Live attenuated vaccine strain of plague bacillus and application thereof as lung delivery vaccine
A plague vaccine, plague technology, applied in vaccines, medical preparations containing active ingredients, DNA/RNA vaccination, etc.
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Embodiment 1
[0024] Example 1. Obtaining Y. pestis EV-B-SHVΔpla
[0025] After sequencing, the pla gene in Y. pestis EV-B-SHV is shown in sequence 1 of the sequence table.
[0026] Y. pestis EV-B-SHV was used as the starting strain to prepare Y. pestis EV-B-SHVΔpla through homologous recombination. It was verified by sequencing that, compared with the genomic DNA of Y. pestis EV-B-SHV, the difference of Y. pestis EV-B-SHVΔpla was only the deletion of nucleotides 7-906 in the DNA molecule shown in sequence 1 of the sequence table. DNA molecules shown.
Embodiment 2
[0027] Example 2. Preparation of Bacteria Liquid
[0028] 1. Thaw the glycerin strains stored at -80°C, and inoculate 20μl in 20ml brain heart infusion broth (BHI), culture at 26°C, 200rpm with shaking for 36h.
[0029] 2. After completing step 1, take 1ml of bacterial solution, inoculate it into 20ml of brain heart extract broth, and cultivate to OD at 26℃ and 200rpm with shaking 600nm = 1.0.
[0030] 3. After completing step 2, take 200μl of bacterial solution, inoculate it into 20ml of brain heart extract broth, and culture with shaking at 26℃ and 200rpm to OD 600nm =1.0, then shake culture at 37°C and 200rpm for 3h.
[0031] 4. After completing step 3, centrifuge at 3000g for 10 minutes, collect the bacterial pellet, and resuspend it in normal saline containing 0.05% poloxamer to obtain OD 600nm = 1.0 of the bacterial solution.
[0032] 5. Take the bacterial liquid obtained in step 4, spread it on a brain-heart extract broth plate after gradient dilution, invert the culture at 26...
Embodiment 3
[0038] Example 3. Comparison of the virulence of Y. pestis EV-B-SHV and Y. pestis EV-B-SHVΔpla
[0039] SPF BALB / c female mice aged 6-8 weeks were divided into 3 groups, each with 40 mice.
[0040] The first group: The EV-B-SHVΔpla prepared in Example 2 was administered by pulmonary delivery (50μl per mouse, with a bacterial content of 5×10 6 CFU).
[0041] The second group: The EV-B-SHV bacterial solution prepared in Example 2 was administered by pulmonary delivery (50μl per mouse, with a bacterial content of 5×10 6 CFU).
[0042] The third group: The EV-LZ bacterial solution prepared in Example 2 was administered by pulmonary delivery (50μl per mouse, with a bacterial content of 5×10 6 CFU).
[0043] Bacterial liquid is administered by lung delivery, that is, a handheld liquid aerosol lung delivery device is inserted into the trachea and delivered to the lungs.
[0044] After lung delivery, the death of mice in each group was recorded for 14 consecutive days, and survival curves were d...
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