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A kind of production method of tuberculin pure protein derivative

A technology of protein derivatives and tuberculin, which is applied in the field of biological protein preparation, can solve the problems that the production conditions cannot meet the production requirements of GMP, the health of production operators is seriously threatened, and the safety protection level is high, so as to shorten the preparation time, Improve production efficiency and product quality, the effect of low safety level requirements

Active Publication Date: 2022-05-10
成都可恩生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the strain of this product is the virulent strain of Mycobacterium tuberculosis CMCC93009 (H37Rv), which is a second-class pathogenic microorganism, which poses a great threat to the health of production operators, has a high level of safety protection, and has a great potential threat to the environment. If a safety accident occurs , will be a local ecological disaster
[0005] With the development of science and technology and the improvement of safety requirements, the production conditions of this strain declared in the early stage can no longer meet the production requirements of GMP

Method used

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  • A kind of production method of tuberculin pure protein derivative
  • A kind of production method of tuberculin pure protein derivative
  • A kind of production method of tuberculin pure protein derivative

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] A production method of tuberculin pure protein derivatives, comprising the steps of:

[0048] A. Seed Lot Recovery

[0049] Confirm that the attenuated Mycobacterium tuberculosis strain ATCC25177 strain tube has no damage, open the seed batch, dilute the strain with Sutong medium, and mix evenly; use Sutong potato soup solid slant medium, with the inoculation amount of 0.5-5mg / branch , covering the entire slant of the medium; culturing for 3-4 weeks at a temperature of 36-38°C to obtain a seed batch recovery culture;

[0050] B. Seed Lot Expansion

[0051] Take the revived culture of the seed batch, add Sutong medium, elute, grind, centrifuge at 15000g for 5min, centrifuge to discard the supernatant, weigh, and calculate the concentration of the bacterial solution; add Sutong medium, dilute the bacterial solution; Sutong potato soup solid slant medium, the inoculation amount is not less than 0.5mg / branch, and the entire medium slant is covered; cultivated at a tempera...

Embodiment 2

[0074] A production method of tuberculin pure protein derivatives, comprising the steps of:

[0075] A. Seed Lot Recovery

[0076] Confirm that the appearance of the attenuated Mycobacterium tuberculosis strain CMCC93020 strain tube is not damaged, open the seed batch, dilute the strain to a concentration of 10 mg / mL in Sutong medium, and mix evenly; use Sutong potato soup solid slant medium to inoculate the bacteria Amount of 2.5mg / branch, covering the entire slant of the medium; culturing for 3 weeks at a temperature of 38°C to obtain a seed batch of resuscitated culture;

[0077] B. Seed Lot Expansion

[0078] Take the revived culture of the seed batch, add Sutong medium, elute, grind, centrifuge at 15000g for 5min, centrifuge to discard the supernatant, weigh, and calculate the concentration of the bacterial solution; add Sutong medium, and dilute the bacterial solution to 100mg / mL; Sutong Potato Soup solid slant medium was used, and the inoculation amount was 25mg / branch...

Embodiment 3

[0101] Based on Example 1, this example discusses the control conditions in the production process to further illustrate the technical solution.

[0102] (1) Sutong Potato Soup Solid Slant Medium Amplified Inoculum Amount

[0103] Set the inoculated amount of 0.5mg / branch, 1mg / branch, 2.5mg / branch and 5mg / branch. Under the same culture conditions, the results obtained are shown in Table 1 below, and it is known that: with the increase of inoculated bacteria amount, the The yield is increasing; the specific amount of inoculum can be determined according to the production scale, passage times and amplification quality;

[0104] Table 1

[0105] Inoculation amount Bacteria yield (mg) 0.5mg / bottle 118 1mg / bottle 168 2.5mg / bottle 235 5mg / bottle 365

[0106] (2) Sutong potato soup solid slant medium expansion culture time

[0107] Set the culture time for 3 weeks and 4 weeks. Under the same culture conditions, the results obtained are shown ...

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Abstract

The invention discloses a production method of tuberculin pure protein derivatives, belonging to the technical field of biological protein preparation. The attenuated Mycobacterium tuberculosis strain ATCC25177 and / or CMCC93020 is used as the strain, and the finished product of tuberculin pure protein derivative is obtained through amplification, inactivation, salting out and sedimentation, sterilization and filtration, inspection, dilution and subpackaging. Solve the defects in the prior art that the strains are harmful to operators and the environment, and require high production conditions (P3 production workshop), and finally obtain a strain that can be used for clinical diagnosis of tuberculosis, screening of BCG vaccination targets, and body immune response after BCG vaccination Proteins used in surveillance and epidemiological surveillance.

Description

technical field [0001] The present invention relates to a production method of tuberculin pure protein derivatives, in particular to a method for producing tuberculin pure protein derivatives with attenuated Mycobacterium tuberculosis strain ATCC25177 or / and CMCC93020 in attenuated Mycobacterium tuberculosis strain H37Ra The invention belongs to the technical field of biological protein preparation. Background technique [0002] Tuberculosis is a chronic infectious disease that seriously endangers human and animal health. Mycobacterium tuberculosis is the pathogenic bacteria of tuberculosis. In 2019, the global death toll from tuberculosis was 1.4 million, and tuberculosis is still the world's number one killer of infectious diseases. According to the 2020 global tuberculosis annual report released by the WTO: the number of global tuberculosis cases in 2019 is estimated to be about 10 million (8.9-11 million); among them, India is 2.64 million, accounting for 26%; Indonesia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00C07K1/34C07K1/30C12R1/32
CPCC12P21/00C07K1/303C07K1/34
Inventor 孙超伟
Owner 成都可恩生物科技有限公司
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