CN101190332A - A bivalent oral vaccine emulsified with vibrio eelium oil and its preparation - Google Patents

Abstract

The invention relates to a fish vaccine, in particular to a vibrio anguillarum oil emulsified bivalent oral vaccine and its preparation. Specifically, the vaccine is composed of an oil phase and a water phase, and the water phase is Tween-80, which is preserved by adding formaldehyde-inactivated Listonella genus Listonella MacDonellet Collwell's two subtype strains of Listonella anguillarum Vibrioanguillarum O1 serotype Bacterial suspension in the General Microbiology Center of China Microbiological Culture Collection Management Committee (CGMCC No.1870 and CGMCC No.1871); the oil phase is: Span-80, aluminum stearate and white mineral oil with a kinematic viscosity of 7-13; The volume ratio of the water phase and the oil phase is 1:1.5-2; the bacteria suspension in the water phase is 94%-98% by volume percentage. The oil-emulsified bivalent oral vaccine prepared by using two subtype strains of Vibrio anguillarum O1 serotype can broaden the vaccine protection spectrum, increase the immune synergy of the vaccine, and induce non-specific and specific immune responses in the intestinal tract of aquaculture animals. and provide a protective effect. The materials used for preparing the oil-emulsified bivalent oral vaccine of the invention have low cost.

Description

A bivalent oral vaccine emulsified with vibrio eelium oil and its preparation

technical field

The invention relates to a fish vaccine, in particular to a vibrio anguillarum oil emulsified bivalent oral vaccine and its preparation.

Background technique

Oral vaccines commonly used in aquaculture are prepared by using biodegradable substances (commonly used polyvinyl alcohol, acrylic resin, alginate or liposomes) to coat a capsule or whole-cell vaccine of a pathogenic strain. A serotype against pathogenic bacteria can protect farmed animals with a narrow immune protection spectrum, and the cost of biodegradable materials used is high.

Contents of the invention

The purpose of the present invention is to provide a kind of oil-emulsified bivalent oral vaccine against different serotypes of Listonia anguillarum and its preparation in order to overcome the shortcomings of narrow immune protection spectrum and high material cost in oral immunization of aquaculture animals in the prior art .

To achieve the above object, the technical scheme adopted in the present invention is as follows:

Morphological characteristics of Listonia anguillaris serotype 01 subtype strain CGMCC No.1870(M3):

The colony of the strain in 2216E medium for 24 hours was round, translucent, with smooth surface and neat edges, with a diameter of about 1.5mm; the colony in TCBS medium was yellow, with a diameter of about 3mm; Gram staining was negative and short rod-shaped. Can move. There is a very long polar single flagella and periphyme, and the cell size is 0.8～1.0×1.5～1.7μm.

The oxidase reaction of the M3 strain was positive, sensitive to the Vibrio inhibitor 0/129 (2,4-diamino-6,7-diisopropylpteridine phosphate, 150ug/mL), no acid was produced in the fermentation of glucose, no pigment

Morphological characteristics of Listonia anguillaris serotype 01 subtype strain CGMCC No.1871 (SMP1):

The colony of the strain in the 2216E medium for 24 hours was round, translucent, smooth and neat. Does not grow on TCBS medium. Gram-negative, curved rod-shaped, motile, with a very long polar single flagella.

The oxidase reaction of SMP1 strain is positive, sensitive to Vibrio inhibitor 0/129 (2,4-diamino-6,7-diisopropylpteridine phosphate, 150ug/ml), fermenting glucose to produce acid but not gas, It does not produce pigment and is negative for urease. According to these characteristics and combined with the morphological characteristics, it can be determined that SMP1 is a Vibrio bacterium of the Vibrio family.

The characteristics of the physiological and biochemical tests are shown in Table 1,

+: positive, -: negative.

