CN101024080A - 一种海豚链球菌白油佐剂灭活疫苗及其制备方法 - Google Patents
一种海豚链球菌白油佐剂灭活疫苗及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种海豚链球菌白油佐剂灭活疫苗及其制备方法，其包括(1)分别从海水养殖或淡水养殖的病鱼体内分离出海豚链球菌；(2)对分离得到的海豚链球菌进行扩大培养，灭活，浓缩，得到灭活浓缩菌液；(3)将96％灭活浓缩菌液和4％灭菌吐温－80混合振荡，使吐温－80完全溶解为止，作为水相；94％的10号白油和6％的司本－80混合液中加入2％的硬脂酸铝作为稳定剂，此混合物混合搅拌加热至透明，高压灭菌处理后作为油相；按水相∶油相＝3∶7的比例，高速搅拌乳化，在终止搅拌前加入终浓度为0.01％的柳硫汞溶液，即得。本发明的灭活疫苗对链球菌病具有很好的防治效果，相对保护率在93.8％以上，完全能满足实际生产的需要。
Description
技术领域
本发明涉及一种灭活疫苗，具体涉及一种海豚链球菌白油佐剂灭活疫苗及其制备方法。
背景技术
海豚链球菌(Streptococcus iniae)属于链球菌科的链球菌属，呈圆形或卵圆形，成对或成不同长度的链状，无鞭毛，不运动，有荚膜，不形成芽孢，革兰氏染色阳性，兼性厌氧。S.iniae最初由Pier(1976)从亚马逊淡水海豚皮下脓肿中分离并命名的。1997年，Weinstein等证明S.iniae是一种条件性人畜共患病病原菌，它不仅能感染几乎所有的海、淡水鱼类，如罗非鱼、鲈、鲷、石斑、三纹鱼、虹鳟等，还可感染人类，引起蜂窝组织炎，心内膜炎。
S.iniae在感染易感鱼品种如罗非鱼、尖吻鲈、条纹杂交鲈等之后，一周内可引起50％以上的鱼死亡，有的甚至高达95％。该病也可以导致罗非鱼呈每天少量死亡但持续时间长、最终造成重大经济损失的慢性流行性疾患。据Shoemaker估计，全球每年因该病造成养殖鱼类的经济损失超过1.5亿美元。该病在各主要鱼类养殖国家均有发生，但温带和热带、亚热带地区尤其严重。
对鱼类链球菌病防治，目前主要依赖抗生素药物，但抗药性和药物残留问题极大限制了抗生素的应用。由此，我们必须探索疫苗预防该病的途径。目前，国外对该菌的疫苗研究已经积累了丰富的经验，Eldar，A.等从1995年起就开始研究利用S.iniae全菌及其提取的蛋白制备成灭活疫苗，并且成功应用于红鳟鱼的链球菌病防治；Klesius等2000年将S.iniae的单一苗和复合苗通过腹腔和肌肉注射到罗非鱼中，使鱼体死亡率较对照组显著降低。Phillip H.Klesius等利用异源S.iniae菌株(包括海水菌株与淡水菌株)按一定比例混合制备成多价疫苗对罗非鱼进行免疫，并利用其制备疫苗的菌株进行攻毒，获得63.1％(淡水菌株攻毒)和87.3％(海水菌株攻毒)的相对保护率。
然而，国外相关报道中，疫苗佐剂大都为佛氏佐剂，此佐剂分为不完全佐剂和完全佐剂，要联合应用。佛氏佐剂为经典油佐剂，尤其当抗原剂量较小时，能够获得最佳的佐剂作用。但是这种佐剂存在如下缺点：①引起局部组织损伤，影响肉质；②完全佛氏佐剂含有分枝杆菌，使结核菌素试验呈阳性反应；③该佐剂的成本太高，难以在生产实际中推广应用。迄今为止，还未见到我国的关于鱼类链球菌疫苗的研究开发报道。而海豚链球菌病已在广东沿海地区养殖鱼类中时有爆发，对我国水产养殖业是一个巨大的潜在威胁。
发明内容
本发明的目的在于针对现有技术存在的问题，提供一种海豚链球菌白油佐剂灭活疫苗。
本发明的另一个目的是提供上述海豚链球菌白油佐剂灭活疫苗的制备方法。
本发明的海豚链球菌白油佐剂灭活疫苗的制备方法，包括如下步骤：
(1)分别从海水养殖或淡水养殖的病鱼体内分离出海豚链球菌；从海水养殖的病鱼体内分离出的海豚链球菌(S.iniae)，命名为HD-1；从淡水养殖的病鱼体内分离出的海豚链球菌(S.iniae)，命名为TBY-1。
(2)对分离得到的海豚链球菌进行扩大培养，灭活，浓缩，得到灭活浓缩菌液；
