WO2011079627A1 - Multi-component adjuvant of immersion vaccine for fish, and its application and using method - Google Patents

Multi-component adjuvant of immersion vaccine for fish, and its application and using method Download PDF

Info

Publication number
WO2011079627A1
WO2011079627A1 PCT/CN2010/076978 CN2010076978W WO2011079627A1 WO 2011079627 A1 WO2011079627 A1 WO 2011079627A1 CN 2010076978 W CN2010076978 W CN 2010076978W WO 2011079627 A1 WO2011079627 A1 WO 2011079627A1
Authority
WO
WIPO (PCT)
Prior art keywords
fish
vaccine
adjuvant
saponin
triton
Prior art date
Application number
PCT/CN2010/076978
Other languages
French (fr)
Chinese (zh)
Inventor
王秀华
黄倢
王玉娟
赵伟伟
Original Assignee
中国水产科学研究院黄海水产研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国水产科学研究院黄海水产研究所 filed Critical 中国水产科学研究院黄海水产研究所
Publication of WO2011079627A1 publication Critical patent/WO2011079627A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS

Definitions

  • the invention relates to a marine biological disease immunodeficiency control technology, in particular to a multi-combination adjuvant for immunizing fish with a immersion vaccine, and an application and method thereof.
  • the object of the present invention is to provide a highly effective multi-group adjuvant for fish immersion vaccines.
  • N-acetylcysteine (NAC) and Triton X-100 (TRTTONX-100) have the ability to dissolve mucus on fish, and saponin has improved mucus immunity.
  • NAC N-acetylcysteine
  • TRTTONX-100 Triton X-100
  • the effect of cellular immunity, while racemic anisodamine (also known as 654-2) can relax muscles and facilitate the penetration of vaccine antigens into the body.
  • the four adjuvants listened to and combined with the 3 ⁇ 41 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ , , , , , , il 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654
  • the multi-combination adjuvant for fish immersion vaccine of the present invention includes NAC, TRITON X-100 saponin and 654-2, wherein the range of NAC, TRITON X-100 saponin and 654-2 is 0.1-10 Mg L, 0.1-10 mg L, 0.1-50 mg L, 1-60
  • the present invention has a concentration of 0.1-10 mg/L, 0.1-10 mg L, 0.1-50 mg L, 1-60 mg L of NAC, TRITON X-100, saponin and 654-2 immunomulti-adjuvant adjuvant. Synergistic effects in the immersion vaccine of seawater-stained water fish to achieve high efficiency immunity -3 ⁇ 4 ⁇ 4.
  • the shed method of the present invention is as follows:
  • the fish to be immunized are transferred into a fine aqueous solution of NAC and TRITON X-100 in a concentration range of 0.1-10 mg/l, pre-soaked for 1-20 min, and then added with a concentration of 0.1-50 saponins and used.
  • Vaccine soak for 1 to 20 minutes, and then into the vaccine soaking solution to force the concentration of 1-60 654-2 to l-10min to complete the j3 ⁇ 43 ⁇ 4 epidemic.
  • the positive of the present invention lies in: using NAC, TRITON X-100, saponin, 654-2 multiple agents as a vaccine combination adjuvant can significantly improve the immunity of the vaccine - 3 ⁇ 43 ⁇ 4 is significantly better than a single adjuvant; Simple, n, safe for the shed, has no effect on the survival rate of the fish and the raw meal. > And the process and the process are not necessary, and it is also harmless to the ringworm.
  • the multi-combination adjuvant of the fine immersion vaccine of the present invention is characterized in that the multi-component adjuvant as a fish immersion vaccine includes NAC, TRITON X-100, saponin and 654-2, wherein NAC, TRITON X-100, saponin
  • the range of glucosides of glucosides and 654-2 is 0.1-10 mg L, 0.1-10 mg/L, 0.1-50 mg L, and l-60 mg/L, respectively.
  • the present invention has a concentration of 0.1-10 mg/L, 0.1-10 mg L, 0.1-50 mg L, 1-60 mg L of NAC, TRITON X-100, saponin and 654-2 immunomulti-adjuvant adjuvant. Synergistic effects in the immersion vaccine of seawater-stained water fish to achieve high efficiency immunity -3 ⁇ 4 ⁇ 4.
  • the shed method of the present invention is as follows:
  • the fish to be immunized are transferred into a fine aqueous solution of NAC and TRITON X-100 in a concentration range of 0.1-10 mg/l, pre-soaked for 1-20 min, and then added with a concentration of 0.1-50 saponins and used.
  • Vaccine immersion immunization for 1-20 min, then add 654-2 with a concentration of 1-60 to the vaccine soaking solution to maintain l-10 min.
  • dissolved oxygen in the water when i3 ⁇ 4 ⁇ 7m or more, ⁇ female $1 ⁇ 2 ⁇ 2 °C. After the game is immune, it is difficult to move the fish into the dance.
  • NAC and TRTTON-100 are used in a concentration range of 0.1-10 mg/L, and the soaking time is 3-50 min; the concentration of saponin is 0.1-50 mg L, the soaking time is 2-30 min; The concentration range is l-60mg L, and the soaking time is l-10min.
  • the ship is used for marine fish, 3 ⁇ 4feK fish and semi-fish.
  • Vaccine preparation Vanguillarum was inoculated into 2216E liquid medium, cultured at 28 ° C for 24 h; centrifuged at 4000 ip for 20 min at 4 ° C, and the cells were collected; the collected cells were placed in sterile PBS, pH 7.2, Made of 1.0 ⁇ 108 cells/mL of Vibrio suspension, 0.5% After being inactivated at 28 °C for 48 hours, it was used as a vaccine for i. It was stored at 4 °C. The inactivated cells were inoculated on TSB solid plate, and cultured at 28 °C for 24 hours and 48 hours. .
  • Immersion immune adjuvant NAC, TRITON X-100 saponin, 654-2, shed concentration of 0.1mg L, 0.1mg L, 50mg L, lmg L.
  • Group i Inch immunization group, no adjuvant vaccine soaking immunization group, Zuoqi vaccine immersion sensitivity, injection blank control group, soaking blank control group, each group was parallel, each group 3x30 fiber.
  • Table 1 uses NAC, TRITON X-100, saponin, 654-2 as the combination of Zuoqi warm bubble to avoid immune immunization relative to ⁇ 3 ⁇ 4 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 0 death rate 0 / 0 RPS% average RPS%
  • Example 2 Application of the present invention to a concentration of 5 m, 5 m, lm, 30 mg L of NAC, TRITON X-100 saponin, 654-2 multi-adjuvant adjuvant and inactivated P. aeruginosa vaccine to Luo Fei Fish soaking immunity 1 material:
  • Aeromonas punctatus was inoculated into LB liquid medium and cultured at 28 ° C for 24 h ; The bacteria were collected by centrifugation at 4000 ipm for 20 min at C; the collected cells were placed in a sterile sterilized solution, pH 7.2, and made into a suspension of 1.0 X 109 cells/mL, and 0.5% formaldehyde was inactivated at 28 ° C. After 48 hours, the test vaccine was stored at 4 ° C. At the same time, the inactivated cells were inoculated on LB solid plates, and cultured at 28 ° C for 24 h and 48 h, and it was confirmed that no cells grew.
  • Immersion immunoadjuvant NAC, TRITON X-100 saponin, 654-2 combination adjuvant, 5m, 5mg L lmg/L 30mg ⁇ L.