The vaccine consists of an oil phase and a water phase. The water phase is added with two subtypes of Listonella MacDonelletCollwell's Listonella anguillarum Vibrio anguillarum 01 serotype. The strains are preserved in the General Microbiology Center of the China Committee for the Collection of Microorganisms (CGMCC No.1870 and CGMCC No.1871) bacterial suspension.

The water phase is Tween-80, 94%-98% by volume of two subtype bacterial suspensions of Listonia anguillaris 01 serotype strains and 2%-6% Tween-80; oil phase It is: Span-80, aluminum stearate and white mineral oil with a kinematic viscosity of 7-13; the components in the oil phase are: 92%-96% by volume of white mineral oil with a kinematic viscosity of 7-13 and 4% -8% Span-80 and 1.5%-2.5% aluminum stearate according to the total mass volume ratio of the oil phase, wherein the water phase and the oil phase are 1: 1.5-2 by volume, and the two kinds of bacterial suspensions The concentration of each solution is 1-6×10 ⁹ cells/ml.

How the vaccine is prepared:

1) Prepare the water phase of the emulsified vaccine: add Tween-80 to the two subtype strains CGMCC of Listonella MacDonellet Collwell serotype 01 serotype Listonella MacDonellet Collwell after the rejuvenation culture and formaldehyde inactivation In the bacterial solution of No.1870 and CGMCC No.1871, stir and mix well to obtain the aqueous phase of the emulsified vaccine; the bacterial suspension accounts for 94%-98% of the aqueous phase volume ratio, and Tween-80 accounts for 2% of the aqueous phase volume ratio. %-6%;

2) Prepare the oil phase of the emulsified vaccine: take 92%-96% white mineral oil with a kinematic viscosity of 7-13, 4%-8% Span-80 and 1.5%- 2.5% aluminum stearate, mixed with it, heated to dissolve the aluminum stearate, to obtain the oily phase of the emulsified vaccine;

3) Preparation of emulsified vaccine: slowly add 1 volume of water phase to 1.5-2 volumes of oil phase, and magnetically stir at 1000-1500 rpm for 10-20 minutes to obtain a water-in-oil emulsified bivalent vaccine.

The two strains were rejuvenated and cultivated to a concentration greater than 10 ⁹ cells/ml.

The present invention has advantages as follows:

1. The present invention adopts the oil-emulsified bivalent oral vaccine prepared by the two subtype strains of Listonia eel 01 serotype, which can broaden the vaccine protection spectrum, increase the immune synergistic effect of the vaccine, and induce non-specificity and Specific immune response and provide a protective effect.

2. To prepare the oil-emulsified bivalent oral vaccine of the present invention, the cost of materials used is low.

Description of drawings

Fig. 1 is a morphological diagram of Listonia anguillarum M3 strain of the present invention.

Fig. 2 is a morphological diagram of the Listonia anguillarum SMP1 strain of the present invention.

Fig. 3 is a diagram showing the change of enteric lysozyme activity in turbot after immunization with the vaccine of the present invention.

Fig. 4 is a graph showing the change of anti-protease activity in the hindgut of turbot after immunization with the vaccine of the present invention.

Fig. 5 is a graph showing the changes in titer of anti-Listonia anguillarum M3 antibody in the hindgut of turbot after immunization with the vaccine of the present invention.

Fig. 6 is a graph showing the titer change of anti-Listonia anguillarum SMP1 antibody in turbot hindgut after immunization with the vaccine of the present invention.

Fig. 7 is a graph showing the mortality of turbot infected with Listeria anguillarum M3 after immunizing turbot with the vaccine of the present invention.

Fig. 8 is a graph showing the mortality of turbot infected with Listonia anguillarum SMP1 after immunizing turbot with the vaccine of the present invention.

Fig. 9 is a diagram of antibodies detected on turbot hindgut slices after immunizing turbot with the vaccine of the present invention.

Fig. 10 is a diagram of antibodies detected on turbot hindgut slices after immunization with unemulsified vaccine.

Fig. 11 is a section view of the turbot hindgut tissue without using the vaccine of the present invention.

