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Separation and purification process for acellular whooping cough antigen albumen

A technology for separation and purification of antigenic proteins, applied in the direction of bacterial antigenic components, chemical instruments and methods, antibacterial drugs, etc., to achieve the effect of low price

Inactive Publication Date: 2004-06-16
YOULIKAI BIOLOGICAL TECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although domestic perlite has sufficient supply, cheap price (1.5-3.0 RMB / kg), and stable product performance, it is only used as a filter aid at present, and there is no domestic report on the use of perlite to separate and purify proteins

Method used

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  • Separation and purification process for acellular whooping cough antigen albumen
  • Separation and purification process for acellular whooping cough antigen albumen
  • Separation and purification process for acellular whooping cough antigen albumen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Select domestic edible medium-speed perlite (Inner Mongolia Chifeng Perlite Factory), containing SiO 2 72%, Al 2 o 3 10%, use a standard sieve to remove perlite with too small and too large particles, and obtain a particle range of 50-125 μm. Rinse the sieved perlite particles with distilled water, let stand to remove suspended solids and suspensions, and load them into a chromatographic column (Beijing Binda Yingchuang Technology Co., Ltd., inner diameter 210mm) with a packing height of 300mm after repeated repetitions. Rinse the perlite chromatography column with 5 times of water for injection (WFI).

[0038] Open the freeze-dried ampoule of Bordetella pertussis CS strain, sprinkle the contents in sterile water, transfer it to semi-integrated activated carbon agar medium with a pipette, and incubate at 37°C for 72 hours . The organisms were subcultured on the above-mentioned medium and cultured at 37°C for 48 hours, thus amplified for 3-4 generations.

[0039] ...

Embodiment 2

[0043] Select domestic edible medium-speed perlite (Xinyang Perlite Factory, Henan Province), containing SiO 2 80%, Al 2 o 3 20%, use a standard sieve to remove the perlite with too small and too large particles, and obtain a particle range of 125-250 μm. Rinse the sieved perlite particles with distilled water, let stand to remove suspended solids and suspensions, and load them into a chromatographic column (Beijing Binda Yingchuang Technology Co., Ltd., inner diameter 210mm) with a packing height of 300mm after repeated repetitions. Rinse the perlite chromatography column with 5 times of water for injection (WFI).

[0044] Open the freeze-dried ampoule of Bordetella pertussis CS strain, sprinkle the contents in sterile water, transfer it to semi-integrated activated carbon agar medium with a pipette, and incubate at 37°C for 72 hours . The organisms were subcultured on the above-mentioned medium and cultured at 37°C for 48 hours, thus amplified for 3-4 generations.

[...

Embodiment 3

[0049] The hemagglutination activity of the collected solution of the three ultraviolet light absorption peaks of the eluate of the perlite chromatography column was measured respectively.

[0050] Collect 5ml of fresh gander blood, add 10ml of 0.9% NaCl solution, centrifuge to discard the supernatant, and then wash with 10ml of 0.9% NaCl solution for a total of 3 times. Then use 0.9% NaCl solution to make goose blood into 0.8% erythrocyte suspension.

[0051] Add 40 μl of 0.9% normal saline to the hemagglutination plate, add 40 μl of the sample to be tested for doubling dilution, and finally add 40 μl of 0.8% red blood cell suspension, shake fully, and place at room temperature for 30 minutes. The experimental results are listed in Table 1. It can be seen from Table 1 that the pertussis antigen peak 2 and peak 3 collected liquids separated and purified from perlite have higher hemagglutination activity, and the hemagglutination activity of the peak 2 collected liquid reaches ...

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Abstract

The invention relates to a method for further separating and purifying pertussis antigen for the supernatant fluid of pertussis bacilliculture liquid, wherein the screen selected domestic pearlstone is used as column chromatography material in the process, the sample is concentrated for conductance adjustment processing and elution condition optimized for further segregation and purification, to obtain the Filamentous hamagglutmin (FHA) and pertassis toxin (PT), the polyacrylamide gel electrophoresis (SDS-PAGE) evaluation has shown that its purity is as high as 85-95%.

Description

technical field [0001] The invention relates to a process for separating and purifying acellular pertussis antigen by column chromatography, in particular to a method for using screened domestic perlite as a column chromatography material to conduct a one-step process on the supernatant of Bordetella pertussis culture fluid. A method for separating and purifying pertussis antigen. Background technique [0002] Pertussis has a high morbidity and mortality rate among children under the age of four. According to statistics, 40 million children are infected every year, and about 950,000 children die of the disease because they have not been vaccinated against pertussis. Worldwide, approximately one billion doses of pertussis vaccine are administered each year. Among them, there are more than 80 million purified acellular pertussis vaccines. [0003] Whole-cell pertussis vaccines have been used in various countries for decades. Due to the side effects of varying degrees of feve...

Claims

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Application Information

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IPC IPC(8): A61K39/10A61P31/04C07K14/23
Inventor 孙世章李贵凡宫慧梅孙雪南
Owner YOULIKAI BIOLOGICAL TECH BEIJING
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