Abstract

The invention provides a multi-component adjuvant of immersion vaccine for fish, comprising N-acetylcysteine, Triton X-100, saponin and raceanisodamine. The working concentrations of said components are 0.1-10 mg/l, 0.1-10 mg/l, 0.1-50 mg/l and 1-60 mg/l respectively. The application of said adjuvant in immersion immunization for fish and the using method thereof are also provided.

Description

删浸泡鶴多组合佐剂及其应用和删方法  Decomposed soaked crane multi-component adjuvant and its application and deletion method
技术领域 Technical field
本发明涉及海洋生物疾病免疫防治技术， 具体是鱼用浸泡疫苗免疫多组合佐剂及其应用和 方 法。  The invention relates to a marine biological disease immunodeficiency control technology, in particular to a multi-combination adjuvant for immunizing fish with a immersion vaccine, and an application and method thereof.
背景技术 Background technique
我国 量据世界首位， ί建类的 «防治技术研 ¾g较墁，开发的渔用疾病防治产品不 能满^ ^的需求， 特别在渔用疫苗开发与应用方面， 目前可供 ^通華翻勺产品不多。 因此 難中鱼类细菌性疾病治疗过多依柳離生素，其结果是长期的应用抗生素不仅会降 产品的质量， 还会导»菌的耐药性增强， 给以后的疾病控制增加艘。 国外在鱼«病控制方面已普遍删疫苗免 疫的方法， 包括注射用疫苗、浸泡用疫苗及口服疫苗等。 国内的鱼用疫苗产品制擁中类不多， 在鱼类疫 苗研究方面多侧重于注射用疫苗。鱼类注射用疫苗对疾病预防 ¾¾明显， 但注射免虹作量大、工作效 率低， 且会对鱼体造成伤害。而鱼类浸删疫苗荷在 ¾缺点， 但单纯的棚疫苗浸泡 不佳。  According to the world's first quantity, the research and development technology of the ίjian class is relatively embarrassing, and the development of fishery disease prevention and control products cannot meet the demand, especially in the development and application of fishery vaccines. There are not many products. Therefore, it is difficult to treat fish bacterial diseases with too much lycopene. As a result, long-term application of antibiotics will not only reduce the quality of the products, but also increase the drug resistance of the bacteria, and increase the control of disease in the future. In foreign countries, vaccines have been widely used to eliminate vaccines, including vaccines for injection, vaccines for infusion, and oral vaccines. There are not many domestic fish vaccine products, and most of them focus on vaccines for injection. Vaccines for fish injections are obvious for disease prevention, but injections are free of rainbows, work inefficient, and cause damage to fish. The fish immersion vaccine has a disadvantage of 3⁄4, but the simple shed vaccine is poorly soaked.
目前， 有 类浸泡免疫用疫苗产品因受浸泡免疫¾¾较差的限制， 开发的产品不多。 国内已开发 的鱼类浸泡疫苗佐剂有莨菪碱、葡聚糖、 MS1312, ¾¾i正实， 棚后免疫 { 户$¾不理想。而国外的 鱼类浸泡免疫佐剂多为保密配方。基于以上现状， 我国的鱼类浸泡疫苗研 处于缓險展状态， 限 制阻碍了产业的发展。 因 共鱼类浸泡疫苗佐剂， 提高鱼类浸泡疫苗 ¾¾， 对推动水产舞細疫苗制 剂产业的快裁展是非常必要的。  At present, there are few products developed for vaccine products for immersion immunization due to poor tolerance of immersion immunity. Domestically developed fish immersion vaccine adjuvants are saponin, dextran, MS1312, 3⁄43⁄4i, and post-shed immunity { household $3⁄4 is not ideal. Foreign fish soaking immune adjuvants are mostly secret formulas. Based on the above situation, China's fish immersion vaccine research is in a state of accelerated risk, and restrictions have hindered the development of the industry. The promotion of fish immersion vaccines due to co-fish immersion vaccine adjuvants is necessary to promote the rapid development of the aquatic dance vaccine industry.
发明内容 Summary of the invention
本发明的目的是为鱼类浸 疫苗 共一种高效的多组^ ^疫佐剂。  The object of the present invention is to provide a highly effective multi-group adjuvant for fish immersion vaccines.
