文献类型：学位论文

中文题名：罗非鱼海豚链球菌疫苗研制及胞外产物特性分析

英文题名：Vaccine Preparation and Characterization of Extracellular Products from S.iniae Isolated from Tilapia

作者：张旭丽[1];

机构：[1]广东海洋大学;

导师：吴灶和;广东海洋大学|简纪常;广东海洋大学

授予学位：硕士

语种：中文

中文关键词：海豚链球菌/(Streptococccus iniae/);鉴定;胞外产物;致病性;疫苗;免疫保护

外文关键词：Streptococcus iniae; indentify; ECP; pathogenicity; vaccine; immunoprotection

中文摘要：海豚链球菌/(Streptococcus iniae/)病是水生动物的一种重要细菌性疾病,可感染多种海水和淡水鱼类,在世界许多国家均有该病的暴发和流行,对水产养殖业造成了巨大危害。近年来我国海豚链球菌病也频频爆发,目前已经感染了牙鲆、罗非鱼、美国红鱼等。 本实验从患病的罗非鱼体内经分离纯化得到一菌株,通过Biolog以及16SrRNA基因PCR鉴定该菌为海豚链球菌,并对罗非鱼进行了致病性实验,得出海豚链球菌对罗非鱼的半致死量为6.81×10~7cfu//mL。药敏试验结果显示该菌对四环素、青霉素G、环丙沙星、新生霉素、头孢呋肟、呋喃妥因、氧哌嗪青霉素、强力霉素等较为敏感,而对新霉素、卡那霉素不敏感。 由于该菌对培养基营养要求极高,同时为以后疫苗基础作准备,本实验对海豚链球菌的培养进行了优化研究,主要优化了液体培养基BHI的条件,得出海豚链球菌的培养基最适pH为7 ~ 8,最适培养温度为30℃,振荡培养对海豚链球菌的生长影响不明显。培养基优化研究表明,在BHI培养基中添加10 mg//mL的酵母粉和2 mg//mL的葡萄糖即可在对数期获得最大细菌量。同时也得出海豚链球菌的生长曲线,即:0 ~ 4 h为生长延迟期,4 ~ 24h为对数生长期,24 ~ 48 h为稳定期,随后进入衰亡期。 通过琼脂平板扩散法、酪蛋白底物法研究了海豚链球菌胞外产物/(ECP/)的一些理化特性以及化学试剂对胞外产物酶/(ECPase/)的影响,将海豚链球菌ECP注射到罗非鱼体中检验其致病性,通过一维和双向电泳技术分析胞外产物蛋白点,并自制了鼠抗海豚链球菌用其对海豚链球菌胞外产物的免疫原性进行分析,结果显示海豚链球菌ECP具有淀粉酶、蛋白酶、卵磷脂酶、明胶酶、脂肪酶以及溶血的活性,但不具有脲酶活性。EDTA、DTT、PMSF可以使ECPase的活性下降至72.4/%、77.6/%、72.4/%,而Cu~/(2+/)、Ca~/(2+/)、K~+、Mg~/(2+/)对ECPase的活性有抑制作用, Co~/(2+/)、Fe~/(3+/)、Mn~/(2+/)对其有一定程度的激活作用;ECPase对热稳定性较好,碱性条件下活性较高,其最适温度为55℃。经腹腔注射感染罗非鱼,发现对罗非鱼有致病性,且随ECP浓度的升高罗非鱼的死亡率也升高。在SDS-PAGE图中可发现ECP主要有12条条带,主要集中在28-68kDa之间,在双向电泳图中,海豚链球菌的ECP主要条带集中在26-95kDa之间,海豚链球菌在pH为4-7时有120个点。western-blot结果主要得出4条免疫条带,分子量分别为26kDa、37 kDa、95 kDa、97 kDa。 同时研制了海豚链球菌福尔马林灭活疫苗以及白油佐剂疫苗,并用这两种疫苗分别腹腔注射罗非鱼/(80g-100g/),每尾注射0.2mL,对照组注射无菌生理盐水,结果显示各免疫组对罗非鱼的溶菌酶活性、SOD活力、血清抗菌活力以及血清抗体效价水平均显著高于对照组。免疫八周后腹腔注射海豚链球菌菌液2×10~8cfu//ml的剂量,观察15天后得出水剂组的相对保护率为65.43/%,而油剂组的相对保护率为88.89/%,表明这两种疫苗均有助于罗非鱼抵抗海豚链球菌的能力。