(3)将96％灭活浓缩菌液和4％灭菌吐温-80混合振荡，使吐温-80完全溶解为止，作为水相；94％的10号白油和6％的司本-80混合液中加入2％的硬脂酸铝作为稳定剂，此混合物混合搅拌加热至透明，高压灭菌处理后作为油相；按水相∶油相＝3∶7的比例，高速搅拌乳化，在终止搅拌前加入终浓度为0.01％的柳硫汞溶液，即制备成海豚链球菌白油佐剂灭活疫苗。
在上述制备方法中，步骤(1)所述灭活的方法为加入甲醛溶液灭活。
在上述制备方法中，步骤(3)所述高速搅拌乳化的速度为13000r/min，时间为5分钟，重复3次。
与现有技术相比，本发明具有如下有益效果：本发明的海豚链球菌白油佐剂灭活疫苗免疫尼罗罗非鱼，HD-1(海水强毒标准株)和TBY-1(淡水强毒标准株)免疫组的相对保护率(RPS)分别为93.8％和100％。可见，本发明的海豚链球菌白油佐剂灭活疫苗对链球菌病具有很好的防治效果，相对保护率在93.8％以上，完全能满足实际生产的需要。
附图说明
图1为海豚链球菌在BHI固体培养基上生长所呈现的单菌落；
图2为尼罗罗非鱼感染海豚链球菌的症状；
图3为海豚链球菌革兰氏染色的菌体图；
图4为海豚链球菌白油佐剂灭活疫苗免疫尼罗罗非鱼后，用对照强毒株攻毒后疫苗保护性折线图。
具体实施方式
实施例1
1.冷冻菌种复苏及扩大培养
将-70℃保存的冻存强毒株标准菌种HD-1和TBY-1，BHI固体培养基划线28℃培养48h(见图1)，挑单菌落接种于10mL液体BHI液体培养基中，28℃振荡培养48h；再将此培养物全部接种于大剂量BHI液体培养基中，28℃振荡培养48h。然后对培养物进行梯度稀释涂板计数。海豚链球菌革兰氏染色的菌体图片如图3。强毒株标准菌种HD-1和TBY-1的生理、生化试验见表1。
表1海豚链球菌HD-1，TBY-1生理、生化试验结果
| 标准株(ATCC29178) | HD-1 | TBY-1 | ||
| 生长 | 10℃6.5％NaCl45℃溶血运动性 | ---N- | ---β- | ---α- |
| 发酵产酸 | 甘露醇蔗糖山梨醇水杨苷棉子糖木糖乳糖麦芽糖阿拉伯糖 | ++----++- | ++----++- | -+---+++- |
| 水解 | 精氨酸马尿酸七叶苷淀粉 | +--+ | +-++ | +--+ |
| 脱羧 | 精氨酸鸟氨酸赖氨酸 | +-- | +-- | -+- |
| 其他 | V-PDNA酶尿素酶H2S过氧化氢酶 | ----- | ----- | ----- |
注：“+”为阳性，“-”为阴性，“N”为不确定
2.菌体抗原的灭活
当大剂量BHI液体培养基中菌体浓度达到108 CFU/mL时，BHI液体培养基中按终体积的0.3％加入甲醛溶液28℃灭活24h。
3.灭活菌体抗原的收集及浓缩
灭活后菌液8000rpm离心30min(eppendrof centrifuge 5810R)，弃上清夜，加PBS液振荡混匀(MSI Minishaker)同条件离心3次去处甲醛。最后离心菌泥中加入0.1倍原灭活菌液体积的PBS液振荡混匀。
4.疫苗的制备
以96％标准强毒株浓缩菌液和4％灭菌吐温-80混合振荡，使吐温-80完全溶解为止，作为水相；94％的10号白油和6％的司本-80混合液中加入2％的硬脂酸铝作为稳定剂，此混合物混合搅拌加热至透明，高压灭菌处理后作为油相。按水相∶油相＝3∶7的比例，以13000rpm高速搅拌乳化5min，重复3次，在终止搅拌前加入终浓度为0.01％的柳硫汞溶液，即制备成海豚链球菌白油佐剂灭活疫苗。
5.疫苗的安全性和质量检测
5.1疫苗无菌检验
取少量制备好的疫苗用涂抹棒涂于BHI固体培养板上28℃恒温培养48h，无任何菌落生长。
5.2疫苗质量检测
取制备的疫苗1滴轻轻滴在水中，静止观测30min没有发现油滴散开；考马斯亮兰R250(水溶性染料)和苏丹II(油溶性染料)检测，疫苗为苏丹II染料颜色，疫苗为油包水剂型；取制备疫苗3000rmp离心15min未见分层现象。
5.3疫苗安全性检测
取制备的疫苗腹腔注射健康尼罗罗非鱼。分成4个组，每组10尾，同时设活菌直接注射对照组和注射PBS对照组，连续观测14d。直接注射活菌组尼罗罗非鱼死亡率在70％以上，PBS对照组和疫苗组尼罗罗非鱼死亡率小于5％(考虑注射机械损伤)。尼罗罗非鱼在感染海豚链球菌后的症状见图2，从图2中可见，尼罗罗非鱼身体出现明显的“C”弯曲，眼部出现明显突出混浊显现。