鱼^ it用浸泡疫苗免疫， 不 喿作方便节省时间， 而且相对于注射免 寸鱼的损伤小， m & 用浸泡疫苗免疫是一种最佳的免疫方案。働用浸泡疫苗免疫时， 在通常不加浸泡疫苗佐剂或特异免疫 增强剂的情况下， 鱼体仅仅¾¾思部擲虫疫苗抗原， 身体表面其他的可以 «免疫反应的部位多被体表 的粘麵覆盖， 导致可 m疫苗抗原的细胞不能有效觀到疫苗抗原， 因此棚»去除鱼謹夜的佐 齐喊分， 可以提高鱼类浸泡疫苗免疫的 ¾¾。研 ¾¾離研究发现， N-乙酰半胱氨酸（简称 NAC)、 曲拉通 X-100 (即 TRTTONX-100)具有溶解鱼体表粘液的能力，皂角苷具有提高鱼类粘液免疫力与细胞 免疫的功效，而消旋山莨菪碱（又称 654-2)可^ 体肌肉松弛， 易于疫苗抗原向体内的渗入。四种佐剂 聽且合 ¾1牟决了鱼体粘液 苗抗原擲虫鱼徹原 il细胞的问题， 皂角苷提高了鱼体粘液 免疫与细胞免疫力， 同时 654-2舰体肌肉松弛， 力膙了抗原渗藤体的 ¾¾。该四种佐剂组合有效提 高了鱼类浸泡疫苗免疫 ¾¾， ■证实具 剂的功能， 且安^ ¾¾i正实在 ^范围内对鱼类及环 境安全。因此确定 NAC、 TRTTONX-100,皂角苷、 654-2佐剂组合为高¾ ^浸泡疫苗免疫多组合佐剂。  Fish ^ it is immunized with immersion vaccine, it is convenient to save time, and it is less effective than injection of squid. m & immunization with immersion vaccine is the best immunization program. When immunizing with a immersion vaccine, the fish body is only 3⁄43⁄4% of the worm vaccine antigen in the absence of immersion vaccine adjuvant or specific immune enhancer, and other parts of the body surface that can be immune-reactive are Covering the sticky surface, the cells that can cause the m vaccine antigen can not effectively see the vaccine antigen, so the sheds to remove the fish from the Zuo Qi shouting, can improve the immunization of fish vaccination -3⁄4⁄4. Research has shown that N-acetylcysteine (NAC) and Triton X-100 (TRTTONX-100) have the ability to dissolve mucus on fish, and saponin has improved mucus immunity. The effect of cellular immunity, while racemic anisodamine (also known as 654-2) can relax muscles and facilitate the penetration of vaccine antigens into the body. The four adjuvants listened to and combined with the 3⁄41 牟 牟 鱼 鱼 鱼 鱼 鱼 鱼 鱼 鱼 鱼 鱼 鱼 鱼 鱼 鱼 鱼 鱼 , , , , , , , il 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 654 It licked the 3⁄43⁄4 of the antigen. The four adjuvant combinations effectively enhance the immunization of fish immersion vaccines. ■ Confirm the function of the agent, and the safety is safe for fish and the environment. Therefore, the combination of NAC, TRTTONX-100, saponin, and 654-2 adjuvant was determined to be a high-complex adjuvant adjuvant.
因此， 本发明鱼用浸泡疫苗多组合佐剂包括 NAC、 TRITON X-100 皂角苷和 654-2， 其中 NAC、 TRITON X-100 皂角苷和 654-2的棚 ¾¾g范围分别为 0.1— 10 mg L、 0.1— 10 mg L、 0.1— 50 mg L、 1-60 本发明的浓度分别在 0.1— 10 mg/L、0.1— 10 mg L、0.1— 50 mg L、 1-60 mg L的 NAC、 TRITON X- 100、 皂角苷和 654-2免疫多组合佐剂，細¾括海纖炎水鱼类的浸泡疫苗中协同作用达到高效免疫 ¾¾。 Therefore, the multi-combination adjuvant for fish immersion vaccine of the present invention includes NAC, TRITON X-100 saponin and 654-2, wherein the range of NAC, TRITON X-100 saponin and 654-2 is 0.1-10 Mg L, 0.1-10 mg L, 0.1-50 mg L, 1-60 The present invention has a concentration of 0.1-10 mg/L, 0.1-10 mg L, 0.1-50 mg L, 1-60 mg L of NAC, TRITON X-100, saponin and 654-2 immunomulti-adjuvant adjuvant. Synergistic effects in the immersion vaccine of seawater-stained water fish to achieve high efficiency immunity -3⁄4⁄4.
本发明的棚方法如下：  The shed method of the present invention is as follows:
(1)首先删体表完 的待免麵类， 体難在每尾 2g以上；  (1) First of all, the face class that is finished after the body form is deleted, and the body is difficult to be more than 2g per tail;
(2)将待免疫的鱼移入浓度范围均为 0.1—lOmg/l的 NAC、 TRITON X- 100混^ 細水溶液中， 预浸泡 1一 20min， 之后加入浓度 0.1— 50 的皂角苷及待用疫苗， 浸泡 1一 20min， 再向疫苗浸泡液 中力口入浓度 1—60 的 654— 2待 l-10min即完 j¾¾疫。  (2) The fish to be immunized are transferred into a fine aqueous solution of NAC and TRITON X-100 in a concentration range of 0.1-10 mg/l, pre-soaked for 1-20 min, and then added with a concentration of 0.1-50 saponins and used. Vaccine, soak for 1 to 20 minutes, and then into the vaccine soaking solution to force the concentration of 1-60 654-2 to l-10min to complete the j3⁄43⁄4 epidemic.
本发明的积极 在于： 用 NAC、 TRITON X-100,皂角苷、 654-2多组^^剂作为疫苗组合佐剂 后可以显著提高疫苗的免疫 ¾¾， ¾¾显著好于单一佐剂； 棚方法简便， n, 棚安全， 对鱼 的存活率与生 ¾¾g没有影响> 而且不需要额汐卜的 与工序， 对环 喿作人员同样无害。  The positive of the present invention lies in: using NAC, TRITON X-100, saponin, 654-2 multiple agents as a vaccine combination adjuvant can significantly improve the immunity of the vaccine - 3⁄43⁄4 is significantly better than a single adjuvant; Simple, n, safe for the shed, has no effect on the survival rate of the fish and the raw meal. > And the process and the process are not necessary, and it is also harmless to the ringworm.
具体猫方式 Specific cat way
