嗜水气单胞菌外膜蛋白W 基因的表达及其免疫原性分析

Expression and immunogenicity analysis of the outer membrane protein W gene of Aeromonas hydrophila

摘要:

从患暴发性败血病的草鱼病灶处分离鉴定了嗜水气单胞菌(Aeromonas hydrophila) Wp3 菌株。以其基因组DNA 为模板扩增外膜蛋白W 基因(OmpW), 该基因全长为865 bp, 开放式阅读框(ORF)为615 bp, 与标准株ATCC7966 的OmpW 基因的同源性为99.8%。根据ORF 序列设计引物扩增OmpW 成熟肽编码序列并将其插入到表达载体pQE30 中, 转化大肠杆菌, 经诱导可表达分子量为24.7 kD 的带His 标签的融合外膜蛋白His-W。用此融合蛋白免疫草鱼, 所得草鱼血清经Elisa 分析显示呈现阳性反应, 说明重组蛋白能诱导产生抗体。采用实时荧光定量PCR 分析草鱼头肾组织IgM 基因表达水平的变化, 结果显示免疫组IgM 的表达量均明显高于空白组, 其中低浓度免疫组(2 μg/g)与空白对照组的差异显著(P<0.05), 说明融合蛋白可使草鱼产生良好的免疫应答并上调抗体基因表达、产生高效抗体。保护性实验显示, 不同免疫剂量均可使免疫组获得较高保护率(57%?86%)。本研究结果显示, 重组嗜水气单胞菌外膜蛋白W 可作为草鱼嗜水气单胞菌基因工程亚单位疫苗。

Abstract:

The complete gene sequence of the outer membrane protein W (OmpW) was amplified from the genomic DNA of Aeromonas hydrophila Wp3 strain which was isolated from grass carp suffered hemorrhage. The OmpW gene was 865 bp in length, containing an open reading frame (ORF) of 615 bp. The cloned gene possessed high similarity with that of the standard strain ATCC7966 (99.8%). The sequence encoding the mature peptide of OmpW was amplified and inserted into the expression vector pQE30. The recombinant vector was transformed into E. coli, and a 24.7 kD recombinant fusion protein His-W was expressed. Elisa analysis of the serum of grass carp immunized with His-W showed a positive immune reaction, suggesting production of the antibodies. Total RNA of the head kidney of the immunized grass carp was extracted and mRNA level of IgM was analyzed by qRT-PCR. Expression levels of IgM gene in the immunized group were higher than those of the control group, and significant difference (P<0.05) was found in the lowest protein concentration group (2 μg/g). These results showed that His-W was able to efficiently induce the immunized grass carp to produce antibodies. Protection experiments showed that the immunized groups had higher survival rates than those of the control groups (57%?86%). This study suggested that the recombinant protein His-W was a candidate vaccine for grass carp A. hydrophila disease.