溶藻弧菌鞭毛蛋白flab基因的克隆及原核表达 - 水产学报.doc

溶藻弧菌HY9901鞭毛蛋白flaB基因的克隆及原核表达 梁海鹰1,2,3,4，夏立群2，吴灶和1,2,3，简纪常2,3，鲁义善2,3 （1.中国科学院南海海洋研究所，广东 广州 510301 2.广东海洋大学水产学院，广东 湛江 524025； 3.广东省水产经济动物病原生物学及流行病学重点实验室，广东 湛江 524088 4.中国科学院研究生院，北京 100049） 摘要:参照GenBank上登录的弧菌鞭毛丝蛋白flaB基因序列设计引物，PCR扩增溶藻弧菌HY9901株的flaB 全基因，序列分析结果显示该基因全长1134bp，编码377个氨基酸，与GenBank中其它弧菌的同源基因序列比较显示，溶藻弧菌flaB 基因与副溶血弧菌flaB 基因的同源性最高(92%) 。将该基因定向克隆到原核表达载体pET32a中，在大肠杆菌BL21 (DE3)中超量表达出带His-tag的融合蛋白，大小与预期分子量一致，而未经IPTG诱导的重组菌和空载体没有得到表达。将表达融合蛋白免疫SPF级小鼠制备了多克隆抗血清，Westernblot结果表明鼠抗FlaB血清与FlaB重组蛋白发生特异性免疫反应。为下一步进行融合蛋白免疫原性的研究以及疫苗的制备奠定了基础。 关键词: 溶藻弧菌；鞭毛蛋白；flaB；原核表达 Cloning and prokaryotic expression of flaB gene from Vibrio alginolyticus strain HY9901，the causative agent of vibriosis in Lutjanus sanguineus LIANG Hai-ying1,2,3,4,XIA Li-qun2,WU Zao-he1，2,3,JIAN Ji-chang2,3,LU Yi-shan2,3 （1．South China Sea Institute of Oceanology,Chinese Academy of Science,Guangzhou 510301,China; 2. Fisheries College, Guangdong Ocean University, Zhanjiang 524025, China； Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals，Zhanjiang 524025, China 4.Graduate University of Chinese Academy of Science,Beijing 100049,China) Abstract : To investigate the possibility of flaB as candidate antigen for vaccine production , primers were designed based on flaB gene sequences published in GenBank. The flaB gene of V.algonilyticus HY9901 was amplified by PCR and the PCR product was linked into pMD19-T vector .Sequence analysis revealed that flaB gene contains 1134bp,which putatively encodes 377 deduced amino acids. When compared with homologs in other vibrios from GenBank ,the flaB gene of V. algonilyticus had the highest homology with that of V.parahaemolytus (92 %). The flaB gene was linked into pET32a expression vector, and the expression of His-FlaB in E.coli BL21 (DE3) resulted in the induction of a 60.6kDa band in accordance with the molecular weight. As temperature, IPTG concentration and induction time were optimized, the soluble recombinant pro