High-concentration bacterial ghost vaccine inactivation method

A high-concentration, inactivated technology applied in the biological field

Pending Publication Date: 2022-07-29
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

[0005] The object of the present invention is to provide a method for inactivating high-concentration ghost vaccines, which is used to solve the problem of completely inactivating live bacteria in the preparation process of high-concentration ghost vaccines and maintaining the complete structure and immunogenicity of bacteria

Method used

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  • High-concentration bacterial ghost vaccine inactivation method
  • High-concentration bacterial ghost vaccine inactivation method
  • High-concentration bacterial ghost vaccine inactivation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A19B

[0039] Example 1 A19BG strain inactivation method

[0040] 1. Basic seed propagation

[0041] Take out the constructed strain from the -80°C ultra-low temperature refrigerator, place it in a 37°C constant temperature incubator to thaw, and use a disposable inoculation loop to streak it on a Brucella agar plate (kana plate 100μg / ml) after thawing at 28°C. Cultivate for 5 to 7 days.

[0042] 2 Primary Seed Preparation

[0043] Pick a single colony and put it into 10ml of Brucella broth for 24h at 28°C, 160r / min.

[0044] 3 Secondary Seed Preparation

[0045]Take the prepared first-class seeds and add them to a 500ml conical flask containing 200ml of Brucella broth medium at a 2% inoculation ratio. Take 100mg / mL kanamycin and add it to the culture at a ratio of 1:1000. medium, 28°C, 300r / min, and cultured for 24h.

[0046] 4 Antigen culture

[0047] Put the bacterial liquid in the seed tank into the fermenter, take 100mg / mL kanamycin, add it to the Brucella broth at a ratio...

Embodiment 2

[0057] Example 2 Selection of culture duration and pouring time interval

[0058] Adopt the method of multiplying and enlarging culture in Example 1, take samples every 8h to carry out OD 600 Value detection, the growth curve of A19BG strain was measured, and the results are shown in figure 2 .

[0059] The results showed that the bacterial concentration reached 4.7 × 10 after 48 hours of culture. 10 CFU / ml.

[0060] Then the pair concentration is 4.7×10 10 The bacterial liquid of CFU / ml was inactivated and counted. The specific test groups and test results are shown in Table 2.

[0061] Table 2 OD values ​​of different pouring time intervals

[0062]

[0063] The results in Table 2 show that the antigen can be completely inactivated when the inactivation interval is 8h, 12h, and 24h within a certain inactivation culture time. On the basis of the time (8 hours), the interval between pouring tanks can be appropriately extended to ensure that the removal effect of bact...

Embodiment 3

[0064] Example 3 Inactivation effect of different bacterial liquid concentrations

[0065] Using the propagation, amplification and inactivation culture methods in Example 1-2, the A19BG strain was cultured with antigen, and the OD was sampled every 8h. 600 value detection, and the concentration of the bacterial solution reaches 5.0 × 10 10 CFU / ml, 5.5×10 10 CFU / ml, 6.0×10 10 When CFU / ml, the bacterial liquid is inactivated and counted to verify the inactivation effect of the present invention. The specific detection results are shown in Table 3.

[0066] Table 3 Inactivation effects of different bacterial concentrations

[0067]

[0068] The results show that the inactivation method of the present invention can kill bacteria with a concentration of 5.0×10 10 CFU / ml, 5.5×10 10 CFU / ml, 6.0×10 10 CFU / ml bacterial solution was completely inactivated.

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Abstract

The invention relates to a high-concentration bacterial ghost vaccine inactivation method which specifically comprises the following steps: a) after bacterial culture is finished, raising the temperature of a tank body, and carrying out inactivation culture; and b) replacing the fermentation tank once every 8-72 hours. The bacterial liquid obtained by the method provided by the invention does not contain live bacteria at all, and the treated bacterial strain has good 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 inactivating bacterial ghosts. Background technique [0002] Bacterial ghosts (BGs) are bacterial bodies without cytoplasm and nucleic acid. They are bacterial empty shells with complete bacterial structure formed after Gram-negative bacteria are lysed. Denaturation, the shadow retains the exact same bacterial cell membrane structure and related antigenic proteins as live bacteria, so that it can retain the immunogenicity similar to live bacteria, and can also stimulate the body to produce humoral immunity and cellular immunity at the same time, and its cross-protection ability is better than Inactivated vaccine. At the same time, because BGs do not contain genetic material, only bacterial shells exist, so there is no possibility of returning to the virus, restoring virulence, and no toxic side effects. The safety is far superior to that of live vaccines. [0003] At p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/00A61K39/10A61P31/04C12R1/01
CPCC12N1/005A61K39/098A61P31/04A61L2202/21A61K2039/521
Inventor 贺笋赵海龙何传雨吴冬玲刘梦志杨启林王雨朦霍新亮余洪磊
Owner 天康制药股份有限公司
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