Method for removing pertussis component pilin 2/3 endotoxin

A technology of pili protein and whooping cough, applied in the field of biopharmaceuticals, can solve the problems of affecting the safety of vaccines, easy introduction of affinity groups, loss of target proteins, etc., and achieve the effect of maintaining biological activity and recovery rate

Active Publication Date: 2019-10-22
CHANGCHUN BCHT BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Methods such as gel affinity can be used to remove endotoxin. In actual use, we found that although this method is simple and only needs to use endotoxin affinity gel in the purification process, the effect of removing endotoxin is not ideal, and it is easy to introduce affinity and groups, affecting the safety of vaccines
The prior art provides another purification method of FIM2 / 3 (CN97197609.0), the steps are relatively cumbersome, and it includes two ultrafiltration steps and two column chromatography before and after, respectively SepharoseCL6B and PEI silica gel column, according to the prior art It can be seen that too many purification steps may cause more loss of target protein

Method used

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  • Method for removing pertussis component pilin 2/3 endotoxin
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  • Method for removing pertussis component pilin 2/3 endotoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: method of the present invention

[0038] 1) Open a strain of Bacillus pertussis and inoculate it on the ginger medium, cultivate it at 36°C for 3 days, transfer it to the activated carbon agar medium, cultivate it at 36°C for 40 hours, and inoculate it in 5L pertussis liquid medium (MSS), The shake flask was cultivated for 20 hours, and as fermentor seeds, it was put into a 300L fermentor (working volume 150L) for cultivation, and harvested after cultivating for 34 hours. The harvested fermentation broth was separated from supernatant and pertussis bacteria using a continuous flow centrifuge.

[0039]2) After dissolving the pertussis cells obtained in step 1 with pH 7.0, 0.02 MPBS buffer solution containing 2M urea, stir at 2-8° C. for 30 min.

[0040] 3) Centrifuge (10000 rpm, 30 min) to collect the supernatant, heat at 80° C. for 30 min, and then centrifuge (10000 rpm, 30 min) to collect the supernatant. PEG8000 was added to the clarified supernatant t...

Embodiment 2

[0044] Embodiment 2: method of the present invention

[0045] 1) Open a strain of Bacillus pertussis and inoculate it on the ginger medium, culture it at 36.5°C for 3 days, transfer it to the activated carbon agar medium, cultivate it at 36.5°C for 42 hours, and inoculate it in 5L pertussis liquid medium (MSS), Cultivate in shake flasks for 20-24 hours, put them into a 300L fermenter (working volume: 150L) as fermenter seeds, and cultivate them for 35 hours before harvesting. The harvested fermentation broth was separated from supernatant and pertussis bacteria using a continuous flow centrifuge.

[0046] 2) After dissolving the pertussis cells obtained in step 1 with pH 7.2, 0.02 MPBS buffer solution containing 4M urea, stir at 2-8° C. for 30 min.

[0047] 3) Centrifuge (10000 rpm, 30 min) to collect the supernatant, heat at 80° C. for 30 min, and then centrifuge (10000 rpm, 30 min) to collect the supernatant. PEG8000 was added to the clarified supernatant to a final concen...

Embodiment 3

[0051] Embodiment 3: method of the present invention

[0052] 1) Open a strain of Bacillus pertussis and inoculate it in ginger medium, culture it at 37°C for 3 days, transfer it to activated carbon agar medium and culture it at 37°C for 43 hours, inoculate it in 5L pertussis liquid medium (MSS), shake The bottle was cultivated for 23 hours, and used as fermenter seeds, put into a 300L fermenter (working volume 150L) for cultivation, and harvested after cultivating for 35 hours. The harvested fermentation broth was separated from supernatant and pertussis bacteria using a continuous flow centrifuge.

[0053] 2) After dissolving the pertussis cells obtained in step 1 with pH 7.4 and 0.02M PBS buffer containing 6M urea, stir at 2-8°C for 30 min.

[0054] 3) Centrifuge (10000 rpm, 30 min) to collect the supernatant, heat at 80° C. for 30 min, and then centrifuge (10000 rpm, 30 min) to collect the supernatant. Add PEG8000 to the clarified supernatant to a final concentration of ...

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Abstract

The invention relates to the technical field of biologic pharmacy, and discloses a method for removing pertussis component pilin 2/3 endotoxin. The method comprises the steps that after bordetella pertussis is fermented and cultured, thalli are centrifugally collected, thalli are extracted through a urea phosphate buffer solution, a supernate is centrifugally collected, and PEG-8000 and ammonium sulfate are used for performing precipitation; precipitates are extracted by the phosphate buffer solution, then, a supernate is centrifugally collected, a Q-Sepharose chromatographic column is adoptedfor processing, and a phosphate buffer solution containing sodium chloride is adopted for eluting the Fim 2/3 component. By combining PEG8000 precipitating, ammonia sulfate precipitating and Q-Sepharose chromatographic column processing are combined, endotoxin in pilin 2/3 is removed through repeated means, on the premise of not introducing allogenic materials, the effective ingredient Fim 2/3 biologic activity and recycling rate are kept, and meanwhile the endotoxin in Fim 2/3 can be removed to 10 EU/mg or lower.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a method for removing pilin 2 / 3 endotoxin, a component of whooping cough. Background technique [0002] Bacillus pertussis is Bachybacterium ovale, belonging to the genus Bordetella, and Gram stain is negative. Obligate aerobic, high nutritional requirements in the initial isolation culture, need to be cultured on Baojiang medium to grow. After incubation, small, smooth, raised, opaque colonies surrounded by an indistinct haemolytic ring were seen. In addition to the capsule and cell wall lipopolysaccharide, there are many biologically active factors related to pathogenicity. Pertussis exotoxin is the main pathogenic factor, which can induce the body's long-lasting immunity and has a variety of biological activities, such as improving the sensitivity of mice to histamine and serotonin, promoting leukocytosis, inhibiting the function of macrophages, Damage to the cil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/23C07K1/36C07K1/30C07K1/16C12R1/01
CPCC07K14/23
Inventor 王长永王梦舒徐艳艳梁洪野闫慧原秀娟
Owner CHANGCHUN BCHT BIOTECH
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