外文摘要：Streptococcus iniae is known to be a major bacterial disease in mariculture animal systems. Disease outbreaks attributed to S.iniae has been reported in some mariculture aquatic animal farm production. In recent years , mass mortalities of many cultured kinds of maricultured fishes,such as Paralichthys olivaceus, tilapia and Sciaenops ocellatus have occurred frequently infected by S.iniae in china . In this study, we isolated one strain from the diseased tilapia collected from the coast of the South China Sea, and identified it as S.iniae by biology system and 16SrRNA PCR. The pathogenicity of S.iniae to tilapia was also tested and its 50/% lethal dose /(LD50/) value in tilapia was 6.81×10~7cfu//mL. In addition, S.iniae was more senstive to Tetracycline、Benzylpenicillin G、Ciprofloxacin、Novobiocin、Cefuroxime、Piperacillin、Doxycycline et al, but resistant to Neomycin、Kanamycin. S.iniae has high nutritional requirement and we have to make a preparation for the development of vaccine , so we optimized its culture conditions. There have some factors that effect the growth of S iniae, for example, pH, gaseous environment, temperature and growth factors in the fluid culture medium were studied to optimize its growth parameters. The results indicated that the initial pH 7 - 8 and the 30 degree’s temperature were optimal for the S.iniae, while whether there was dissolved oxygen or not had small effect on its growth. The maximal bacteria’s concentration could be achieved by supplying 10 mg//mL yeast powder and 2 mg//mL glucose in the BHI. And we also got its growth curve : 0-4h is growth delay phase, 4-24h is logarithmic phase, 24-48h stable phase, and then is decline phase. The biologic activities of S.iniae extracellular products were studied. The results showed that S.iniae ECP has amylase, protease, lecitinase, gelatinase and lipase activities, but no urease activity. It could dissolve the blood cell of tilapia , which can get the haemolysis potency of 1:64. The ECP had good stablity , and the optimal temperature was 55℃. Some chemical agents had also many effects on the ECP, whose results showed that the EDTA、PMSF、DTT could reduce the activities of ECPase to 72.4/%、77.6/%、72.4/% respectivly, and Cu~/(2+/)、Ca~/(2+/)、K~+、Mg~/(2+/) could reduce the activities of ECPase, while Fe~/(3+/), Co~/(2+/), Mn~/(2+/) could activate its activities. The pathogenicity of ECP to tilapia was tested, which showed S.iniae ECP has toxicity to tilapia and the mortality will raise with the increase of ECP concentration. We analyzed the main protein of ECP by SDS-PAGE and two dimensional electrophoresis. The SDS-PAGE analysis exhibited that the main protein of the ECP were arranged between 26 kDa and 95kDa, while the two dimensional electrophoresis showed 120 spots. In addition, we studied the ECP of western-blot with rat anti S.iniae ,which showed four proteins ,whose molecular weight were 26kDa、37 kDa、95 kDa、97 kDa respectively. The formalin killed S.iniae vaccine and white oil adjuvant vaccine were prepared and used to vaccinate the tilapia for eight weeks. After immunization the fish were challenged by intraperitioneal injection of S.iniae. The tilapia immunized with vaccine showed that there were significantly higher serum bacteriolytic activities, lysozyme activities ,SOD,and antibody activity than those fish in the control group. And the formalin killed S.iniae vaccine and white oil adjuvant vaccine showed high immunoprotection with RPS of 65.43/%, 88.89/% respectively.

年份：2010