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Production process of high-titer tuberculin skin test diagnostic reagent PPD (Purified Protein Derivative)

A production process, tuberculin technology, applied in the field of immunology, can solve the problem of inability to solve the problem of fine bands, the complexity of PPD production process, the technical difficulty and the definition of components, etc.

Active Publication Date: 2019-01-11
BEIJING SANROAD BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that the 9.7KDa protein has strong DTH activity, but it is not a bacterial component of Mycobacterium tuberculosis, so the PPD production process is complicated, and the technical difficulty is related to the definition of components
The initial evaluation of standard PPD-S2 was using SDS-PAGE, but as expected, this method did not resolve the problem of fine bands, but it is certain that protein inactivation and degradation occurred during the production process

Method used

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  • Production process of high-titer tuberculin skin test diagnostic reagent PPD (Purified Protein Derivative)
  • Production process of high-titer tuberculin skin test diagnostic reagent PPD (Purified Protein Derivative)
  • Production process of high-titer tuberculin skin test diagnostic reagent PPD (Purified Protein Derivative)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 bacterial culture

[0047] (1) 1 seed of recovery work was inoculated into 5 Roche's egg culture medium, and cultured for 2 weeks. After the bacteria grew into dry, wrinkled and slightly light yellow bacterial film, the growth of the bacterial lawn could be observed at this time: the end of the culture After that, each 1st-generation strain was first suspended with 5ml of sodium chloride 0.9% solution, collected all the bacterial liquid, and sampled for acid-fast staining. Acid-fast staining was positive (bacteria were stained red). Biochemical reaction: Nitrate reduction reaction is positive, resulting in red; urease reaction is positive, showing red; heat-resistant catalase reaction should be negative, no bubbles are generated in 10-20 minutes; polysorbate 80 hydrolysis reaction does not change color. Each medium tube was resuspended with 2ml of physiological saline to prepare a bacterial solution, and a total of 10ml (1st generation) was collected. The bac...

Embodiment 3

[0061] Example 3 Precipitation

[0062] A 40% trichloroacetic acid solution was prepared, added to the harvest solution at a ratio of 1:10, with a final concentration of 4%, and allowed to stand at room temperature for 2 hours. After shaking well, centrifuge at 4°C and 8000rpm for 20min. The precipitate was collected and weighed 450 g.

[0063]Precipitate 450g is dissolved with 3150ml of 0.9% sodium chloride solution, adjusts the pH value with 1M sodium hydroxide, adds ammonium sulfate according to the amount of precipitation dissolution, makes its concentration reach 60% saturation. Add 390g×3.15L=1228.5g of ammonium sulfate, let stand at 2-8°C overnight (16 hours), centrifuge at 8000rpm for 20min at 4°C, and collect 365g of precipitate. Add 2555ml of 0.9% sodium chloride solution to dissolve the precipitate. Add 40% trichloroacetic acid to a final concentration of 4%, precipitate protein, centrifuge at 8000 rpm, collect the precipitate, and weigh 310 g. The precipitation...

Embodiment 5

[0066] Embodiment 5 sterile filtration

[0067] Filter and clarify the dialysate through a 1.2μm~0.45μm microporous membrane, take a 10ml sample after filtration to determine the microbial limit, and then sterilize and filter through a 0.45μm~0.22μm filter membrane to obtain the stock solution. Harvest a total of 1800ml, take a sample for testing, and test items For protein content, polysaccharide content, nucleic acid content, sterility test. The sterilization and filtration time should be controlled within 4 hours. The test result shows that the protein recovery rate is 60%, and the recovery rate%=(liquid stock solution ml×liquid stock solution protein content mg / ml) / (filtrate volume ml×filtrate protein content mg / ml), wherein the protein content is 1.75 mg / ml.

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Abstract

The invention discloses a production process of a high-titer tuberculin skin test diagnostic reagent PPD (Purified Protein Derivative). The production process comprises bacterial culture, inactivationand precipitation. The bacterial culture specifically comprises the following process steps: culturing and developing a working seed culture into dry agglomerated light-yellow bacterial lawn, performing microscopic examination on the bacterial lawn as bacillus brevis that is micro bent and round in two ends; and performing two-generation culture to obtain wrinkled light yellow mycoderm, performing passage culture for three generations when the mycoderm overspreads the surface of the culture medium and the culture solution is clarified and transparent, so as to form multi-wrinkled light yellowmycoderm, and performing four-generation culture for 8-10 weeks. According to the production process of the high-titer PPD capable of enriching two antigens such as ESAT-6 and CFP10, the PPD preparedby the process disclosed by the invention has high titer, and the content of the ESAT-6 and CFP10 is high.

Description

technical field [0001] The invention relates to the field of immunology, and relates to the preparation of a skin test reagent, in particular to a method for preparing a highly efficient pure protein derivative of tuberculin. Background technique [0002] Tuberculosis is a serious bacterial infection caused by the bacterium Mycobacterium tuberculosis, usually affecting the lungs. The disease is spread from person to person by droplets from the throat and lungs of people with active respiratory disease. In healthy people, Mycobacterium tuberculosis infection usually causes no symptoms because the immune system works to fend off the bacteria. Symptoms of active tuberculosis are cough, sometimes with phlegm or blood, chest pain, weakness, weight loss, fever and night sweats. Tuberculosis can be treated with a six-month course of antibiotics. Tuberculosis is the ninth leading cause of death globally and the leading cause of death from a single pathogen, higher than HIV / AIDS. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P21/02C12R1/32
CPCC12N1/20C12P21/02
Inventor 高正伦党银慧王翠芳李明静叶晓明王宁
Owner BEIJING SANROAD BIOLOGICAL PROD CO LTD
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