文献类型：期刊文献

中文题名：哈维氏弧菌外膜蛋白OmpW基因克隆、表达及DNA疫苗的免疫效果

英文题名：Cloning,expression and DNA vaccine analysis of OmpW of Vibrio harveyi ZJ 0607

作者：黄浦江[1,2,3];黄郁葱[1,2,3];简纪常[1,2,3];吴灶和[2,3,4];鲁义善[1,2,3];张雪利[1,2,3]

机构：[1]广东海洋大学水产学院;[2]广东海洋大学广东省水产经济动物病原生物学及流行病学重点实验室;[3]广东海洋大学广东省高等学校水产经济动物病害控制重点实验室;[4]仲恺农业工程学院

年份：2013

卷号：37

期号：12

起止页码：1839

中文期刊名：水产学报

外文期刊名：Journal of Fisheries of China

收录：CSTPCD、、北大核心2011、Scopus、北大核心、CSCD、CSCD2013_2014

基金：国家自然科学基金项目(41240041);广东省科技厅国际合作项目(2012B050600029);国家科技支撑计划(2012BAD17B02;2012BAD17B03)

语种：中文

中文关键词：哈维氏弧菌;外膜蛋白;OmpW;免疫原性;DNA疫苗

外文关键词：Vibrio harveyi; outer membrane protein ; OmpW ; immunogenicity ; DNA vaccine

中文摘要：参照GenBank上登录的哈维氏弧菌外膜蛋白OmpW基因序列，设计引物扩增OmpW的开放阅读框，序列分析结果显示该基因全长645bp。将其定向克隆到原核表达载体pET-32a（＋），在大肠杆菌BL21中成功表达出带His-tag的融合蛋白，融合蛋白分子量约为43．8ku，且主要以包涵体形式存在。优化的表达条件为37oC，IPTG浓度0．1mmol／L，诱导时间5h。将纯化的融合蛋白免疫SPF昆明小鼠制备多克隆抗体，ELISA方法检测抗体效价达1：30000，Western．Blot结果表明鼠抗OmpW血清能与诱导后的重组蛋白发生特异反应，提示OmpW可能是哈维氏弧菌的一种重要保护性抗原。为进一步研究哈维氏弧菌外膜蛋白OmpW基因DNA疫苗对红笛鲷的免疫保护效果，构建了真核表达重组质粒pcDNA-OmpW，然后将真核质粒免疫接种红笛鲷。PCR结果显示，免疫接种后第7—28天，在红笛鲷的肌肉、头肾、肝脏和脾脏组织中均存在质粒分布；RT．PCR结果显示，免疫接种后第7～28天，红笛鲷上述组织均有目的基因的表达。ELISA结果表明，红笛鲷体内血清产生了抗OmpW蛋白的高效价抗体。Western．Blot分析表明，DNA疫苗免疫后红笛鲷体内表达了相应的目的蛋白，并诱导产生了相应抗体。攻毒实验结果表明，免疫保护率高达60％，可见pcDNA-OmpW可作为红笛鲷预防哈维氏弧菌感染的有效候选疫苗之一。

外文摘要：According to OmpW gene sequence published in GenBank, primers were designed and the DNA fragment of about 645 bp（encoding 214 amino acids）was amplified by PCR from genomic DNA of Vibrio Harveyi ZJ0607. The full length product was then cloned into the prokaryotic expression vector pET-32a （ ＋ ） for protein expression in Escherichia coli strain BL21 （ DE3 ）. The molecular weight of expression fusion protein OmpW was about 43.8 ku. The recombinant protein was highly expressed under induction conditions of exposure at 37 ℃ ,in 0.1 mmol/L of IPTG for 5 h. The recombinant protein was purified by the best expression condition,purified and used as antigen to immunize the Kunming-mice,and the antibody titer reached 1 ： 30 000. The result of the Western-blot revealed that specific antigen-antibody reaction occurred between the antiserum and its corresponding recombinant fusion protein. In order to study the immunogenic and protective effects of DNA vaccine,plasmid DNA encoding outer membrane protein OmpW gene was used as a DNA vaccine to immunize red snapper（Lutjanus sanguineus）. PCR results indicated that pcDNA-OmpW was distributed in muscle, head kidney, liver, spleen 7 -28 days after vaccination. RT-PCR results indicated that the OmpW gene was expressed in all above tissues of vaccinated fish 7 - 28 days after vaccination. Red snapper immunized with DNA vaccine showed higher serum antibody and corresponding recombinant fusion protein by ELISA and Western-blot. In addition, fish immunized with DNA vaccine developed a protective response to live Vibrio harveyi challenged 35 days post-inoculation, as demonstrated by increased survival of vaccinated fish over the control fish. This study indicates that pcDNA-OmpW is an effective vaccine candidate against Vibrio harveyi infection.