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.

Inactive Publication Date: 2019-08-16
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are problems with the safety of such vaccine strains. A case of a pgm site-deleted vaccine strain has been reported in an American laboratory and resulted in the death of a patient with hyperferemia. Therefore, it is necessary to prepare a live attenuated mouse YI vaccine strain with better safety.

Method used

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  • Live attenuated vaccine strain of plague bacillus and application thereof as lung delivery vaccine
  • Live attenuated vaccine strain of plague bacillus and application thereof as lung delivery vaccine
  • Live attenuated vaccine strain of plague bacillus and application thereof as lung delivery vaccine

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a live attenuated vaccine strain of plague bacillus and an application thereof as a lung delivery vaccine. The invention discloses plague bacillus EV-B-SHV[delta]pla; comparedwith a genomic DNA of plague bacillus EV-B-SHV, the plague bacillus EV-B-SHV[delta]pla has the difference only in that a DNA molecule shown in nucleotide at 7th-906th site of a DNA molecule shown in asequence 1 of a sequence table is knocked out. The invention also discloses an application of the plague bacillus EV-B-SHV[delta]pla in preparation of a plague vaccine. The plague vaccine is given bya lung delivery way. The invention also discloses the plague vaccine; the active ingredient of the plague vaccine is the plague bacillus EV-B-SHV[delta]pla. The detection results of specific antibodies, cytokines and the bacterial load amount show that the plague bacillus EV-B-SHV[delta]pla can be used as a vaccine strain for immunization inoculation by the lung delivery way.

Description

Technical field [0001] The invention relates to a live attenuated Yersinia pestis vaccine strain and its application as a lung delivery vaccine. Background technique [0002] Plague is a natural epidemic disease that spreads among rodents and is spread by fleas. It has caused three worldwide pandemics in history, claiming hundreds of millions of lives. Yersinia pestis is the pathogen of plague. It is a Gram-negative bacterium belonging to Yersinia enterobacteriaceae. It can cause three main syndromes, pneumonic plague, sepsis, and bubonic plague. Pneumonic plague is spread by aerosol droplets. The main cause of sepsis is the bite of an infected flea. Without treatment, the mortality rate can reach 100%. The bubonic plague is also spread by fleas, and failure to take treatment can lead to a fatality rate of 40-60%. People who come into contact with the blood and tissues of infected animals, or are exposed to an environment with bacterial aerosols, may be infected, and even lea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K39/02A61P31/04C12R1/01
CPCA61K9/007A61K39/0291A61K2039/522A61K2039/53A61K2039/544A61P31/04C07K14/24Y02A50/30
Inventor 冯俊霞杨慧盈周冬生孙岩松邱业峰法云智高波邓颖颖杨文慧殷喆熊小路于学东赵月娥焦俊胡凌飞
Owner ACADEMY OF MILITARY MEDICAL SCI
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