副溶血弧菌TDH的原核表达及单克隆抗体的制备

Expression of TDH from Vibrio parahaemolyticus and Preparation of Monoclonal Antibody against TDH

为实现副溶血性弧菌tdh基因的原核表达,采用TCBS选择培养基从贝类中筛选副溶血性弧菌疑似菌株;根据Gen Bank上已有的tdh基因序列,设计并人工合成引物,通过PCR技术鉴定副溶血性弧菌并扩增tdh基因;酶切后定向插入到p ET-28a表达载体中,构建重组表达质粒p ET-28a-tdh,转入E.coli Rosetta中,在IPTG诱导下进行TDH蛋白表达。为制备耐热直接溶血毒素TDH单克隆抗体,用纯化的蛋白作为免疫原免疫BALB/c小鼠,成功用原核载体表达的TDH蛋白为免疫原,制得一株能稳定分泌单克隆抗体的杂交瘤细胞,命名为T9N10。获取腹水并经Ni-NTA Resin亲和柱纯化,其稳定分泌的单克隆抗体经鉴定为Ig G1,相对分子质量约为146 000,并表现出较强的特异性。本研究为开发副溶血弧菌免疫学快速检测和深入的研究奠定良好的物质基础。

To express of tdh gene in E.coli,TCBS medium was used to select the suspected strains of Vibrio parahaemolyticus （VP）.Primers were designed and synthesized to identify VP and amplify tdh gene based on the gene sequence from GenBank.The PCR gene product was ligated into prokaryotic expression vector pET-28a,and then the recombinant plasmids pET-28a-tdh was transformed into E.coli Rosetta.TDH protein was expressed by IPTG induction.To prepare monoclonal antibody against TDH （thermostable direct hemolysin） produced by VP,the purified TDH was used to immune BALB/c mice.The hybrid tumor cells named as T9N10 were prepared to secrete monoclonal antibodies by conventional prepared techniques.The secreted monoclonal antibodies are IgG1,which was 146 000 weight,and showed strong specificity.Results provide new insights to immunological rapid detection and research.