本发明細浸泡疫苗多组合佐齐唭特征在于:作为鱼类浸泡疫苗的多组合佐剂包括， NAC、 TRITON X-100、皂角苷和 654-2,其中 NAC、 TRITON X-100、皂角苷和 654-2的棚 ¾¾g范围分别为 0.1— 10 mg L、 0.1— 10 mg/L、 0.1— 50 mg L、 l-60 mg/L。  The multi-combination adjuvant of the fine immersion vaccine of the present invention is characterized in that the multi-component adjuvant as a fish immersion vaccine includes NAC, TRITON X-100, saponin and 654-2, wherein NAC, TRITON X-100, saponin The range of glucosides of glucosides and 654-2 is 0.1-10 mg L, 0.1-10 mg/L, 0.1-50 mg L, and l-60 mg/L, respectively.
±¾的^乙酰半胱氨酸、 曲拉通 χ-ιοο、皂角苷、消旋山 m¾碱的赚优于 目当于化学椒。 本发明的浓度分别在 0.1— 10 mg/L、0.1— 10 mg L、0.1— 50 mg L、 1-60 mg L的 NAC、 TRITON X- 100、 皂角苷和 654-2免疫多组合佐剂，細¾括海纖炎水鱼类的浸泡疫苗中协同作用达到高效免疫 ¾¾。  ±3⁄4 of acetylcysteine, Triton χ-ιοο, saponin, racemic m3⁄4 base earned better than the chemical pepper. The present invention has a concentration of 0.1-10 mg/L, 0.1-10 mg L, 0.1-50 mg L, 1-60 mg L of NAC, TRITON X-100, saponin and 654-2 immunomulti-adjuvant adjuvant. Synergistic effects in the immersion vaccine of seawater-stained water fish to achieve high efficiency immunity -3⁄4⁄4.
本发明的棚方法如下：  The shed method of the present invention is as follows:
(1)首先删体表完 的待免麵类， 体難在每尾 2g以上；  (1) First of all, the face class that is finished after the body form is deleted, and the body is difficult to be more than 2g per tail;
(2)将待免疫的鱼移入浓度范围均为 0.1—lOmg/l的 NAC、 TRITON X- 100混^ 細水溶液中， 预浸泡 1一 20min， 之后加入浓度 0.1— 50 的皂角苷及待用疫苗， 浸泡免疫 1一 20min， 再向疫苗浸 泡液中加入浓度 1—60 的 654— 2维持 l-10min即完 j¾¾疫。 为了保证免疫 ¾¾， 浸泡时水中溶解 氧 i¾^ 7m 以上， ^雌 $½±2°C内。完戯泡免疫后， 小心将鱼移入舞 纖行難即可。  (2) The fish to be immunized are transferred into a fine aqueous solution of NAC and TRITON X-100 in a concentration range of 0.1-10 mg/l, pre-soaked for 1-20 min, and then added with a concentration of 0.1-50 saponins and used. Vaccine, immersion immunization for 1-20 min, then add 654-2 with a concentration of 1-60 to the vaccine soaking solution to maintain l-10 min. In order to ensure immunity 3⁄43⁄4, dissolved oxygen in the water when i3⁄4^ 7m or more, ^ female $1⁄2 ± 2 °C. After the game is immune, it is difficult to move the fish into the dance.
本发明中 NAC、 TRTTON-100的使用浓度范围均为 0.1— 10mg/L， 浸泡时间均为 3-50min; 皂角苷 的浓度范围为 0.1-50mg L， 浸泡时间 2— 30min; 645-2的浓度范围为 l-60mg L， 浸泡时间 l-10min。并 舰用于海水鱼类、 ¾feK鱼类及半 鱼类。  In the present invention, NAC and TRTTON-100 are used in a concentration range of 0.1-10 mg/L, and the soaking time is 3-50 min; the concentration of saponin is 0.1-50 mg L, the soaking time is 2-30 min; The concentration range is l-60mg L, and the soaking time is l-10min. The ship is used for marine fish, 3⁄4feK fish and semi-fish.
以下 实施例寸本发明作进一步说明。  The invention is further illustrated by the following examples.
实施例 1:細本发明的滅分别为 0.1mg L、 0.1mg L、 50mg/L、 lmg L的 NAC、 TRITON X-100 皂角苷、 654-2多组合佐剂和灭¾|！弧菌 (Vibrio. Anguillarum)疫苗对? 浸泡免疫  EXAMPLES 1: Fines of the present invention were 0.1 mg L, 0.1 mg L, 50 mg/L, 1 mg L of NAC, TRITON X-100 saponin, 654-2 multi-adjuvant adjuvant and deuterium! Vibrio. Anguillarum vaccine pair? Immune immersion
1材料：  1 material:
(1) ¾¾用舰難管理：綳健 5g的 «3¾ ， i 前暂养 1周， i 用海水稳定 20-23°C， pH8.1-8.2, 30， i 期间投喂配 料， 日投喂 2次， 24小时纖充气， 、 難。  (1) 3⁄43⁄4 with shipwreck management: 綳健 5g «33⁄4 , i held for 1 week before, i stabilized with seawater 20-23 ° C, pH 8.1-8.2, 30, i during feeding period, daily feeding 2 times, 24 hours of fiber inflating, difficult.
(2)疫苗制备： 将 Vanguillarum接种至 2216E液体培养基， 28°C培养 24h; 4°C下 4000ipm离心 20min, 收集菌体；将收集的菌体置于灭菌的 PBS， pH7.2中，制成 1.0 χ 108 cells/mL的弧菌悬液， 0.5% 甲醛 28°C下灭活 48h后为 i離用疫苗， 4°C储存； 同时将灭活后的菌体接种于 TSB固体平板， 置 28°C 下恒温培养 24h、 48h， 确定无謹长出。 (2) Vaccine preparation: Vanguillarum was inoculated into 2216E liquid medium, cultured at 28 ° C for 24 h; centrifuged at 4000 ip for 20 min at 4 ° C, and the cells were collected; the collected cells were placed in sterile PBS, pH 7.2, Made of 1.0 χ 108 cells/mL of Vibrio suspension, 0.5% After being inactivated at 28 °C for 48 hours, it was used as a vaccine for i. It was stored at 4 °C. The inactivated cells were inoculated on TSB solid plate, and cultured at 28 °C for 24 hours and 48 hours. .
