摘要
根据已发表的哈维氏弧菌（Vibrio harveyi）外膜蛋白OmpU的基因序列设计1对特异性引物,应用聚合酶链式反应（PCR）方法,自哈维氏弧菌致病菌株基因组中扩增获得一段约1 000 bp的序列,构建重组载体pMD18-T-OmpU;测序结果表明,该基因与已发表的哈维氏弧菌外膜蛋白OmpU序列存在98%以上的相似性,该序列在GenBank上的登录号为FJ919231。构建表达质粒pET-30a（＋）-OmpU后转化至大肠杆菌E.coliBL21（DE3）,在IPTG诱导下实现高效表达,SDS-PAGE显示重组蛋白分子质量约为41 ku,与预期基本相符。重组蛋白以包涵体形式表达。以脲素法得到初步纯化的重组蛋白,以50μg/mL的剂量注射免疫大黄鱼幼鱼,经间接ELISA法检测了4-8周后特异性抗体的效价,同时检测了注射后4周的免疫保护率。结果表明,4-8周内特异性抗体效价持续升高,达到log28.0以上,4周时的相对免疫保护率为85%。试验结果显示了重组哈维氏弧菌外膜蛋白OmpU具有良好的免疫原性,可作为亚单位疫苗的开发对象。
According to OmpU gene sequence published in GenBank,primers were designed and the DNA fragment of about 1 000 bp was amplified by PCR from genomic DNA of Vibrio Harveyi ZJ2008,isolated from disease Pseudosciaena crocea.The gene was cloned into pMD18-T vector and sequenced,then submitted to GenBank with an accession number FJ919231.BlastN result showed that the sequence shared identity of 99.0% with OmpU of Vibrio harveyi.The sequence was subcloned into pET-30a resulted with the recombinant plasmid pET30a-OmpU which was further transferred into host bacteria E.coli BL21（DE3） with IPTG induction for expression.SDS-PAGE profiles showed the expected MW of the recombinant OmpU was around 41 ku.The protein was expressed in the form of insolvable fusion bodies and was preliminary purified by urea method.Forty pieces of large yellow croaker were individually immunized with 50 μg of the recombinant protein by intraperitoneal injection.Four weeks after vaccination,ten pieces were artificially challenged by intraperitoneal injection with live bacteria and the mortality was recorded in the following 14 d.Relative percentage of the survival rate of the immunized group reached 85%.Specific antibody level was detected by indirect ELISA after 4-8 weeks post vaccination.Resulting an increase of antibody titers to the immunized recombinant protein.The present study demonstrated strong immunogenicity of the recombinant OmpU.OmpU of V.harveyi should be given enough attention for their potential application in vaccine development.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2010年第3期346-350,共5页
Journal of Huazhong Agricultural University
基金
浙江省自然科学基金项目(Y308408)
宁波市科技局择优委托项目(2007C10037)资助
