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

Extraction method for escherichia coli pilus antigen used for preparing yolk antibody, and method for preparing yolk antibody

A technology of Escherichia coli and extraction method, which is applied in the preparation of egg yolk antibody and the field of improved Escherichia coli fimbriae extraction, can solve the problems of toxicity of host cells, easy damage of bacterial cell walls, endotoxin shock and disseminated intravascular coagulation. , to achieve the effect of good antigenicity

Active Publication Date: 2014-09-03
SHENZHEN NEPTUNUS INTERLONG BIO TECHN HLDG
View PDF5 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is because even without vigorous heating and stirring, the bacterial cell wall is easily damaged, thereby producing a large amount of endotoxin
Although endotoxin is not highly antigenic, it is toxic to host cells and can cause symptoms such as fever, microcirculation disturbance, endotoxin shock, and disseminated intravascular coagulation, which will affect the immune effect

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
  • Extraction method for escherichia coli pilus antigen used for preparing yolk antibody, and method for preparing yolk antibody
  • Extraction method for escherichia coli pilus antigen used for preparing yolk antibody, and method for preparing yolk antibody
  • Extraction method for escherichia coli pilus antigen used for preparing yolk antibody, and method for preparing yolk antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] [Example 1] Enterotoxin Escherichia coli K99 pili extraction

[0030] Primary Seed Preparation

[0031] Prepare 100mL modified Minca culture based on a 500mL Erlenmeyer flask, and sterilize at 116°C for 30min. After cooling, inoculate K99 with an inoculum size of 2‰, culture at 37°C for 12-18h, and rotate at 200rpm.

[0032] Secondary Seed Preparation

[0033] Prepare two 500mL modified Minca cultures based on two 2000mL Erlenmeyer flasks, and sterilize at 116°C for 30min. After cooling, inoculate K99 primary seed solution with an inoculum size of 2‰, cultivate at 37°C for 12-18h, and rotate at 200rpm.

[0034] 15L Fermentation Tank High Density Fermentation

[0035] Prepare 15L of modified Minca culture-based fermenter, add an appropriate amount of antifoaming agent, and sterilize at 116°C for 30 minutes. After the sterilization is completed, when the culture temperature in the tank is 37°C, keep it for 12-24 hours for sterility test. After passing the sterility ...

Embodiment 2

[0040] [Example 2] Enterotoxin Escherichia coli 987P pili extraction

[0041] The fermentation method of 987P is the same as that of K99. After the bacteria are collected, the pili protein is extracted by the following method:

[0042] 1. Weigh 100-150g of 987P fermented cells with a wet weight, add PBS (containing 2M urea) to dissolve (the volume ratio of cells to PBS is 1:10), and stir well with a stirrer. The fully dissolved bacteria were homogenized 5 times by a pressure breaker. The homogenate was processed by centrifugation at 8000rpm for 30min to remove the precipitate. The supernatant was then centrifuged at 18000rpm / 30min to remove the precipitate. The final supernatant was purified by 4FF chromatography column.

[0043]2. Equilibrate 2-3 volumes of 4FF agarose gel column with PBS (containing 2M urea) at a flow rate of 8ml / min, and turn on the UV detector at the same time. After the baseline is stable, measure 100ml of the crude pili extract, start loading the sam...

Embodiment 3

[0044] [Example 3] Removal of endotoxin

[0045] The sample solution obtained by the above method was added into acetone (under ice bath condition) at a volume ratio of 1:1, stirred for 10 min, centrifuged at 10,000 rpm for 20 min to remove the supernatant. Collect the precipitate, add PBS and acetone at a volume ratio of 1:9, stir in an ice bath for 10 min, and centrifuge at 10,000 rpm to discard the supernatant. After the acetone volatilized completely, the precipitate was redissolved in PBS to obtain the pili antigen. The change of endotoxin content was determined by the gel method limulus reagent, image 3 It can be seen that after the acetone treatment, the highest dilution factor at which the bacterial endotoxin reacted positively with the Limulus reagent decreased significantly, that is, the endotoxin content was significantly reduced, and no adverse reactions occurred after immunization of laying hens, and the obtained antigen can be used for immunization of laying he...

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

No PUM Login to View More

Abstract

The invention provides an extraction method for an escherichia coli pilus antigen used for preparing a yolk antibody. The extraction method comprises the steps of (1) culturing escherichia coli, collecting thalli, breaking and dissolving the thalli and preparing a primary extract of escherichia coli thalli; 2) purifying the primary extract by a 4FF gel chromatographic column to obtain a purified solution containing escherichia coli pilus protein; and 3) removing endotoxin in the purifiedsolution of escherichia coli pilus protein by using acetone precipitation to obtain the escherichia coli pilus antigen. Pilus protein with relatively high purity is obtained by utilizing gel filtration chromatography; and factors such as endotoxin released by the thalli which are possible to interfere an immune effect are removed. The prepared subunit vaccine immunized laying hens gain high-titer antibodies; and new ideals are provided for preventing and treating related diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an improved method for extracting Escherichia coli pili; [0002] The invention also relates to a preparation method of egg yolk antibody. Background technique [0003] Escherichia coli (Escherichia coli) is a kind of normal bacteria that parasitizes the intestines of animals on the one hand, and on the other hand, it also shows certain pathogenicity and causes various diseases. Pathogenic Escherichia coli can cause diarrhea and sepsis in humans and animals, especially for young animals. In large-scale pig production, Escherichia coli infection can cause yellow scour, pullorum, edema, etc. of piglets, and the morbidity and mortality account for 56.2% and 24.7% of all piglet diarrhea, which brings no small damage to the breeding industry. Economic losses. [0004] Enterotoxigenic Escherichia coli (ETEC) is a major pathogen that causes diarrhea in piglets. It can be divi...

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): C07K14/245C07K1/36C07K1/30C07K1/34C07K16/12C07K16/02
CPCC07K14/245C07K16/02C07K16/1232C07K2317/11
Inventor 陈娟赵秦孙盼柴向东王荣刘晓璐刘超岳静孟美玲
Owner SHENZHEN NEPTUNUS INTERLONG BIO TECHN HLDG
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