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为了探讨草鱼出血病病毒(GCRV)VP6亚单位疫苗对草鱼出血病的免疫保护作用,将vp6基因克隆进原核表达载体pET-28a(+),构建了重组质粒pET28a(+)-VP6。SDS-PAGE和Western-blotting显示,重组VP6蛋白的分子量约为43 ku,主要以包涵体形式存在,重组蛋白占菌体总蛋白的20%左右;Ni2+柱纯化后的重组蛋白,以每尾500μg肌肉注射免疫草鱼(14～20 cm,60～120 g),并在第14、21、28、49、70天通过间接凝集反应检测抗体水平,结果显示,免疫注射后第14天可检测到鱼体产生的特异性抗体,第21天达到最高峰,第70天仍可检测到抗体的存在;免疫注射第21天人工接种GCRV,免疫后的草鱼对出血病的保护力达100%。
In order to investigate the protective effect of VP6 subunit vaccine of grass carp hemorrhage virus (GCRV) on grass carp hemorrhagic disease, the recombinant plasmid pET28a (+) - VP6 was constructed by cloning vp6 gene into prokaryotic expression vector pET-28a (+). SDS-PAGE and Western-blotting showed that the molecular weight of the recombinant VP6 protein was about 43 ku, mainly in the form of inclusion bodies. The recombinant protein accounted for about 20% of the total bacterial proteins. The recombinant protein purified with Ni2 + Muscle was immunized with grass carp (14-20 cm, 60-120 g), and antibody levels were detected by indirect agglutination reaction on days 14, 21, 28, 49, 70. The results showed that fish could be detected on the 14th day after immunization The specific antibody produced in the body reached the peak on the 21st day and the antibody was still detectable on the 70th day. On the 21st day of immunization, GCRV was inoculated manually, and the immunized grass carp protected the hemorrhagic disease by 100%.