IOCAS-IR  > 海洋环流与波动重点实验室
鱼类疫苗的研究:I: 海水鱼类病原菌乳化口服疫苗的研究;II:迟缓爱德华氏菌和嗜水气单胞菌外膜蛋白疫苗的研究
其他题名Studies of fish vaccines: I: Immune response and efficacy of oral-oil-emulsified vaccines against marine fish bacterial pathogens; II: Immune response and efficacy of outer membrane proteins of Edwardsiella tarda and Aeromonas hydrophila
肖鹏
学位类型博士
2008-06-15
学位授予单位中国科学院海洋研究所
学位授予地点海洋研究所
关键词鳗弧菌 嗜水气单胞菌 迟缓爱德华氏菌 链球菌 口服疫苗 蛋白抗原
摘要本论文的目的是研究几种病原菌口服疫苗接种鱼类的免疫效果,并从常见病原菌株中筛选几个具有较好保护效果的蛋白抗原,利用口服免疫的方式,接种养殖动物,并检测免疫效果。 以10号白油为有机溶剂,采用搅拌与均浆方法制备鳗弧菌M3和SMP1的油乳化二价疫苗,用饵料包埋后以口服途径免疫养殖大菱鲆,评价免疫大菱鲆的免疫应答和疫苗的保护效果。结果显示,以油乳化和未油乳化疫苗分别连续口服免疫大菱鲆一周后,在后肠组织,乳化疫苗刺激产生的非特异性活力、特异性抗体水平均高于未乳化疫苗;而在血清,两种疫苗引起的两种酶的活力、SMP1抗体水平没有变化,但在乳化疫苗免疫的大菱鲆检测到明显高于未免疫对照大菱鲆的M3抗体水平。大菱鲆后肠组织原位杂交结果显示,口服免疫的大菱鲆后肠褶皱有IgM抗体的产生和分布。其中,乳化疫苗免疫大菱鲆的IgM抗体的产生和分布水平高于未乳化疫苗免疫的大菱鲆。攻毒实验显示,乳化疫苗免疫的大菱鲆对M3和SMP1的感染分别获得100%和50%的免疫保护率,而未乳化疫苗获得的免疫保护率分别为57.9%和0%,表明乳化疫苗比未乳化疫苗更有效地保护大菱鲆、抵抗病原的感染。在乳化疫苗免疫持续期的研究中,免疫的大菱鲆后肠在免疫后120天仍能检测到抗体效价,在免疫后90天还能观察到一定的免疫保护效果。免疫30天、60天、90天和120天的大菱鲆分别获得100%、66.7%、36.7%和13.3%的免疫保护力。 以鳗弧菌M3和SMP1、链球菌CF、迟缓爱德华菌SMW7作为细菌抗原制备油乳化多价口服疫苗和轮虫携带疫苗,口服途径免疫养殖大菱鲆与大菱鲆初孵仔鱼。结果显示,在免疫大菱鲆后肠可检测到抗M3抗体水平的提高(P<0.05),而在其胆汁、鳃、中肠、体表黏液、前肠与血清中抗体效价变化与对照组没有显示出差异;没有检测到免疫大菱鲆后肠抗SMP1、SMW7、CF抗体效价。M3浸泡攻毒实验显示,口服免疫的大菱鲆获得了100%的免疫保护力;在M3注射攻毒和SMP1、CF、SMW7浸泡攻毒大菱鲆的实验中,在每个攻毒组中,免疫组大菱鲆开始死亡的时间都要比对照组有不同程度的延迟,但攻毒大菱鲆都发生死亡,不能显示出与对照组的差异。轮虫携带免疫的结果显示,免疫的大菱鲆初孵仔鱼并未获得较好的保护效果,与对照大菱鲆没有体现出差异。 从致病性病嗜水气单胞菌(Aeromonas hydrophila)LSA34克隆并表达ahaI基因和gapA基因,从迟缓爱德华菌(Edwardsiella tarda)LSE40克隆并表达eseB,将所得蛋白分别通过腹腔注射途径免疫大菱鲆,检测蛋白的免疫原性和免疫保护。结果在免疫后7天就可以检测到AhaI、GapA蛋白免疫组大菱鲆产生的抗体,至第40天可以检测到明显的保护性抗体,之后抗体效价增加明显,直至第60天时达到最高值。EseB免疫的大菱鲆第一次免疫后15天就有较高的抗体效价产生,明显高于对照组大菱鲆血清抗体效价,到距第一次免疫60天时,抗体效价达到最高值。攻毒实验显示,与对照组相比,AhaI免疫组和GapA免疫组对LSA34感染的免疫保护力分别为80%和100%;AhaI免疫组和GapA免疫组对LSE40感染的免疫保护力分别为30%和10%。,而对照组牙鲆对人工攻毒不具有保护力。以AhaI和GapA作为疫苗免疫大菱鲆,使大菱鲆获得了对嗜水气单胞菌LSA34较高的免疫保护;而对迟缓爱德华氏菌LSE40的交叉保护能力没有明显提高。EseB免疫的大菱鲆在攻毒实验中并没有显示出较好的保护效果,与对照组相比,只是在死亡时间上有所延迟。 以从致病性嗜水气单胞菌中克隆的ahaI和gapA基因表达出的蛋白为蛋白抗原制备油乳化疫苗,用饵料包埋后以口服途径免疫养殖牙鲆,评价免疫牙鲆的免疫应答和疫苗的保护效果。结果显示,以油乳化和未油乳化疫苗分别免疫牙鲆一周后,在后肠组织,AhaI和GapA乳化疫苗免疫组牙鲆检测到抗体,且分别高于AhaI和GapA未乳化疫苗免疫的牙鲆;而在血清,GapA的两种疫苗引起的GapA抗体水平没有变化;但在AhaI乳化疫苗免疫的牙鲆第21天和第35天的血清中检测到高于未免疫对照牙鲆的AhaI抗体水平,AhaI未乳化疫苗免疫牙鲆血清对照组相比没有检测到AhaI抗体水平的变化。
其他摘要This research aimed to evaluate the immune response and efficacy of several vaccine preparations, including oral-oil-emulsified vaccines against infections of some important marine fish bacterial pathogens, and some outer membrane proteins against infection of E. tarda and A. hydrophila. An oil-emulsified bivalent vaccine was prepared comprised of inactivated bacterial cells of pathogenic Vibrio anguillarum strains M3 and SMP1. The oil-emulsified bivalent vaccine was embedded in the feed pellet and administered to cultured turbot (Scophthalmus maximus) by the route of oral. The protection against M3 and SMP1 was evaluated, and several parameters of the specific and non-specific immune response were characterized in the vaccinated and control fish. Vaccination administration for 7 days elicited higher protection against M3 and SMP1 (with relative percentage survival RPS of 100% and 50%, respectively) in the group of oil-emulsified bivalent vaccine than in the non-oil-emulsified bivalent vaccine (with RPS of 57.9% and 0%, respectively). The protection was correlated with the activation of mucosa and humoral immune response in the hindgut of the group of oil-emulsified bivalent vaccine, which showed more increasing lysozyme and antiprotease activities and higher levels of antibody against M3 and SMP1 (p<0.05), and moderately increasimg antibody titres against M3 in the