虹鳟鱼造血器官坏死病病毒(IHNV)m基因的表达及免疫原性分析

Expression and Immunogenicity Assay of m Gene of Infectious Hematopoietic Necrosis Virus(IHNV)

根据NCBI中报道的造血器官坏死病病毒(IHNV)m基因序列设计特异性引物,提取病变组织中的RNA后,通过RT-PCR扩增得到m基因,大小约为570bp.与pET-22b连接,构建表达质粒pET-22b-m,转化大肠杆菌BL21(DE3)得到重组菌株BL21(DE3)(pET-22b-m).挑取阳性克隆子,IPTG诱导,经SDS-PAGE验证,在21.6ku处有目的蛋白表达;Western blot表明目的蛋白与相应抗体具有很好的反应性;免疫小鼠后,经ELISA测定血清中抗体效价,为1∶128 000;虹鳟鱼攻毒试验,发现重组菌株BL21(DE3)(pET-22b-m)表达的蛋白对虹鳟的最高保护率为68%.本试验结果为研制造血器官坏死病病毒基因工程疫苗奠定了坚实的基础.

A pair of specific primers was designed according to the sequence of m gene of infectious hematopoietic necrosis virus （IHNV） reported by NCBI. RNA was extracted from diseased ilsh by lnzol, and a fragment of appoximate 570 bp was obtained by RT-PCR. The PCR product was then ligated to the prokaryotic expression vector pET-22b to construct the recombinant plasmid pET-22b-rn, and positive transformants were used to transform Escherichia coli BL21 （DE3）. The expression of the recombinant gene was induced by IPTG,and results of SDS-PAGE confirmed the expression of target protein with the molecular weight of 21.6 ku. Western blotting showed that the protein had good reactivity toward the cor- responding antibody. The antibody titer of immunized mice was measured by enzyme-linked immunosorbent assay （ELISA） to be 1：128 000, and the highest protection rate of the protein expressed by the recombinant E. coli BL21 （DE3） （pET-22b-m） was shown as 68% through challenge assay on rainbow trouts. There-fore,this study has laid a foundation for development of effective genetically engineered vaccine for infec- tious hematopoietic necrosis.