(3)浸泡免疫佐剂： NAC、 TRITON X-100 皂角苷、 654-2， 棚浓度分别为 0.1mg L、 0.1mg L、 50mg L、 lmg L。  (3) Immersion immune adjuvant: NAC, TRITON X-100 saponin, 654-2, shed concentration of 0.1mg L, 0.1mg L, 50mg L, lmg L.
2 方法  2 methods
(1) i 分组： 寸免疫组、无佐剂疫苗浸泡免疫组、佐齐 ^疫苗浸泡免敏、注射空白对 照组， 浸泡空白对照组， 每组 个平行， 每组 3x30纖。  (1) Group i: Inch immunization group, no adjuvant vaccine soaking immunization group, Zuoqi vaccine immersion sensitivity, injection blank control group, soaking blank control group, each group was parallel, each group 3x30 fiber.
(2)免疫方法： 鱼暂养 1周后， 注射免疫组每 j feJH空注 ¾ί农度为 1.0 X 108 cells/mL的 Vanguillarum疫苗 0.1ml; 无佐剂疫苗浸泡免慘则海水配制疫苗浓度 1.0 χ 107 cells/mL， 浸泡 llmin; 佐齐 ^疫苗浸泡免疫勸用 NAC、 TRITON X-100的混合溶液浸泡待她 20min，之后加入疫苗及皂角苷 维持 lmin, 再加入 654-2维持 lOmin; 注射空白对照组与浸泡空白对照分别用 PBS溶翻空龍和海水 浸泡， 方法相同。  (2) Immunization method: After the fish was kept for 1 week, the injected immunization group was given a dose of 1.0×108 cells/mL of Vanguillarum vaccine 0.1 ml per j feJH; no adjuvant vaccine soaked in the seawater. χ 107 cells / mL, soaked llmin; Zuo Qi ^ vaccine soaking immunization with NAC, TRITON X-100 mixed solution soaked for 20min, then add vaccine and saponin for 1min, then add 654-2 to maintain lOmin; The blank control group and the immersion blank control were immersed in PBS and seawater, respectively, in the same manner.
(3)免疫后攻毒： 免疫 30天后各删鳗弧菌 Y anguillarum攻毒， 攻毒采用注射感染， 齐隱为 5.0xl06cells/尾。  (3) After the immunization, the challenge: After 30 days of immunization, each of the Vibrio cholerae Y anguillarum was challenged, and the infection was injected with injection, and the ambiguity was 5.0xl06cells/tail.
3 i 结果  3 i results
攻毒 15天后， 统计侧勺存活率， 根据公式 RPS=(1—免敏死亡辆照组死亡率)着 /0。计算 免疫相当 率。结果见表 1 After 15 days of challenge, the survival rate of the side scoop was counted according to the formula RPS=(1—the death rate of the death-free death group)/ 0 . Calculate the immune equivalent rate. The results are shown in Table 1.
表 1应用 NAC、 TRITON X-100,皂角苷、 654-2为组合佐齐暖泡免^ I的免疫相对 ί¾ί户率 翻 攻毒尾数 死 数 死亡率0 /0 RPS% 平均 RPS% Table 1 uses NAC, TRITON X-100, saponin, 654-2 as the combination of Zuoqi warm bubble to avoid immune immunization relative to ί3⁄4 户 翻 翻 毒 毒 毒 死亡率0 death rate 0 / 0 RPS% average RPS%
20 1 5 94.91525  20 1 5 94.91525
注射免敏 20 0 0 100 98.30508  Injection-free sensitivity 20 0 0 100 98.30508
20 0 0 100  20 0 0 100
20 12 60 38.98305  20 12 60 38.98305
20 15 75 23.72881 27.11864 20 15 75 23.72881 27.11864
20 16 80 18.64407 20 16 80 18.64407
20 3 15 84.74576  20 3 15 84.74576
浸泡免麵含佐  Soaking free face
20 4 20 79.66102 83.05085 剂） 20 3 15 84.74576  20 4 20 79.66102 83.05085 agent) 20 3 15 84.74576
20 19 95  20 19 95
注射 PBS组 20 19 95  Injection PBS group 20 19 95
20 20 100  20 20 100
20 20 100  20 20 100
浸泡海 20 20 100  Soaking the sea 20 20 100
20 19 95  20 19 95
结果表明， 单画曼弧菌灭活疫苗浸泡免^ I， 免疫相对 率低仅为 27%， 棚佐剂 NAC、 TRITON X-100,皂角苷、 654-2组合佐剂后与疫苗 浸泡免疫相对 户率提高到 83%， 免疫 { 户¾¾ 明显， 证明細本发明可以显著提高浸泡疫苗对海水鱼的免疫相对{ 户率。  The results showed that the inoculation of Vibrio single bacterium was inactivated, and the relative rate of immunity was only 27%. The adjuvant adjuvant NAC, TRITON X-100, saponin, 654-2 adjuvant adjuvant and vaccine immersion immunity The relative household rate increased to 83%, and the immunization {house 3⁄43⁄4 was obvious, which proves that the invention can significantly improve the immunity of the immersion vaccine to marine fish.
实施例 2: 应用本发明的浓度分别为 5m 、 5m 、 lm 、 30mg L的 NAC、 TRITON X-100 皂角苷、 654-2多组合佐剂和灭活点状气单胞菌疫苗对罗非鱼浸泡免疫 1材料： Example 2: Application of the present invention to a concentration of 5 m, 5 m, lm, 30 mg L of NAC, TRITON X-100 saponin, 654-2 multi-adjuvant adjuvant and inactivated P. aeruginosa vaccine to Luo Fei Fish soaking immunity 1 material:
( 1 ) ¾¾用&¾辦直管理： Jmi 3g髓罗非鱼， 前暂养 10天， «ffl¾feK¾¾g 27-28°C, pH7.1-7.2, 期间投喂配^料， 日投喂 2次， 日 ifeR3( /。， 24小时遊卖充气。  (1) 3⁄43⁄4 with &3⁄4 management: Jmi 3g tilapia, for the first 10 days, «ffl3⁄4feK3⁄43⁄4g 27-28°C, pH7.1-7.2, during feeding, feeding 2 times a day, DayifeR3 (/., 24 hour tour to sell inflatable.
