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Preparation method of bacterial ghost

A technology of ghost and bacteria, applied in the field of preparation of bacterial ghost, can solve the problems of restricting vaccination and application, difficult to remove live bacteria, impact on vaccine safety, etc. Effect

Pending Publication Date: 2022-01-07
天康制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most important way to prepare bacterial ghosts is genetic engineering, that is, to form pores on the bacterial cell membrane through bacterial lysis genes (such as the cleavage gene E of phage phiX174), so that the contents of bacterial cells are discharged to form bacteria shadows. The method retains the natural structure of the bacterial surface antigenic determinant, which is beneficial to the immunogenicity of the vaccine, but its cracking method is relatively mild, and it is difficult to achieve the purpose of removing all live bacteria, and the bacterial residue has a great impact on the safety of the vaccine. Significantly limit the vaccination and application of vaccines

Method used

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  • Preparation method of bacterial ghost
  • Preparation method of bacterial ghost
  • Preparation method of bacterial ghost

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] 1.1 Lysis gene strain construction

[0073] 1.1.1 Construction of pGEM-2E plasmid

[0074] Using the pUC57-E plasmid as a template, use the E gene amplification primers E-F, E-R for amplification. The reaction conditions are: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 56°C annealing for 30 seconds, and 72°C extension for 30 seconds. 30 cycles, 72°C extension for 10 minutes. A 50 μl reaction system is used, and the specific components are as follows:

[0075] Table 2 E gene amplification PCR reaction system

[0076]

[0077] Recover the E gene with an agarose gel recovery kit, connect the E gene to the pMD18-TSimple vector overnight at 4°C, and transform it into DH5α competent cells. Take 100 μl of the competent cell and carrier mixture and spread it on a sample containing Amp (100 μg / ml) on a TSA plate, cultured at 36-38°C for 24 hours, and the specific connection information is shown in Table 3.

[0078] Table 3 Connection system inf...

Embodiment 2

[0101] Example 2H 2 o 2 Treatment concentration and timing

[0102] Adopt processing method, parameter and detection method among the embodiment 1, adjust different concentration of hydrogen peroxide and processing time to concentration be 10 10 The CFU / ml bacterial solution was used for the treatment experiment and the detection of the number of viable bacteria. The specific test groups and detection results are shown in Tables 6 and 7.

[0103] Table 6 OD value of different concentrations of hydrogen peroxide at different treatment times

[0104]

[0105] Table 7 The live bacteria detection results of different concentrations of hydrogen peroxide and different treatment times

[0106]

[0107] From the test results in the table above, it can be seen that when H 2 o 2 When the concentration is 0.005%, the treatment time can not be completely removed from 12 hours to 60 hours; when the concentration is 0.01%-0.5%, and the treatment time is more than 24 hours, the li...

Embodiment 3

[0108] Example 3H 2 o 2 Processing temperature selection

[0109] Adopt processing method, parameter and detection method among the embodiment 1, adopt the H of final concentration 0.1% 2 o 2 The test group adjusted the different treatment temperature to concentration as 2×10 10 The CFU / ml bacterial solution was used for the treatment experiment and the detection of the number of viable bacteria. The specific test groups and detection results are shown in Table 8.

[0110] Table 8 Viable bacteria detection results under different treatment temperatures

[0111] Processing temperature (℃) OD 600

[0112] The above test results show that 0.1% H 2 o 2 Two additions at intervals of 4 hours cannot effectively remove live bacteria at 30°C and 32°C, but a large number of live bacteria can still be detected; at 35°C-45°C, the number of live bacteria is greatly reduced, and live bacteria can be almost completely removed , among them, under the condition of 38°C-43°...

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Abstract

The invention relates to a preparation method of a bacterial ghost. The preparation method specifically comprises the following steps: a) treating bacteria by using plasmids containing a splitting gene E to obtain bacterial liquid; and b) treating the bacterial liquid with H2O2. The bacterial liquid treated by the method provided by the invention completely does not contain live bacteria, and the treated bacterial strain has obvious holes on the surface, is empty-shell-shaped, complete in form and good in immunocompetence, can achieve the effect of completely removing the live bacteria in the high-concentration bacterial liquid, and can be applied to large-scale industrial production of vaccines.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing bacterial shadows. Background technique [0002] Bacterial ghosts (BGs) is a bacterial body without cytoplasm and nucleic acid. It is a bacterial shell with a complete bacterial structure formed after Gram-negative bacteria are lysed. Electron microscopy analysis shows that BGs retains the complete structure. The bacterial shell is empty, and the basic components of its outer membrane are retained, including the entire cell surface structure such as bacterial outer membrane protein, adhesin, lipopolysaccharide (LPS) and peptidoglycan layer. While inactive, this bacterium still retains the surface antigen components and structure of natural bacteria, has good immunogenicity, can enter the body in the form of living bacteria in a way similar to natural infection, and is easy to be macrophages in the body Phagocytosis by cells, followed by presentation by dendrit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N1/06C12N15/74C12N15/33C12R1/01
CPCC12N1/20C12N1/06C12N15/74C07K14/005C12N2795/14222
Inventor 贺笋何传雨潘毅平赵海龙刘梦志吴冬玲李明奇
Owner 天康制药股份有限公司