血热病毒(EHDV)L3与S7基因片段插入至昆虫杆状病毒穿梭载体pFastBac-Dual中,构建重组质粒pFastBac-Dualvp3-vp7,并通过转座获得重组杆状病毒质粒Bacmid-vp3-vp7,随后转染Sf9细胞获得重组杆状病毒rBac-vp3-vp7。通过重组杆状病毒感染Sf9细胞共表达VP3和VP7蛋白,并利用蔗糖梯度离心进行纯化,两者组装形成CLPs。负染后通过透射电镜观察纯化产物的形态,Western-blotting验证其反应原性,免疫小鼠检验其免疫原性。结果显示,透射电镜下可以观察到纯化产物中含有大量的与EHDV形态相似的直径为60~70 nm的中空颗粒;以绵羊EHDV阳性血清为一抗的Western-blotting可以检测到大小分别为100 ku和38 ku的2条蛋白条带,分别与EHDV VP3和VP7蛋白大小一致;间接ELISA结果显示,3次免疫后可刺激产生的EHDV特异性抗体效价可达1∶12 800。上述结果表明,本研究获得了具有典型形态、良好反应原性和免疫原性的EHDV核心样颗粒(CLPs)。To synthesize epizootic hemorrhagic disease virus(EHDV) core-like particles(CLPs),the L3 and S7 genes of EHDV,which encode the two major core proteins VP3 and VP7 of the virus,were inserted into a baculovirus dual-expression transfer vector,and then p Fast Bac-dual-vp3-vp7 was constructed.By transfected Sf9 cells with Bacmid-vp3-vp7,the recombinant baculoviruses named r Bac-vp3-vp7 was generated. In the Sf9 cells infected by r Bac-vp3-vp7,VP3 and VP7 were co-expressed and assembled EHDV CLPs. EHDV CLPs were purified by sucrose density-gradient centrifugation. Under the electron microscope,these particles had been observed to be similar to authentic EHDV cores.Besides,EHDV CLPs had the same antigenicity with EHDV were confirmed by Western-blotting tests.After three times of inoculation with EHDV CLPs,the anti-EHDV antibody titer was 1 ∶ 12 800.The results showed that EHDV CLPs were constructed successfully,and had good reactivity and immunogenicity.This study will be the basis for assembly of virus-like particles and preparation of EHDV VLPs vaccine.

关 键 词：鹿流行性出血热病毒 VP3蛋白 VP7蛋白 核心样颗粒 

分 类 号：S852.659.4[农业科学—基础兽医学]