Listonella MacDonellet Collwell's Listonella anguillarum Vibrioanguillarum 01 serotype subtype strain M3 is preserved in the General Microbiology Center No. 1870 of the China Committee for the Collection of Microorganisms.

Listonella MacDonellet Collwell's Listonella anguillarum Vibrioanguillarum 01 serotype subtype strain SMP1 is preserved in the General Microbiology Center No. 1871 of the China Committee for the Collection of Microorganisms.

Detailed ways

The present invention will be further described below according to the accompanying drawings and examples.

Example 1

Listonella MacDonellet Collwell's Listonella anguillarum Vibrioanguillarum 01 serotype subtype strain M3 is preserved in the General Microbiology Center No. 1870 of the China Committee for the Collection of Microorganisms.

Listonella MacDonellet Collwell's Listonella anguillarum Vibrioanguillarum 01 serotype subtype strain SMP1 is preserved in the General Microbiology Center No. 1871 of the China Committee for the Collection of Microorganisms.

Morphological characteristics of strain M3:

The colony of the strain in 2216E medium for 24 hours was round, translucent, with smooth surface and neat edges, with a diameter of about 1.5mm; the colony in TCBS medium was yellow, with a diameter of about 3mm; Gram staining was negative and short rod-shaped. Can move. There is a very long polar single flagella and perigenetic pili, and the cell size is 0.8-1.0×1.5-1.7 μm (see Figure 1).

Strain M3 is positive, sensitive to Vibrio inhibitor 0/129 (2,4-diamino-6,7-diisopropylpteridine phosphate, 150ug/mL), does not produce acid or pigment when fermenting glucose

Morphological characteristics of strain SMP1:

The colony of the strain in the 2216E medium for 24 hours was round, translucent, smooth and neat. Does not grow on TCBS medium. Gram-negative, curved rod-shaped, motile, with a very long polar single flagella (see Figure 2).

The strain SMP1 oxidase was positive, sensitive to Vibrio inhibitor 0/129 (2,4-diamino-6,7-diisopropylpteridine phosphate, 150ug/ml), fermented glucose to produce acid but not gas, It does not produce pigment and is negative for urease. According to these characteristics and combined with the morphological characteristics, it can be determined that the strain SMP1 belongs to the genus Vibrio in the Vibrio family.

Rejuvenate the two subtype strains M3 and SMP1 of Listonia eelensis 01 serotype: connect M3 and SMP1 to liquid test tubes respectively, shake at 28°C overnight, wash the bacterial liquid by centrifugation with PBS with pH 7.4, until the bacterial cells The concentration was 10 ⁸ cells/ml, turbot was injected intraperitoneally, and the dead turbot was dissected after injection, and the viscera were sliced and sampled to obtain two rejuvenated bacteria; the two subtype strains M3 and SMP1 after rejuvenation were picked. Bacterial colonies were inoculated into tryptic peptone broth medium containing 1.5% NaCl, cultured on a shaker at 28°C at 200 rpm for 24-48 hours, added formaldehyde at a final concentration of 0.3% to inactivate at room temperature for 48 hours, and then 5000 rpm Centrifuge for 10 minutes, wash with pH7.4 buffer solution PBS, and wash three times in total so that the concentrations of the two strains in the suspension are respectively 10 ⁹ cells/ml. Components of Tryptic Soybean Broth Medium: 18g TSB, 9g NaCl, 600ml distilled water, adjust pH to 7.4, autoclave at 117°C for 35 minutes.

1) Prepare the water phase of the emulsified vaccine: add Tween-80 to the two subtypes of Listonia anguillaris serotype 01 serotype 01 inactivated by formaldehyde at concentrations of 10 ⁹ cells/ml after rejuvenation culture, Stir and mix well to obtain the aqueous phase of the emulsified vaccine; wherein 19.2 ml of the bacterial suspension is 96% by volume, and 0.8 ml of Tween-80 is 4% by volume.