serum. Hindgut-situ-hybridization also indicated that the IgM were produced and distributed with higher level in the lamina of hindgut of fish administered by oil-emulsified bivalent vaccine. The oil-emulsified bivalent vaccine in the present study is applicable in the cultured fish against the infection of V. anguillarum. Inactivated bacteria cells of M3 and SMP1, CF(Streptococcus sp.) and SMW7(Edwardsiella tarda) were used as antigens to prepare for the polyvalent oral-oil-emulsified vaccine. The cultured fingerling turbot were immunized with the polyvalent vaccine by oral route. Results showed that antibody level against M3 increased (P<0.05) in hindgut, while the antibody titer in bile, gill, mid-gut, surface mucus, foregut and serum had no change in the immunized turbot. No antibody titres against SMP1, CF and SMW7 were detected in the above tissues of the immunized fish. The RPS was 100% when challenged with M3 by immersion route but no protection was detected when challenged by the i.p. route. Although the mortalities were delayed in turbot challenged immersely with SMP1, CF and SMW7, respectively, all the fish died during the observation period. Meanwhile, live rotifer vaccine was prepared using the rotifers feed by cells of M3, SMP1, CF and SMW7. The newly hatched larvae turbot were immunized with the live rotifer vaccine by oral route, but no visible protection were detected against M3, SMP1, CF and SMW7. The genes of ahaI and gapA of Aeromonas hydrophila LSA34, and eseB of Edwardsiella tarda LSE40 were cloned and expressed in E. coli. The expressed proteins were used to immunize turbots by intraperitoneal (IP) injection route. Specific antibodies were detected in the immunized turbot, and challenged tests showed that proteins of AhaI and GapA could confer higher RPS (80% and 100% respectively) against infection of A. hydrophila, but lower cross prection against E. tarda challenge (RPS of 30% and 10% respectively). Although high antibody titres could be detected in the turbot immunized with eseB, but no visible protection against the infection of E.tarda was detected. Based on the results obtained above, proteins of AhaI and GapA were used for antigens to prepare oil-emulsified vaccine for further evaluation their efficacy. Japanese flounder fish were immunized with the oil- emulsified vaccine and non-oil- emulsified vaccine respectively, via the administration of oral route. Results showed that specific antibody titres were detected in hinds of both groups of fish, however, the antibody titre in group of oil- emulsified fish were higher than that of non-oil- emulsified fish. In contrast, antibody levels produced by GapA oil-emulsified vaccine and GapA normal vaccine had no difference in the serum. The serum antibody levels against AhaI in the group of oil-emulsified fish were higher than those of the non-immunized control fish in 21-day and 35-day, respectively; while the serum antibody levels against AhaI in the group of non-oil-emulsified fish showed no difference from those of non-immunized control fish.
页数138
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/493
专题海洋环流与波动重点实验室
推荐引用方式
GB/T 7714
肖鹏. 鱼类疫苗的研究:I: 海水鱼类病原菌乳化口服疫苗的研究;II:迟缓爱德华氏菌和嗜水气单胞菌外膜蛋白疫苗的研究[D]. 海洋研究所. 中国科学院海洋研究所,2008.
条目包含的文件
文件名称/大小 文献类型 版本类型 开放类型 使用许可
10001_20061800681201(2532KB) 限制开放--浏览
个性服务
推荐该条目
保存到收藏夹
查看访问统计
导出为Endnote文件
谷歌学术
谷歌学术中相似的文章
[肖鹏]的文章
百度学术
百度学术中相似的文章
[肖鹏]的文章
必应学术
必应学术中相似的文章
[肖鹏]的文章
相关权益政策
暂无数据
收藏/分享
文件名: 10001_200618006812014肖鹏_paper.pdf
格式: Adobe PDF
此文件暂不支持浏览
所有评论 (0)
暂无评论
 

除非特别说明,本系统中所有内容都受版权保护,并保留所有权利。