草鱼出血病病毒VP6蛋白的原核表达、纯化及免疫效果

Prokaryotic expression,purification and immune efficacy of VP6 protein of grass carp reovirus

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 study the immuno-protective efficacy of GCRV-VP6 subunit vaccine,the recombinant expression vector pET28a（＋）-VP6 was constructed by cloning vp6 gene of GCRV into the prokaryotic expression plasmid pET-28a（＋）.The results of SDS-PAGE and Western-blotting showed that the molecular mass of the recombinant VP6 protein was approximately 43 ku.The recombinant protein reached 20% of the total bacterial proteins,and was mainly in the form of insoluble bodies.The grass carp（60-120 g in body weight and 14-20 cm in body length）was immunized with 500 μg purified VP6 protein by Ni2＋ affinity chromatography and antibody titers in the blood of fish were determined by means of indirect agglutination reaction on the 14th,21st,28th,49th and 70th days post-vaccination.The results showed that the specific antibody could be detected on the 14th day,reached peak on the 21st day and could be still detected on the 70th day after immunization.The fish in vaccinated group and control group were challenged with GCRV on 21st days after vaccine delivery and the relative survival rate in the vaccined group was 100%.These results provided important academic foundation for research and development of GCRV genetic vaccine.