Attenuated live vaccine of vibrion from eel

A technology of live attenuated vaccine and Vibrio anguillarum, which is applied in the direction of bacteria, antibacterial drugs, antibody medical components, etc., can solve the problems of low level, inconvenient administration, unstable immune response, etc., and achieve good control effect, The effect of low toxicity and low immune protection rate

Inactive Publication Date: 2005-06-08
上海浩思海洋生物科技有限公司
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Inactivated vaccines often have the following defects: incomplete protective antigens, unstable and low levels of immune responses, even unable to stimulate correct immune responses and cause side effects, inconvenient administration, etc.
In view of this, this type of attenuated vaccine has been identified as a biological product with high environmental safety risks by the approval regulations of biological product safety inspection and management agencies in various countries, including China, and it is difficult to enter the commercial development process

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Preparation of live attenuated and inactivated vaccines against Vibrio anguillarum

[0025] Preparation of live attenuated vaccine preparation: Take 1 loop and store it in LB slant medium (Soy Peptone (Difco) 10g / L, Yeast Extract (Merck) 5g / L, NaCl 30g / L, Agar 18g / L, pH 7.5) ), inoculate 100ml liquid LB seed medium (Soy Peptone (Difco) 10g / L, Yeast Extract (Merck) 5g / L, NaCl 30g / L, pH 7.5) in a 500ml shaker flask containing 100ml of liquid LB seed medium. Incubate with shaking at 28°C (rotational speed 200 rpm). After 12 hours, take 5ml of vigorously growing bacteria liquid (OD=4.0) and inoculate 100ml of fresh iron-rich LB medium (Soy Peptone (Difco) 10g / L, Yeast Extract (Merck) 5g / L, NaCl 30g / L) , Ferric ammonium citrate 0.2-0.5mmol, pH 6.8), incubate at 28°C for 12 hours. Wash with sterile normal saline (0.85% NaCl) and dilute to a certain concentration suspension (10 6 -10 8 Pcs / ml) spare.

[0026] Preparation of inactivated vaccine preparation: Take 1 loop a...

Embodiment 2

[0027] Example 2: Infection test and LD50 for grouper 50 Test for determination of pathogenicity:

[0028] The fish used for the experiment was the grouper provided by the 863 breeding base in Zhanjiang, Guangdong, with an average weight of 13g. Those without abnormalities were kept in the aquarium for more than one week as experimental fish. The experimental fish were randomly divided into groups with 15 fish in each group. 1 / 3 of the rearing water body was replaced with sufficient oxygen every day, and the breeding water temperature was 28°C. No feeding during the whole process. Exclude abnormal individuals before the experiment.

[0029] Each experimental fish was abdominally injected with 0.3ml of the attenuated live vaccine preparation prepared in Example 1 and the wild Vibrio anguilla virulent strain MVM425 preparation. The control group was injected with 0.85% sterile normal saline, and the cumulative number of deaths was recorded within 10 days And mortality, take the aver...

Embodiment 3

[0043] Example 3: Immunity protection test to grouper:

[0044] The experimental groupers were randomly divided into groups, 30 fish per group. The grouper was immunized with the attenuated live vaccine preparation prepared in Example 1 by intraperitoneal injection and immersion. In the injection immunization, the concentration of the prepared vaccine preparation is adjusted to 10 8 Pcs / ml, 0.3ml / tail, intraperitoneal injection. During the immersion immunization, the concentration of the vaccine preparation soaking solution is adjusted to 10 6 Pieces / ml, soaking time is controlled at about 1 minute. The control group was injected with sterile normal saline, 0.3ml / tail. After 15 days, booster immunization was performed by repeating the above-mentioned dose. After another 15 days, each group was challenged with live Vibrio anguillarum strain MVM425: 9×10 8 / ml normal saline preparation, 0.3ml / tail, intraperitoneal injection. Then observe the control group and the immune group, recor...

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PUM

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Abstract

An attenuated living vaccine of eel vibrio for fish is disclosed. Its advantages are low poison, high immunoprotection effect, and no any antibiotic resistance marker.

Description

Technical field [0001] The present invention relates to a live attenuated vaccine for aquaculture fish, in particular to a live attenuated vaccine of Vibiro anguillarum. Background technique [0002] With the gradual development of the marine aquaculture industry, a large-scale outbreak of various diseases followed, and the disease problems have become increasingly prominent, which restricts the further development of the marine aquaculture industry. Disease prevention and control has become the primary problem that should be solved in marine aquaculture. [0003] Vibrio anguilla is an important bacterial pathogen that seriously harms aquaculture fish in my country and the world. It mainly causes vibriosis such as hemorrhagic septicemia in farmed fish. Its main viral causative factor is contained in Vibrio anguillarum. The iron uptake system encoded by pJM1 type plasmids (Crosa, JH, Walsh, CT Genetics and assembly line enzymology of siderophore biosynthe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/104A61P31/04C12N1/21
Inventor 马悦张元兴赵东玲刘琴邵明非何建国黄志坚
Owner 上海浩思海洋生物科技有限公司
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