Method of separating and purifying various antigen ingredients of pertussis

A separation, purification and pertussis technology, which is applied in the field of biomedicine, can solve the problems of destroying the immunogenicity of FHA, loss of PT and FHA, and difficult removal of PT, and achieves the effects of good immunogenicity, good integrity and improved recovery rate.

Active Publication Date: 2018-09-25
WUHAN INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both PT and FHA have strong hydrophobicity, so 2M urea and high-salt solution are required to maintain stability, while cation exchange chromatography requires low conductivity to load samples, so PT and FHA will have a relatively high temperature during the ultrafiltration replacement process. big loss
[0005] Furthermore, PT and FHA are positively charged and have a certain degree of hydrophobicity in a solution environment with a low pH value, so PT in purified FHA is difficult to remove, and FHA needs to be detoxified, which will destroy Immunogenicity of FHA

Method used

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  • Method of separating and purifying various antigen ingredients of pertussis
  • Method of separating and purifying various antigen ingredients of pertussis
  • Method of separating and purifying various antigen ingredients of pertussis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, fermentation culture of Bacillus pertussis

[0063] experiment material:

[0064] Strains: pertussis phase I CS strain, CMCC58003. Sourced from China National Institutes for Food and Drug Control. Pertussis phase I CS strain, CMCC58003. Sourced from China National Institutes for Food and Drug Control.

[0065] Medium: acellular pertussis SSM complete comprehensive medium

[0066] Main equipment: Bioengineering AG fully automatic fermentation system (50L, 500L, 5000L)

[0067] experiment method:

[0068] The working seed batch of pertussis bacillus was opened, inoculated on Jiang's medium containing 15% sheep blood modified bag, cultured at 35°C for 72 hours, then transferred to activated carbon semi-comprehensive medium, and cultivated at 37°C for 48 hours; Generation 3 (activated charcoal semi-comprehensive medium), continue culturing at 37°C for 48 hours, then scrape seeds in 250ml phosphate buffer (i.e. PBS buffer, 137mM NaCl, 2.7mM KCl, 10mM NaCl...

Embodiment 2

[0071] Embodiment 2, the centrifugation of large tank fermented liquid

[0072] Experimental materials and samples: pertussis bacteria liquid 3500L

[0073] Main equipment: disc centrifuge, Alfa Laval CLARA 250

[0074] experiment method:

[0075] Centrifuge 500 liters of pertussis fermentation and harvested bacteria liquid with a disc centrifuge. The centrifuge feed flow rate is 1000-1500L / h, and the slag discharge time is 5-10min. By reducing the feed flow rate and shortening the slag discharge time, the centrifuge The turbidity of the supernatant of the bacterial liquid was ≤30NTU, and the bacterial precipitate was collected.

[0076] test results:

[0077] Harvest 475 liters of bacterial supernatant, with a turbidity value of 11.4 NTU.

Embodiment 3

[0078] Example 3, Separation and Purification of PT Antigen from Fermentation Broth Supernatant

[0079] 1. Ultrafiltration treatment of fermentation broth

[0080] Test materials and samples:

[0081] Ultrafiltration membrane pack: Pellicon2 Biomax 30, Milipore

[0082] Tangential flow treatment system: CUF200SA membrane package ultrafiltration system, Milipore

[0083] Sample: Bacterial fluid harvested from pertussis fermentation, supernatant after centrifugation and separation of bacterial cells, the sample volume is 475 liters.

[0084] experiment method:

[0085] The supernatant after bacterial cell separation was concentrated by ultrafiltration using a polyethersulfone membrane bag with a molecular weight cut-off of 10KD / 30KD, and the pressure at the feed end during the concentration process was kept at 15psi-25psi. When the volume is concentrated to about 50 liters, use 3 to 5 times the volume replacement solution (10mM sodium citrate+2M urea+0.5MNaCl, pH=5.9-6.1) t...

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Abstract

The invention relates to a method of separating and purifying various antigen ingredients of pertussis. The various antigens include PT (pertussis toxin) antigen, FHA (filamentous hemagglutinin) antigen and PRN (pertactin) antigen. The method comprises the steps of (1) subjecting a pertussis strain to fermenting culture to acquire culture supernate and bacterial precipitate separately; (2) isolating and purifying PT antigen from the culture supernate, and separating and purifying PRN antigen and FHA antigen from the bacterial precipitation.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for separating and purifying various antigenic components of pertussis. Background technique [0002] Whooping cough is a severe infectious disease that seriously endangers the health of infants and young children. Vaccination is the most ideal means of prevention and control. Acellular DPT vaccine (DTaP) is the second-generation DPT vaccine, because its side effects are significantly lower than the first-generation whole-bacteria DPT vaccine (DTwP), it has been widely promoted and entered the national immunization plan. The annual market demand is more than 60 million pieces. [0003] DTaP vaccines can be divided into co-purified vaccines and component vaccines (DTacP) according to different production methods: the former adopts relatively backward salting-out and ultracentrifugation techniques to extract effective antigens pertussis toxin (PT) and filamentous hema...

Claims

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Application Information

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IPC IPC(8): C07K14/235C07K1/36C07K1/34C07K1/30C07K1/18
CPCC07K14/235
Inventor 胡业勤田聪潘聪曾赟周昉陈雯朱德武段凯李新国瞿明霞
Owner WUHAN INST OF BIOLOGICAL PROD CO LTD
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