(2)疫苗制备:将点状气单胞菌接种到 LB液体培养基， 28°C培养 24h; 4。C下 4000ipm离心 20min, 收集菌体； 将收集的菌体置于灭菌的生 ¾toK中， pH 7.2中， 制成 1.0 X 109 cells/mL的菌悬液， 0.5%甲 醛 28°C下灭活 48h后为试验用疫苗， 4°C储存； 同时将灭活后的菌体接种于 LB固体平板，置 28°C下恒 温培养 24h、 48h， 确定无纏长出。 (2) Vaccine preparation: Aeromonas punctatus was inoculated into LB liquid medium and cultured at 28 ° C for 24 h ; The bacteria were collected by centrifugation at 4000 ipm for 20 min at C; the collected cells were placed in a sterile sterilized solution, pH 7.2, and made into a suspension of 1.0 X 109 cells/mL, and 0.5% formaldehyde was inactivated at 28 ° C. After 48 hours, the test vaccine was stored at 4 ° C. At the same time, the inactivated cells were inoculated on LB solid plates, and cultured at 28 ° C for 24 h and 48 h, and it was confirmed that no cells grew.
(3 )浸泡免疫佐剂： NAC、 TRITON X-100 皂角苷、 654-2组合佐剂， 棚滅分别为 5m 、 5mg L lmg/L 30mg^L。  (3) Immersion immunoadjuvant: NAC, TRITON X-100 saponin, 654-2 combination adjuvant, 5m, 5mg L lmg/L 30mg^L.
(4)疫苗棚滅： ¾¾ &中疫苗的有謝被为 1.0 x 108 cdls/mL。  (4) Vaccine shed: 3⁄43⁄4 & the vaccine was given 1.0 x 108 cdls/mL.
2 方法  2 methods
( 1 ) 分组： 无佐剂疫苗浸泡免疫组、 ί NAC TRITON X-100,皂角苷、 654-2组^^挤!^ 疫苗浸泡免敏、浸泡空白对照组， 每组 ¾≡个平行， 每组 3x40纖。  (1) Grouping: no adjuvant vaccine soaking immunization group, ί NAC TRITON X-100, saponin, 654-2 group ^^ squeezed! ^ Vaccine soaking, sensitive, soaking blank control group, each group 3⁄4≡ parallel, 3x40 fibers per group.
(2)免疫方法： 鱼暂养 10天后，无佐齐暖泡免疫画海水配制疫苗¾¾浓度 1.0 X 108 cells/mL 后樹寺免疫的鱼移入其中，浸泡 15min;棚佐齐幅包免慘则 NAC、 TRITON X- 100溶液浸泡待她 lOmin,之后加入疫苗及皂角苷维持 10min，再加入 654-2维持 5min;浸泡空白对照只用海水浸泡，方法 相同。 (2) Immunization method: After the fish is kept for 10 days, the vaccine is prepared without water. The concentration of the vaccine is 3⁄43⁄4 concentration 1.0 X 108 cells/mL. The fish immunized from the Houshu Temple are moved into it and soaked for 15 minutes . NAC, TRITON X-100 solution was immersed in her for 10 min, then the vaccine and saponin were added for 10 min, then 654-2 was added for 5 min ; the soaking blank control was only soaked in seawater, the method was the same.
(3 )免疫后攻毒： 免疫 30天后各删点状气单胞難毒， 攻毒翻 纖， 齐糧为 5.0x l07Cdls/尾。 3 i 结果 (3) Attacking after immunization: After 30 days of immunization, each of the singular gas cells is intoxicated, and the virus is retort, and the grain is 5.0x l07 C dls/tail. 3 i results
攻毒 15天后， 统计各组的存活率， 根据公式 RPS=(1—免敏死亡辆照组死亡率) X爾 0。计算 免疫相当 率。结果见表 2 After 15 days of challenge, the survival rate of each group was counted according to the formula RPS = (1 - death rate of death-free death group) X 0 . Calculate the immune equivalent rate. The results are shown in Table 2.
表 2 .各免疫组免疫相对 率统计  Table 2. Statistics of immune relative rate in each immunization group
翻 攻毒尾数 死 数 死亡率 RPS% 平均 RPS%  Turn over the number of attack tails death number mortality RPS% average RPS%
30 12 40 59.32203  30 12 40 59.32203
30 15 50 49.15254 51.41243 30 15 50 49.15254 51.41243
30 16 53.33333 45.76271 30 16 53.33333 45.76271
30 5 16.66667 83.05085  30 5 16.66667 83.05085
浸泡免敏（佐  Soaking and sensitization
30 4 13.33333 86.44068 84.18079 剂）  30 4 13.33333 86.44068 84.18079 agent)
30 5 16.66667 83.05085  30 5 16.66667 83.05085
30 20 66.66667  30 20 66.66667
浸泡海 30 20 66.66667  Soaking the sea 30 20 66.66667
30 19 63.33333  30 19 63.33333
实¾ ^果表明， 单纯用状气单胞菌灭活疫苗浸泡免疫罗非鱼， 免疫相对{ 户率低仅为 51.4%， ί NAC、 TRITON X-100,皂角苷、 654-2组合佐齐暖泡免疫组， 浸泡免疫相对 ί¾ί户率提高到 84.1%， ¾¾ 提高了 30%以上。再次证明細本发明可显著提高浸泡疫苗偷她的免疫  The results show that the tilapia is immersed in the inactivated vaccine against Aeromonas serovar, and the relative immunity is only 51.4%, ί NAC, TRITON X-100, saponin, 654-2 In the warm-bubble immunization group, the immersion immunity increased to 84.1%, and the 3⁄43⁄4 increased by more than 30%. Once again prove that the invention can significantly improve the immunization of the immersion vaccine

