Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for purifying pertussis fimbrillin

A fimbrial protein and pertussis technology, applied in the biological field, can solve the problems of many steps, high production cost, long time consumption, etc., and achieve good repeatability.

Inactive Publication Date: 2021-02-09
WUHAN INST OF BIOLOGICAL PROD CO LTD
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The above method has many steps, takes a long time, and has high production costs. It is necessary to improve and upgrade the production process for large-scale continuous preparation of pili protein

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for purifying pertussis fimbrillin
  • Method for purifying pertussis fimbrillin
  • Method for purifying pertussis fimbrillin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050]Example 1. Fermentation culture and pretreatment of Bordetella pertussis

[0051]The pertussis strain was fermented and cultured, the culture supernatant and the bacterial pellet were harvested, and 10-15 times the volume of the precipitation (wet weight) of the 50 mM PB buffer containing 1 M sodium chloride at pH=8.0 was added to the bacterial pellet and stirred Evenly, leaching at 2~8℃ for 1-5 hours (preferably 1.5 hours). After centrifugation, the leaching supernatant and the leaching precipitate are collected, and the crude PRN solution is separated from the leaching precipitate, and passed through the Capto Adhere layer After the column is separated, the flow-through liquid is collected (for the specific method, please refer to our company's previous patent, patent number CN108570098A).

Embodiment 2

[0052]Example 2. Convective flow-through is firstly salted out with ammonium sulfate and then passed through a Capto Adhere column

[0053]1. Slowly add solid ammonium sulfate to the PRN chromatographic flow-through solution obtained in Example 1 at 2-8°C, so that the final concentration of ammonium sulfate reaches (mass volume ratio W / V) 15-25%, and stir slowly Dissolve completely after 1 hour,

[0054]2. Leave the solution to stand for 16-24 hours, centrifuge at 15000×g for 30 minutes, dissolve the precipitate with 50mM Tris-HCl buffer with a pH of 8.0-9.0, fully dissolve it and centrifuge again. The centrifugal conditions are the same as the above, and collect after centrifugation. Supernatant solution.

[0055]3. Adjust the pH and conductivity of the supernatant solution to 8.0-9.0 and 20-30mS / cm, respectively, and collect the flow-through after passing through the Capto Adhere column to obtain the crude fimbrin (Fim) pure liquid;

Embodiment 3

[0056]Example 3. The flow-through liquid was first collected through a Capto Adhere chromatography column and the flow-through was added with ammonium sulfate salting out

[0057]1. Adjust the pH and conductivity of the flow-through fluid collected in Example 1 to 8.0-9.0 and 20-30 mS / cm, respectively, and collect the flow-through fluid after passing through a Capto Adhere column;

[0058]2. Under the condition of 2-8℃, slowly add ammonium sulfate solid to make the final concentration of ammonium sulfate reach (mass volume ratio W / V) 15-25%, stir slowly for 1 hour and then dissolve completely.

[0059]3. Leave the solution to stand for 16-24 hours, centrifuge at 15000×g for 30 minutes, dissolve the precipitate with 50mM Tris-HCl buffer of pH 8.0-9.0, and centrifuge again after fully dissolving. The centrifugal conditions are the same as the above, and collect after centrifugation. The supernatant solution is used to obtain a crude fimbrin (Fim) pure liquid.

[0060]The SDS-PAGE diagrams of samp...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Conductivityaaaaaaaaaa
Sensitivityaaaaaaaaaa
Login to View More

Abstract

The invention relates to a method for purifying pertussis fimbrillin. The method comprises the following steps: (1) carrying out fermentation culture on bacillus pertussis, then carrying out centrifugation and collecting precipitates, cracking thalli with urea or other suitable methods to obtain a PRN crude pure solution, and collecting a flow-through solution after chromatography; (2) carrying out salting-out cascade hydrophobic anion chromatography on the fimbrillin (Fim) crude pure solution; and (3) regulating the pH value and conductivity of the fimbrillin (Fim) crude pure solution collected in the step (2), making the fimbrillin (Fim) crude pure solution flow through a CHT chromatographic column and performing elution and collection to obtain a Fim antigen.

Description

Technical field[0001]The invention belongs to the field of biotechnology, and specifically relates to a method for purifying pertussis fimbriae protein.Background technique[0002]Whooping cough (pertussis, whooping cough) is a severe respiratory infection caused by the gram-negative bacillus B.pertussis that is extremely harmful to infants and young children. Before vaccination, the disease was widespread all over the world, and its morbidity and mortality were high. The vaccination of whole cell pertussis vaccine (wPV) has allowed the disease to be well controlled, but due to higher adverse reactions after vaccination, it has gradually been adopted by the safer acellular pertussis vaccine aPV (acellular pertussis vaccine aPV). replace. Since 2010, in countries with high vaccine coverage, the incidence of pertussis among adults and adolescents has increased. These infected adults and adolescents act as spreaders and spread the pathogen to susceptible infants and young children who ar...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K14/235C07K1/36C07K1/30C07K1/18C07K1/20C07K1/22A61K39/10A61P31/04
CPCA61K39/099A61P31/04C07K14/235
Inventor 杨晓明杨柏峰田聪潘聪胡源霍梦颖陈雯胡太平施斌李新国段凯朱德武
Owner WUHAN INST OF BIOLOGICAL PROD CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com