兔瘟病毒VP60基因重组真核表达质粒的构建及其免疫原性  

Construction and immunogenicity of recombinant eukaryotic expression plasmid of VP60 gene of rabbit hemorrhagic disease virus

作　　者：鲁忠祥 耿毅[1] 汪开毓[1] 坤清芳 邓梦玲 邓钊彬 余韬[1] 陈成[1] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014

出　　处：《中国兽医学报》2015年第12期1887-1892,共6页Chinese Journal of Veterinary Science

基　　金：国家星火计划资助项目(2012GA810004);四川省科技支撑计划资助项目(2012NZ0005)

摘　　要：采用RT-PCR方法扩增兔病毒性出血症病毒（rabbit hemorrhagic disease virus，RHDV）衣壳蛋白VP60基因，PCR产物纯化后克隆到pMD19-T载体，获得重组质粒pMD19-T-VP60，并进行序列测定。测序正确的pMD19-T-VP60经EcoRI与XhoI双酶切后，将目的片段VP60插入真核表达载体pVAX-1，构建真核表达质粒pVAX1-VP60。将构建好的重组质粒pVAX1-VP60按500fig／只腿部肌肉注射25日龄家兔，同时设pVAX-1组、兔瘟组织灭活苗组和PBS组。分别于免疫后7，14，21，28，35，42d对各组兔静脉采血，间接ELISA法检测血清中特异性抗体水平，结果显示PBS组和pVAX-1组未检测到特异性抗体，pVAX1-VP60组和兔瘟组织灭活苗组家兔都能产生高水平的特异性抗体，在特异性抗体水平高峰期，2组数据差异不显著（P〉0．05）。于免疫后3，7，14，21，28，35，42d随机剖杀pVAX1-VP60组试验兔3只，取心、肝、脾、肺、肾、腿部肌肉，提取组织DNA，进行PCR检测，结果均检测到目的基因VP60的存在。免疫后28d以滴度10^8的RHDV 0.5mL／只腿部肌肉注射攻毒，pVAX-1组、PBS组、免瘟组织灭活苗组、pVAX1-VP60组的相对保护率分别为0％，0％，90％，85％，表明pVAX1-VP60具有良好的免疫效果。The capsid protein VP60 gene of rabbit hemorrhagic disease virus （RHDV） was amplified by RT-PCR and the purified product was linked into the pMD19-T vector. Then the recombi- nant plasmid （pMD19-T-VP60） was sequenced and digested by EcoR I and Xho I to obtain the VP60 gene fragment. The fragment was inserted into the eukaryotic expression vector pVAX-1 to construct nucleotide vaccine plasmid pVAX1-VP60 and identified via PCR and double restriction enzyme digestion. To investigate the immunogenicity of the constructed plasmid pVAX1-VP60,25- day-old healthy rabbits were used to intramuscularly inject （500μg/each）. Meanwhile,three other experimental groups received an intramuscular injection of the same volume of pVAX-1, RHDV tissue inactivated vaccine and PBS, respectively. After immunized 7,14,21,28,35 and 42 d, blood was collected from each rabbit in four groups to test the specific antibody level by indirect EL1SA. The results showed that there was no specific antibody for RHDV in PBS group and pVAX-1 group, but there was a high specific antibody both in pVAX1-VP60 grou.p and RHD tissue inactiva- ted vaccine group,with no significant difference （P〉0.05） between the two groups. For detecting the tissue distribution of the pVAX1-VP60 in rabbits, three rabbits were killed randomly after immunized 3,7,14,21,28,35 and 42 d,and collected heart,liver,spleen,lung,kidney and leg mus- cle {or PCR test. The results showed that the plasmid pVAX1-VP60 could distribute in all these tissues in each period after immunized. The challenge experiment was conducted by intramuscular injection with 0. 5 mL/each rabbit RHDV at 10^8 plaques after immunized 28 days. The relative protection rates were 0%, 0%, 90%, 85% in pVAX-1, PBS, RHDV tissue inactivated vaccine and the pVAX1-VP60 group, respectively. These results suggested that the recombinant eukaryotic expression plasmid pVAX1-VP60 possessed optimun immunity.

关 键 词：兔病毒性出血症病毒 VP60 pVAX1-VP60 核酸疫苗 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学]