Claims

权 利 要 求 书 Claim
1. 一种鱼用浸泡疫苗多组合佐剂， 其特征在于上述的多组合佐剂分别 是 N-乙酰半胱氨酸、 曲拉通 X-100、 皂角苷和消旋山莨菪碱的四种佐剂组 合， 其中 N-乙酰半胱氨酸、 曲拉通 X-100、 皂角苷和消旋山莨菪碱的使用 浓度分别为 0.1— 10 mg/L、 0.1—10 mg/L、 0.1— 50 mg/L和 1—60 mg/L。  A multi-combination adjuvant for fish immersion vaccine, characterized in that the above multi-combination adjuvants are respectively N-acetylcysteine, Triton X-100, saponin and racemic anisodamine Adjuvant combination, wherein N-acetylcysteine, Triton X-100, saponin and racemic anisodamine are used at concentrations of 0.1-10 mg/L, 0.1-10 mg/L, 0.1- 50 mg/L and 1-60 mg/L.
2 .根据权利要求 1所述的鱼用浸泡疫苗多组合佐剂， 其特征在于上述 的 N-乙酰半胱氨酸、 曲拉通 X-100、 皂角苷和消旋山莨菪碱的纯度优于或 相当于化学纯级。  The multi-component combination adjuvant for fish immersion vaccine according to claim 1, characterized in that the above-mentioned N-acetylcysteine, Triton X-100, saponin and racemic anisodamine are excellent in purity. Or equivalent to chemical grade.
3.权利要求 1 所述的鱼用浸泡疫苗多组合佐剂的使用方法， 其特征在 于：  The method for using a fish immersion vaccine multi-component adjuvant according to claim 1, characterized in that:
( 1 ) 首先选用体表完好健康的待免疫鱼类， 体重在每尾 2g以上； (1) First select healthy and healthy fish to be immunized, weighing more than 2g per tail;
( 2 ) 将待免疫的鱼移入浓度范围均为 0.1 10mg/l 的 N-乙酰半胱氨 酸、 曲拉通 X-100混合养殖用水溶液中， 预浸泡 1 20min， 之后加入浓度 0.1—50 mg/L的皂角苷及待用疫苗浸泡 1 20min， 再向疫苗浸泡液中加入 浓度 1 60 mg/L 的消旋山莨菪碱待 l-10min即完成免疫。 (2) The fish to be immunized are transferred to an aqueous solution of N-acetylcysteine and Triton X-100 mixed culture in a concentration range of 0.1 10m g /l, pre-soaked for 20 minutes, and then added to a concentration of 0.1-50. The saponin of mg/L and the vaccine to be used are soaked for 1 20 min, and then the concentration of 1 60 mg/L of racemic anisodamine is added to the vaccine soaking solution for l-10 min to complete the immunization.
4.利用权利要求 1的浓度分别在 0.1— 10 mg/L, 0.1— 10 mg/L, 0.1— 50 mg/L和 1 60 mg/L的 N-乙酰半胱氨酸、曲拉通 X-100和皂角苷和消旋 山莨菪碱多组合免疫佐剂， 应用于包括海水和淡水鱼类的浸泡疫苗中协同 作用达到高效的免疫效果。  4. Using the concentrations of claim 1 at 0.1-10 mg/L, 0.1-10 mg/L, 0.1-50 mg/L and 1 60 mg/L of N-acetylcysteine, Triton X- 100 and saponin and racemic anisodamine multi-combination immunoadjuvant, applied to the immersion vaccine including seawater and freshwater fish to achieve a highly effective immune effect.
PCT/CN2010/076978 2009-12-31 2010-09-16 Multi-component adjuvant of immersion vaccine for fish, and its application and using method WO2011079627A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2009102558261A CN101791401B (en) 2009-12-31 2009-12-31 Fish soaking vaccine multiple-combination adjuvant and application and using method thereof
CN200910255826.1 2009-12-31

