草鱼呼肠孤病毒外衣壳蛋白VP7重组疫苗的构建及其免疫效力评价

为探明呼肠孤病毒(GCRV)外衣壳蛋白VP7基因工程亚单位疫苗产生的抗体效价水平和免疫保护力，用RTPCR方法，从GCRV中扩增到VP7基因片段，并克隆至原核表达载体pET28a(+)中，得到重组质粒pETVP7，酶切鉴定后，将pETVP7转化到大肠杆菌BL21(DE3)中进行诱导表达，产生的重组蛋白经变性和复性后，按0.5、1.0、2.0 mg/尾的注射剂量分为3个免疫组，以等体积的氢氧化铝为佐剂进行注射。经SDSPAGE电泳分析，发现了相对分子质量为34 000的蛋白表达带；Western Blot检测结果显示表达的蛋白具有很好的抗原性；间接凝集试验结果表明，3个免疫组均有抗体产生；攻毒试验结果表明，0.5、1.0、2.0 mg/尾重组蛋白剂量组的保护率分别为90%、85%、90%，对照组为0，表明所构建的疫苗具有较好的免疫作用。

Development and efficacy of a grass carp reovirus (GCRV) outer capsid protein VP7 subunit vaccine

Antibody titer and immunoprotection induced by VP7 subunit vaccine were explored in the present study. The gene of the grass carp reovirus(GCRV) coding the outer capsid protein VP7 was amplified and inserted into prokaryotic expression vector pET-28a(+) to obtain pET-VP7, which was transformed into BL21(DE3) for expressing. SDSPAGE showed that the expressed VP7 protein had a molecular weight of about 34 000 and Western Blot detection indicated good immunoreactivity of the protein. After denaturation and renaturation, the expressed VP7 protein was used to immunize grass carp based on three doses: 0.5, 1.0 and 2.0 mg, which was mixed with an equal volume of aluminum hydroxide adjuvant except the control. Indirect agglutination detection showed that antibody against VP7 protein was produced in the immunized groups. Challenge experiment with GCRV showed that 90%, 85% and 90% protection were achieved in groups respectively immunized with 0.5, 1.0 and 2.0 mg of the expressed VP7 protein and no protection was shown in the control group which demonstrated that the VP7 subunit vaccine made here had good immunoprotection against GCRV.