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

作　　者：刘明智[1,2] 叶星[1] 田园园[1] 马冬梅[1] 张莉莉[1] 迟妍妍[1,2] 邓国成[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所,广东广州510380 [2]上海海洋大学,上海201306

出　　处：《微生物学通报》2011年第3期437-445,共9页Microbiology China

基　　金：广东省重点科技项目(No.2008A020100016);广东省海洋渔业科技项目(No.A200899F01);广州市科技项目(No.2009J1-C021);荔湾区科技项目(No.20084411115)

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(2μg/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%)。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。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 se-quence 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（P0.05） 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.

关 键 词：嗜水气单胞菌 外膜蛋白W 克隆 基因工程表达 免疫原性 IGM 保护率 

分 类 号：S941[农业科学—水产养殖]