溶藻弧菌TssJ基因的原核表达及多克隆抗体制备  

Prokaryotic Expression of the TssJ Gene from Vibrio alginolyticus and Preparation of the Polyclonal Antibody

作　　者：范雪亭[1] 许悦[1] 孙云[1] 周永灿[1] 王世锋[1] 谢珍玉[1] 

机构地区：[1]海南大学海洋学院海南省热带水生生物技术重点实验室,海南海口570228

出　　处：《海南大学学报（自然科学版）》2016年第2期150-154,共5页Natural Science Journal of Hainan University

基　　金：国家自然科学基金(No.41466002;31060360);海南省重大科技项目(No.ZDZX2013014)

摘　　要：Tss J是溶藻弧菌Ⅵ型分泌系统(T6SS)的一个重要基因.为了制备Tss J基因的多克隆抗体,以溶藻弧菌HN08155菌株的DNA为模板,扩增Tss J基因后利用表达载体PET-32a(+)和大肠杆菌BL21(DE3)原核表达,分离纯化后免疫大鼠获得Tss J基因的多克隆抗体.结果显示,37℃、IPTG终浓度1 mmol·L^(-1)是较适宜的表达诱导条件,表达的融合蛋白是存在于细胞内的包涵体,与预期的分子量42 k D一致,并且免疫大鼠后血清的多克隆抗体效价很高,为1∶32 768.制备的溶藻弧菌Tss J高效价多克隆抗体有助于进一步开展Tss J的定位和功能研究.Abstraet：TssJ is an important gene of the type VI secretion system （T6SS） in Vibrio alginolyticus. To prepare the polyclonal antibody against TssJ, TssJ gene was amplified from genomic DNA of V. alginolyticus HN08155 and cloned into pET-32a（ ＋ ） vector. The recombinant plasmid, pET32a-TssJ, was transformed into Escherichia coli BI221 （DE3） for the expression. Then, the purified TssJ recombinant protein was used to immunize SPF rats for preparing polyclonal antibody. The results indicated that the optimization inducing condition in E. coli was at 37 ~C with 1 mmol ~ L-1 IPTG; the recombinant TssJ protein, with a molecular weight of 42 kD as expected, was mainly expressed in the inclusion body; the antibody titer of serum was very high, which was validated to be 1： 32 768 by ELISA. The polyclonal antibody is very helpful for the position location and functional description of TssJ in the future experiment. Keywords： Vibrio alginolyticus ; TssJ; prokaryotic expression; polyclonal antibody

关 键 词：溶藻弧菌 TssJ 原核表达 多克隆抗体 

分 类 号：Q78[生物学—分子生物学] S917.1[农业科学—水产科学]