Publications (1)

Publication Number Publication Date
WO2011079627A1 true WO2011079627A1 (en) 2011-07-07

Family

ID=42584457

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2010/076978 WO2011079627A1 (en) 2009-12-31 2010-09-16 Multi-component adjuvant of immersion vaccine for fish, and its application and using method

Country Status (2)

Country Link
CN (1) CN101791401B (en)
WO (1) WO2011079627A1 (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101791401B (en) * 2009-12-31 2012-04-18 中国水产科学研究院黄海水产研究所 Fish soaking vaccine multiple-combination adjuvant and application and using method thereof
CN105816874A (en) * 2016-03-21 2016-08-03 沈阳工学院 Immersion immunization compound vaccine adjuvant for fish and application method thereof
CN105833266A (en) * 2016-04-26 2016-08-10 肇庆大华农生物药品有限公司 Adjuvant capable of improving immune effect of fish soaking vaccine and fish soaking immunization method
CN109568575A (en) * 2018-12-02 2019-04-05 河南师范大学 It is a kind of enhance Aeromonas hydrophila OmpA vaccine inoculation effect small molecule metabolites adjuvant and its application

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007068907A2 (en) * 2005-12-13 2007-06-21 Glaxosmithkline Biologicals Sa Vaccine compositions comprising a saponin adjuvant
CN101791401A (en) * 2009-12-31 2010-08-04 中国水产科学研究院黄海水产研究所 Fish soaking vaccine multiple-combination adjuvant and application and using method thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1568678A (en) * 2004-05-12 2005-01-26 中国水产科学研究院黄海水产研究所 Madefacting immunization method of fish vaccine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007068907A2 (en) * 2005-12-13 2007-06-21 Glaxosmithkline Biologicals Sa Vaccine compositions comprising a saponin adjuvant
CN101791401A (en) * 2009-12-31 2010-08-04 中国水产科学研究院黄海水产研究所 Fish soaking vaccine multiple-combination adjuvant and application and using method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU ZHENXING ET AL: "Adjuvants used in aquaculture", RESERVOIR FISHERIES, vol. 27, no. 2, 2007, pages 3 - 6 *
ZHANG NIANCI ET AL: "Supplementary effect of henbane on the bathing immunization of summer fingerlings of grass carp (Ctenopharyngodon idellus)", JOURNAL OF ZHEJIANG COLLEGE OF FISHERIES, vol. 9, no. 2, November 1990 (1990-11-01), pages 79 - 84 *

Also Published As

Publication number Publication date
CN101791401B (en) 2012-04-18
CN101791401A (en) 2010-08-04

Similar Documents

Publication Publication Date Title
Tagliabue et al. Vaccine adjuvants: the dream becomes real
ES2257769T3 (en) METHOD FOR THE PREPARATION OF MULTIVALENT VACCINES.
CN102988981B (en) Adjuvant for improving immunization effect of Edwardsiella vaccine and use method of adjuvant
CN102430120A (en) Adjuvant for enhancing fish vaccine immunization effect and application thereof
WO2011079627A1 (en) Multi-component adjuvant of immersion vaccine for fish, and its application and using method
WO2009056629A1 (en) Fish vaccine
JP2012510497A (en) Production method of vaccine
Edelman Prevention and treatment of infectious diarrhea: speculations on the next 10 years
US10314902B2 (en) Outer membrane vesicles and uses thereof
KR100267747B1 (en) Complex immunological preparation containing egg-yolk antibodies, for prevention and treatment of porcine diarrhea caused by enterotoxigenic escherichia coli or porcine epidemic diarrhea virus
CN103202828B (en) Fish immersion immunization method
WO2016119078A1 (en) Combined use of haemophilus parasuis lc strain and lz-20100109 strain
Kataoka et al. Enhanced Induction of Immune Resistance by Concanavalin Abound L1210 Vaccine and an Immunopotentiator Prepared from Coriolus versicolor
TW200936759A (en) Fish vaccine
Callomon et al. Streptomycin and diasone in the treatment of experimental tuberculosis of guinea pigs
CN101791403B (en) Fish soaking vaccine combination adjuvant and application and using method thereof
KR101640716B1 (en) Killed vaccine against scuticociliated ciliate
CN114805554B (en) Refined egg yolk antibody injection for resisting streptococcus suis and haemophilus parasuis and preparation method thereof
CN101791404B (en) Fish soaking vaccine immunity adjuvant and application and using method thereof
CN111569062A (en) Adjuvant for newcastle disease live vaccine and preparation method thereof
CN101791402B (en) Single-component fish vaccine soaking immunologic adjuvant as well as application and use method thereof
CN113559256A (en) Nocardia immunopotentiator and application thereof in preparation of vaccines for pigs
CN104922667B (en) A kind of Antirabic Vaccine and preparation method thereof
WO2023225458A1 (en) Vaccines against moritella viscosa
Chen Vaccines to Prevent Cholera

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10840416

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10840416

Country of ref document: EP

Kind code of ref document: A1