6.制备的疫苗免疫保护力试验
将试验用把尼罗罗非鱼分成实验组和对照组，每组至少30尾且每组至少要3个重复。制备的海豚链球菌强毒株标准疫苗对实验组尼罗罗非鱼以0.1ml/尾腹腔注射方式免疫，初次免疫后第2周加强免疫一次，对照组每次腹腔注射PBS液0.1ml/尾，程序同实验组。坚强免疫后第2周，以8-10倍海豚链球菌LD50(半数致死量)的量同时对实验组和对照组尼罗罗非鱼进行攻毒，实验组免疫相对保护力在90％以上。海豚链球菌白油佐剂灭活疫苗免疫尼罗罗非鱼后，用对照强毒株攻毒后疫苗的保护性折线图如图4所示。
Claims (4)
1.一种海豚链球菌白油佐剂灭活疫苗的制备方法，其特征在于包括如下步骤：
(1)分别从海水养殖或淡水养殖的病鱼体内分离出海豚链球菌；
(2)对分离得到的海豚链球菌进行扩大培养，灭活，浓缩，得到灭活浓缩菌液；
(3)将96％灭活浓缩菌液和4％灭菌吐温-80混合振荡，使吐温-80完全溶解为止，作为水相；94％的10号白油和6％的司本-80混合液中加入2％的硬脂酸铝作为稳定剂，此混合物混合搅拌加热至透明，高压灭菌处理后作为油相；按水相∶油相＝3∶7的比例，高速搅拌乳化，在终止搅拌前加入终浓度为0.01％的柳硫汞溶液，即制备成海豚链球菌白油佐剂灭活疫苗。
2.如权利要求1所述的制备方法，其特征在于步骤(1)所述灭活的方法为加入甲醛溶液灭活。
3.如权利要求1所述的制备方法，其特征在于步骤(3)所述高速搅拌乳化的速度为13000r/min，时间为5分钟，重复3次。
4.一种海豚链球菌白油佐剂灭活疫苗，其特征在于由权利要求1所述方法制备而成。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101890159A (zh) * | 2010-06-23 | 2010-11-24 | 广西壮族自治区水产研究所 | 罗非鱼二联链球菌灭活疫苗的制备方法 |
| CN101586157B (zh) * | 2008-12-18 | 2012-01-11 | 中山大学 | 一种鱼类病原菌海豚链球菌分子诊断试剂盒及检测方法 |
| CN102329746A (zh) * | 2011-08-16 | 2012-01-25 | 武汉科前动物生物制品有限责任公司 | 猪链球菌、副猪嗜血杆菌病二联灭活疫苗及制备方法 |
| CN105481973A (zh) * | 2015-12-11 | 2016-04-13 | 四川农业大学 | 一种用于制备渔用免疫佐剂的多肽及其用途 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101586157B (zh) * | 2008-12-18 | 2012-01-11 | 中山大学 | 一种鱼类病原菌海豚链球菌分子诊断试剂盒及检测方法 |
| CN101890159A (zh) * | 2010-06-23 | 2010-11-24 | 广西壮族自治区水产研究所 | 罗非鱼二联链球菌灭活疫苗的制备方法 |
| CN101890159B (zh) * | 2010-06-23 | 2012-07-25 | 广西壮族自治区水产研究所 | 罗非鱼二联链球菌灭活疫苗的制备方法 |
| CN102329746A (zh) * | 2011-08-16 | 2012-01-25 | 武汉科前动物生物制品有限责任公司 | 猪链球菌、副猪嗜血杆菌病二联灭活疫苗及制备方法 |
| CN105481973A (zh) * | 2015-12-11 | 2016-04-13 | 四川农业大学 | 一种用于制备渔用免疫佐剂的多肽及其用途 |
| CN105481973B (zh) * | 2015-12-11 | 2020-05-05 | 四川农业大学 | 一种用于制备渔用免疫佐剂的多肽及其用途 |
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