2) Prepare the oil phase of the emulsified vaccine: take 28.2ml No. 10 white oil according to the volume percentage of 94%, take 1.8ml Span-80 according to the volume percentage of 6%, and take 0.6g aluminum stearate according to the total mass volume ratio of the oil phase of 2% , mixed them, heated to completely dissolve the aluminum stearate, and sterilized under high pressure at 121° C. for 25 minutes to prepare the oil phase of the emulsified vaccine.

3) Preparation of emulsified vaccine: 20ml of the water phase was slowly added to 30ml of the oil phase at a volume percentage of 1:1.5, and magnetically stirred at a speed of 1000rpm for 10 minutes to obtain a milky white water-in-oil emulsified bivalent vaccine.

Example 2

The difference from Example 1 is:

1) Prepare the water phase of the emulsified vaccine: Add the two subtypes of Listonia anguillaris serotype 01 at concentrations of 4×10 ⁹ cells/ml to Tween-80 after rejuvenation culture , stir and mix well to obtain the aqueous phase of the emulsified vaccine; wherein the bacterial suspension takes 18.8ml according to the volume percentage of 94%, and Tween-80 takes 1.2ml according to the volume percentage of 6%.

2) Prepare the oil phase of the emulsified vaccine: prepare the oil phase of the emulsified vaccine: take 27.6ml No. 10 white oil by volume percentage 92%, take 2.4ml Span-80 by volume percentage 8%, take by volume percentage 1.5% of the total mass volume of the oil phase 0.45g aluminum stearate is mixed, heated to make the aluminum stearate dissolve completely, and sterilized to prepare the oil phase of the emulsified vaccine;

3) Preparation of emulsified vaccine: 20ml of water phase was slowly added to 40ml of oil phase at a volume percentage of 1:2, and magnetically stirred at a speed of 1000rpm for 10 minutes to obtain a milky white water-in-oil emulsified bivalent vaccine.

Example 3

The difference from Example 1 is:

1) Prepare the water phase of the emulsified vaccine: add the two subtypes of Listonia anguillaris serotype 01 at concentrations of 6×10 ⁹ cells/ml after rejuvenation culture to Tween-80 , stir and mix to obtain the aqueous phase of the emulsified vaccine; wherein the bacterial suspension takes 19.6ml according to the volume percentage of 98%, and Tween-80 takes 0.4ml according to the volume percentage of 2%.

2) Prepare the oil phase of the emulsified vaccine: take 28.8ml of No. 10 white oil according to the volume percentage of 92%, take 1.2ml of Span-80 according to the volume percentage of 4%, and take 0.75g of aluminum stearate according to the total mass volume ratio of the oil phase of 2.5% , mix them, heat to make the aluminum stearate dissolve completely, and sterilize to prepare the oil phase of the emulsified vaccine;

3) Preparation of emulsified vaccine: 20ml of water phase was slowly added to 36ml of oil phase at a volume percentage of 1:1.8, and magnetically stirred at a speed of 1000rpm for 10 minutes to obtain a milky white water-in-oil emulsified bivalent vaccine.

Application example 1

Add 50ml of the emulsified vaccine prepared in Example 1 to 300g of fish meal, and use a mixer to stir and mix thoroughly to prepare baits embedded with emulsified vaccine and non-emulsified vaccine respectively. The bait is placed in an oven at 50° C., and baked for 6-10 hours until dry to obtain the fish bait. The bacterial cell content in the fish bait is 10 ⁹ cells/g bait. The temporarily reared turbot were randomly divided into two groups, and they were fed with emulsified vaccine, non-emulsified vaccine and non-vaccinated bait respectively. Feed 0.75g/tail twice a day in the morning and afternoon, and immunize once every 2 days, and immunize once every two weeks, for a total of 3 times. Non-specific and specific immune responses to two antigens were detected in turbot hindgut after immunization. See Figure 3, Figure 4, Figure 5, Figure 6.

