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Method for preparing pure protein derivative of tubercle bacillus by using mycobacterium tuberculosis attenuated strain H37Ra

A technology of Mycobacterium tuberculosis and protein derivatives, applied in microorganism-based methods, peptide preparation methods, biochemical equipment and methods, etc., can solve the production of high safety protection level, ecological disasters, and production conditions that cannot meet GMP. requirements, etc.

Active Publication Date: 2021-05-18
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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  • Method for preparing pure protein derivative of tubercle bacillus by using mycobacterium tuberculosis attenuated strain H37Ra
  • Method for preparing pure protein derivative of tubercle bacillus by using mycobacterium tuberculosis attenuated strain H37Ra
  • Method for preparing pure protein derivative of tubercle bacillus by using mycobacterium tuberculosis attenuated strain H37Ra

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] A method for preparing tuberculin pure protein derivatives with attenuated mycobacterium tuberculosis strain H37Ra, comprising the steps of:

[0045] A. Seed Lot Recovery

[0046] 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 three weeks at a temperature of 36-38°C to obtain a seed batch of revived culture;

[0047] B. Seed Lot Expansion

[0048] 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...

Embodiment 2

[0071] Based on Example 1, this example discusses the control conditions in the production process to further illustrate the technical solution.

[0072] (1) Ammonium sulfate salting out precipitation time

[0073] The ammonium sulfate salting-out precipitation time is set to be 2h, 4h, 6h, 12h and 18h ​​respectively, and the obtained results are shown in Table 1 below, and it is known that: under different precipitation time limits, the difference in the weight of the precipitate collected is not large. Therefore, according to the prepared The efficiency of the method, select the precipitation time 2-4h;

[0074] Table 1

[0075] settling time Insoluble weight (mg) 2h 28.1 4h 27.2 6h 29.3 12h 31.0 18h 28.9

[0076] (2) Final saturation of ammonium sulfate salting out precipitation

[0077] Set the final saturation of ammonium sulfate salting out precipitation to 40%, 60% and 80% respectively, the obtained results are shown in Table 2...

Embodiment 1-2

[0102] Based on Examples 1-2, this example detects the obtained tuberculin pure protein derivatives to further illustrate the technical solution.

[0103] 1. Appearance of tuberculin pure protein derivative stock solution

[0104] Light yellow to brownish yellow clear liquid, without insoluble matter or impurity.

[0105] 2. Purity of stock solution

[0106] Protein detection: detect protein content according to the current edition of "Chinese Pharmacopoeia" (general rule 0731 second method);

[0107] Polysaccharide detection: detect the polysaccharide content according to the current version of "Chinese Pharmacopoeia" (Tubercin Pure Protein Derivatives);

[0108] Nucleic acid detection: detect nucleic acid content according to the current version of "Chinese Pharmacopoeia" (general rule 0401);

[0109] The results obtained are shown in Table 8 below. It is known that the total amount of polysaccharide and nucleic acid contained in 1 mg of tuberculin pure protein derivative...

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Abstract

The invention discloses a method for preparing a pure protein derivative of tubercle bacillus by using a mycobacterium tuberculosis attenuated strain H37Ra, and belongs to the technical field of biological protein preparation. The mycobacterium tuberculosis attenuated strain CMCC93020 (H37Ra) is used as a strain, and a finished product of the pure protein derivative of the tubercle bacillus is obtained through amplification, inactivation, salting-out precipitation, degerming filtration, inspection, dilution and subpackaging. The defects in the prior art that strains have great harm to operators and the environment and have high requirements for production conditions (P3 production workshops) are overcome, and finally the protein which can be used for clinical diagnosis of tuberculosis, screening of bacillus calmette-guerin vaccine inoculation objects, monitoring of organism immune responses after bacillus calmette-guerin vaccine inoculation and epidemiological monitoring is obtained.

Description

technical field [0001] The invention relates to a method for preparing tuberculin pure protein derivatives with the attenuated mycobacterium tuberculosis strain H37Ra, belonging 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 is 846,000, accounting for 8.5%; China is 833,000, accounting for 8.5%. 8.4%. The number of tuberculosis patients in China ranks third in the world, and it is also a...

Claims

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Application Information

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IPC IPC(8): C12P21/02C12N1/20C07K14/35C07K1/30C07K1/34C07K1/36C12R1/32
CPCC12P21/02C12N1/20C07K14/35
Inventor 孙超伟
Owner 成都可恩生物科技有限公司
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