文献类型：期刊文献

中文题名：溶藻弧菌HY9901鞭毛蛋白flaB基因的克隆及原核表达

英文题名：Cloning and prokaryotic expression of flaB gene from Vibrio alginolyticus strain HY9901,the causative agent of vibriosis in Lutjanus sanguineus

作者：梁海鹰[1,2,3,4,5];夏立群[2];吴灶和[1,2,3,4];简纪常[2,3,4];鲁义善[2,3,4]

机构：[1]中国科学院南海海洋研究所;[2]广东海洋大学水产学院;[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;[4]广东省水产经济动物病害控制重点实验室;[5]中国科学院研究生院

年份：2010

期号：1

起止页码：139

中文期刊名：水产学报

外文期刊名：Journal of Fisheries of China

收录：CSTPCD、、Scopus、CSCD2011_2012、北大核心2008、北大核心、CSCD

基金：国家科技支撑计划(2007BAD29B03);广东省科技重大专项(2004A20401001);广东省自然科学基金项目(9151064201000063);广东海洋大学自然科学研究项目(0612187)

语种：中文

中文关键词：溶藻弧菌;鞭毛蛋白;flaB基因;原核表达

外文关键词：Vibrio alginolyticus ; flagellin; flaB gene; prokaryotic expression

中文摘要：参照GenBank上登录的弧菌鞭毛蛋白flaB基因序列设计引物,PCR扩增溶藻弧菌HY9901株的flaB全长基因,序列分析结果显示该基因为1134bp,编码377个氨基酸。与GenBank中其它弧菌的同源基因序列比对显示,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pET-32a(+)中,在大肠杆菌BL21(DE3)中成功表达出带His-tag的融合蛋白,分子量大小与预期一致。优化的表达条件为28℃,0.4mmol/LIPTG浓度诱导10h。用纯化后的融合蛋白免疫SPF级小鼠,制备了多克隆抗体。Western-blotting结果表明鼠抗FlaB血清不仅能与诱导后的重组蛋白发生反应,而且能与天然的溶藻弧菌全菌蛋白发生反应,提示鞭毛蛋白FlaB可能是溶藻弧菌的重要保护性抗原之一,为下一步进行FlaB蛋白免疫原性的研究以及疫苗的制备奠定了基础。

外文摘要：To investigate the possibility offlaB as a candidate antigen for vaccine production, primers were designed based on flaB gene sequences published in GenBank. The flaB gene of Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus sanguineus, was amplified by PCR and cloned into pMD19-T vector. Sequence analysis revealed that flaB gene is 1 134 bp and encodes a putative protein of 377 amino acids. The amino acid sequence of FIaB of V. algonilyticus showed highest identity to V. parahaemolytus （92%）. TheflaB gene was linked into prokaryotic vector pET-32a（ ＋ ）, and the His-naB fusion protein with 60. 5 ku molecular mass was successfully expressed in E. coli BL21. The soluble recombinant protein was highly expressed under induction conditions of exposure to IPTG （0.4 mmol/L） at 28 ℃ for 10 h and successfully purified on Ni^2＋ -IDA column. The purified fusion protein was injected into SPF mice to produce anti-FlaB serum. Western blot analysis revealed that the prepared antiserum not only specifically reacts to the FlaB fusion protein, but also specifically reacts to natural total protein extracted from V. alginolyticus. This result indicates that the FlaB may be one of the important protective antigens of V. alginolyticus, which could provide a basis for further study on the immunogenecity of FlaB and vaccine preparation.