Application example 2

Mix the oil-emulsified bivalent oral vaccine of Listonia anguillaris and the unemulsified vaccine containing the same amount of bacterial antigens with self-made bait respectively, shape and dry to make emulsified and unemulsified Bivalent Listonella anguillarum bivalent For oral vaccine, the bacterial cell content is 10 ⁹ cells/g bait. The temporarily reared turbot were randomly divided into three groups, and they were fed with emulsified vaccine, non-emulsified vaccine and non-vaccinated bait respectively. Feed 0.75g/tail twice a day in the morning and afternoon for 7 consecutive days. And 30 days after the immunization, 20 turbots from the immunization group and 20 turbot from the control group were taken, and the infection experiments were carried out with the strains M3 and SMP1 respectively. The death conditions of each group were shown in Figure 7 and Figure 8, and the hindgut tissue was taken to make tissue slices , antibodies detected by in situ hybridization. See Figure 9, Figure 10, Figure 11.

The immune protection of the emulsified vaccine group against the infection of strains M3 and SMP1 was 100% and 50%, respectively, and that of the non-emulsified immunization group was 57.9% and 0% of the infection of the strains M3 and SMP1.

Claims (4)

1. a vibrio anguilla oil emulsified bivalent oral vaccine, is characterized in that: vaccine is made up of oil phase and water phase, adds the eel Listonella Vibrio anguillarum O1 of Listonella Listonella MacDonellet Collwell in the water phase The two subtypes of serotypes are bacterial suspensions preserved in the General Microbiology Center (CGMCC No.1870 and CGMCC No.1871) of the China Committee for the Collection of Microbial Cultures. 2. Vibrio anguillaris oil emulsified bivalent oral vaccine according to claim 1, characterized in that: the water phase is Tween-80, 94%-98% by volume percentage of Listonia anguillarum O1 serum Bacterial suspension of two subtype strains and 2%-6% Tween-80; the oil phase is: Span-80, aluminum stearate and white mineral oil with a kinematic viscosity of 7-13; each component in the oil phase is : 92%-96% by volume of white mineral oil with a kinematic viscosity of 7-13 and 4%-8% of Span-80 and 1.5%-2.5% of aluminum stearate according to the total mass volume ratio of the oil phase, The volume ratio of the water phase and the oil phase is 1:1.5-2, and the concentrations of the two bacterial suspensions are 1-610 ⁹ cells/ml. 3. a preparation method of Vibrio anguillaris oil emulsified bivalent oral vaccine according to claim 1, characterized in that: 1) Prepare the water phase of the emulsified vaccine: add Tween-80 to two subtype strains CGMCC of Listonella MacDonellet Collwell serotype O1 serotype Listonella MacDonellet Collwell after the rejuvenation culture and formaldehyde inactivation In the bacterial solution of No.1870 and CGMCC No.1871, stir and mix well to obtain the aqueous phase of the emulsified vaccine; the bacterial suspension accounts for 94%-98% of the aqueous phase volume ratio, and Tween-80 accounts for 2% of the aqueous phase volume ratio. %-6%; 2) Prepare the oil phase of the emulsified vaccine: take 92%-96% white mineral oil with a kinematic viscosity of 7-13, 4%-8% Span-80 and 1.5%- 2.5% aluminum stearate, mixed with it, heated to dissolve the aluminum stearate, to obtain the oily phase of the emulsified vaccine; 3) Preparation of emulsified vaccine: slowly add 1 volume of water phase to 1.5-2 volumes of oil phase, and magnetically stir at 1000-1500 rpm for 10-20 minutes to obtain a water-in-oil emulsified bivalent vaccine. 4 . The method for preparing the Vibrio anguillaris oil-emulsified bivalent oral vaccine according to claim 3 , characterized in that: the two strains are rejuvenated and cultured to a concentration greater than 10 ⁹ cells/ml.

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