A kind of staphylococcus aureus and marine mammal vaccine containing the bacteria
A staphylococcal, mammalian technology applied in the field of vaccines can solve problems such as cetacean death
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Embodiment 1
[0032] Embodiment 1, acquisition and preservation of bacterial strains
[0033] 1. The stomata of 12 marine mammals were collected in sterile plastic vials, and white or gray mucus was found in the stomata of some animals. Isolation and purification of Staphylococcus aureus strains from marine mammal stomatal exhalations. Colony morphological characteristics of Staphylococcus aureus (37°C, cultured for 24-72h): (1) Brain-heart infusion agar medium: round colonies with smooth, moist and raised edges, neat opaque and yellow pigment, about 1-2mm in diameter . (2) Blood agar plate (fresh defibrinated sheep blood): On the blood plate, a large smooth raised moist colony is formed, and a completely transparent β-hemolytic ring is formed around the colony. (3) Baird-parker medium: The diameter of the colony is 2mm-3mm, the color is black, the edge is light-colored, there is a cloudy zone around it, and there is a layer of transparent circle around it. The colony has a cream-like to ...
Embodiment 2
[0054] Embodiment 2, the preparation of vaccine
[0055] Thaw the frozen bacterial solution obtained in Step 2 of Example 1 at room temperature, and perform the following steps on the P3 generation strain:
[0056] 1. Antigen preparation
[0057] 1. Take the frozen bacterial solution of the P3 generation DCS5 strain, thaw at room temperature, inoculate BHI liquid medium at a volume ratio of 1% to 10%, and culture at 37°C and 150rpm / min for 5 to 18 hours with shaking. Take the above-mentioned activated bacterial solution and inoculate BHI medium at a ratio of 1% to 10%, culture at 37°C and shake at 150rpm / min for 5 to 18 hours, carry out Gram staining to identify the bacterial solution, and use the method of counting viable bacteria Calculate the number of viable bacteria.
[0058] 2. Add 37-40% formaldehyde solution to the above cultured bacterial solution and make the formaldehyde concentration 0.2%-0.5% (volume ratio, 37°C, 150rpm / min shaking incubation for 36h-48h), then ...
Embodiment 3
[0062] Embodiment 3, the inspection of vaccine
[0063] 1. Dosage Form
[0064] Get a 1ml disposable pipette, draw each vaccine prepared in a small amount of embodiment 2 and drop it on the cold water surface installed on a 90mm disposable plate, except for the first drop, it should not spread.
[0065] 2. The stability of the vaccine
[0066] Take 10 mL of each vaccine prepared in Example 2 and add it to a 10 mL centrifuge tube, centrifuge at 3500 r / min for 15 minutes, and the water precipitated at the bottom of the tube should not be more than 0.5 mL. The vaccines prepared in Example 2 were stored at 2-8°C for 24 months, at 37°C for 1 month or at 25°C for 3 months without discoloration, delamination and demulsification.
[0067] 3. Vaccine sterility test
[0068] 1. Inoculate each vaccine prepared in Example 2 into 50 mL of thioglycolate medium (flow type), and culture at 35-37° C. for 3 days.
[0069] 2. Take the culture obtained in step 1, inoculate 2 TG small tubes, 0...
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