原核表达的兔出血症病毒衣壳蛋白对兔的免疫保护效果

 为了检验大肠杆菌表达的兔出血症病毒（RHDV）衣壳蛋白（VP60）对兔的免疫保护效果，将34只清洁兔随机分成3组：单用免疫佐剂对照组（8只）、单用重组VP60组（8只）和重组VP60加免疫佐剂组（18只），分别经皮下注射，VP60的免疫剂量为每只每次400 μg，免疫2次，间隔7 d。在第2次免疫7 d后，VP60加免疫佐剂组的兔血清抗体水平显著高于单用VP60免疫组（ELISA 1∶64 000 对比1∶4 000 / HI 1∶128 对比1∶16），表明免疫佐剂可明显加速重组VP60诱导抗体产生的进程，用RHDV NJ85强毒攻击，免疫佐剂对照组与单用VP60免疫组的实验兔均全部死亡（8/8和8/8），VP60加免疫佐剂组均全部存活，保护率为100%（18/18）。用RHDV血凝试验（HA）检测，死亡兔的肝脏组织液HA效价大于1∶512，而存活兔的肝脏组织液HA效价小于1∶2，肝脏组织触（涂）片检查和细菌培养均为阴性，证明病死兔系RHDV感染所致。用RHD重组亚单位疫苗在养兔场进行800只兔的田间免疫试验，安全有效，没有出现不良反应。对重组菌进行冻存、复苏、培养、诱导表达和质粒酶切分析，证明VP60基因在宿主菌内保持稳定。

Protection of Rabbit Hemorrhagic Disease Virus by Immunization with the Recombinant Capsid Protein Expressed in E.Coli

The immunogenicity of the recombinant rabbit hemorrhagic disease virus (RHDV) capsid protein (VP60) expressed in E. coli was investigated. Thirty-four clean rabbits were randomly divided into 3 groups and respective subcutaneously injected twice, with 7 days interval, using adjuvant (8 rabbits), the recombinant VP60 (8 rabbits) and VP60 mixed with adjuvant (18 rabbits). The dosage of VP60 protein was 400μg per injection. Titer of the antibody raised by VP60 protein combined adjuvant was much higher than that of VP60 protein only at the 7th day after second injection (ELISA 1∶64 000 vs 1∶4 000 / HI 1∶128 vs 1∶16). The result indicated that adjuvant could accelerate the production of antibody. The immunized animals were challenged with highly virulent NJ85 isolate of RHDV. The mortality rates were 100% (8/8) for the adjuvant control and recombinant VP60 groups. In the meanwhile, the protective rate was 100% (18/18) for VP60 combined with adjuvant group. The death caused by RHDV infection was confirmed by the evidences that HA titer of liver tissue in dead rabbits was over 1∶512 while less than 1∶2 in surviving rabbits, and no bacteria staining in liver tissues smear as well as no bacteria growth in culture. Eight hundred domestic rabbits in rabbit farm immunized with the recombinant subunit vaccine to RHD and the occurrence of RHD and harmful effects due to the vaccination were not observed. Additionally, it was also confirmed that the recombinant E. coli could keep stability in the process of storing, restoring, culture, induced expression and digestion of recombinant plasmid.