Serratia marcescens vaccine and preparation method and application thereof
A Serratia marcescens and bacterin technology, applied in the application field of medicine, can solve the problems of toxic side effects, retention, and insufficient curative effect, and achieve the effects of strong tumor inhibition, strong spleen activation, and stable yield
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Embodiment 1
[0042] Embodiment 1: the screening of bacterial strain
[0043] 1. Screening of starting strains
[0044] The strains are derived from Serratia marcescens preserved in the China General Microorganism Culture Collection and Management Center. The preservation number is: CGMCC1.0589. Under sterile conditions, the original strains are diluted 10 times and then spread on LB slant medium. Incubate at 37°C for 24h. Select a plate with about 20 colonies, pick a single colony with a large, clear boundary, and a milky white color that grows well as the starting strain for ultraviolet mutagenesis, and take its bacterial body in a triangular flask with normal saline and glass beads to make a sterile Bacteria suspension 500mL, 30 ℃, 250rpm water bath 4h, prepared into 10 6 CFU / mL of the mutagenized bacterial suspension, for later use.
[0045] 2. Determination of strain mutagenesis and lethality
[0046] Preheat the UV lamp for 25 minutes, take 10 mL of the mutagenic bacteria suspensi...
Embodiment 2
[0075] Embodiment 2: Optimization of preparation process of Serratia marcescens bacterin vaccine
[0076] During the research, it was found that the pH of LB agar medium, the time of slant culture, the temperature of slant culture, the pH of LB liquid medium, the inoculum size, the ventilation rate, the fermentation temperature and time and the rotation speed had a great influence on the growth and reproduction of bacteria. The preparation process of Serratia bacterin vaccine has been optimized, as follows:
[0077] 1. Screening of LB agar medium pH for spreading slant strains
[0078] According to the selected LB agar medium formula, the dosage of other components remained unchanged, and Serratia marcescens was cultured on LB agar medium with different pH at 37°C for 21 hours.
[0079] The results show that the bacteria can grow and reproduce on the agar medium with pH 6.0-8.0; because the bacteria will produce various acidic metabolites during the growth and metabolism proc...
Embodiment 3
[0113] Embodiment 3: the preparation of Serratia marcescens bacterin injection
[0114] The preparation method of Serratia marcescens injection is as follows:
[0115] (1) Slant-transfer strains: Serratia marcescens (strain SM-1 screened in Example 1) was inoculated on LB agar medium (pH 7.2) in a Kerner flask, and cultured at 37° C. for 21 hours. Take it out and store it in a -80°C refrigerator for later use.
[0116] (2) Cultivation and fermentation: Take 3 single colonies from the original slant strain of Serratia marcescens, mix them evenly, inoculate them on the LB liquid medium in the Erlenmeyer flask, and cultivate and ferment. The conditions are: LB liquid medium pH 7.2, 10% inoculum size, 75% aeration, temperature 37° C., 21 h, rotation speed 180 rpm.
[0117] (3) Bacteria collection, sterilizing and washing: before collecting bacteria, carry out the pure species inspection such as the morphological characteristics of Serratia marcescens, centrifuge at 4000-6000rpm ...
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