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

Cell disruption method for recombinant hansenula polymorpha for expressing enterovirus type 71 antigen and application of method in preparation of hand-foot-mouth disease vaccine

A cell fragmentation and enterovirus technology, applied in the direction of viral antigen components, microorganism-based methods, viruses, etc., can solve the problems of cumbersome operation, low fragmentation rate, and a large amount of heat generation, so as to maintain antigenic activity and improve cell fragmentation rate. , the effect of low equipment requirements

Pending Publication Date: 2020-05-22
SHENZHEN KANGTAI BIOLOGICAL PROD
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The commonly used cell crushing method in the prior art is crushing with a high-pressure homogenizer. This method consumes a lot of energy, is cumbersome to operate, has a low crushing rate, and can only crush one sample at a time.
It is reported in China that zirconia beads are used as the grinding medium to crush Hansenula cells, but the crushing rate can only reach 50-60%, and the crushing process generates a lot of heat and causes zirconium residues

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
  • Cell disruption method for recombinant hansenula polymorpha for expressing enterovirus type 71 antigen and application of method in preparation of hand-foot-mouth disease vaccine
  • Cell disruption method for recombinant hansenula polymorpha for expressing enterovirus type 71 antigen and application of method in preparation of hand-foot-mouth disease vaccine
  • Cell disruption method for recombinant hansenula polymorpha for expressing enterovirus type 71 antigen and application of method in preparation of hand-foot-mouth disease vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] instrument:

[0055] Centrifuge: Eppendorf, model MiniSpin plus

[0056]Cell disruptor: American GLAS-COL vortex shaker

[0057] Electronic balance: Sartorius BT 4202S

[0058] Microscope: XDS-1B inverted microscope

[0059] experiment material:

[0060] Acidified glass beads (Glass beads, Acid-washed): biochemical grade, Sigma, 425-600 μm.

[0061] step:

[0062] Preparation of cell washing buffer (pH8.0), weigh 0.334g disodium hydrogen phosphate, 0.016g sodium dihydrogen phosphate monohydrate, 0.876g sodium chloride, add purified water to fully dissolve and mix, adjust the pH value to 8.0 , set the volume to 100mL;

[0063] Preparation of cell disruption buffer (pH7.4), weigh 0.596g disodium hydrogen phosphate, 0.11g sodium dihydrogen phosphate monohydrate, 2.925g sodium chloride, 0.037g disodium edetate, glycerin 5g, add purified water to fully dissolve and mix, adjust the pH value to 7.4, and dilute to 100mL;

[0064] Collect 0.1 g of recombinant Hansenula y...

Embodiment 2

[0070] instrument:

[0071] Centrifuge: Eppendorf, model MiniSpin plus

[0072] Cell disruptor: American GLAS-COL vortex shaker

[0073] Electronic balance: Sartorius BT 4202S

[0074] experiment material:

[0075] Acidified glass beads (Glass beads, Acid-washed): biochemical grade, Sigma, 425-600 μm.

[0076] step:

[0077] Preparation of cell washing buffer (pH8.0), weigh 16.7 g of disodium hydrogen phosphate, 0.8 g of sodium dihydrogen phosphate monohydrate, and 43.8 g of sodium chloride, add purified water to fully dissolve and mix, and adjust the pH value to 8.0 , set the volume to 5L.

[0078] Preparation of cell disruption buffer (pH7.4), weigh 29.8 g of disodium hydrogen phosphate, 5.5 g of sodium dihydrogen phosphate monohydrate, 146.2 g of sodium chloride, 1.8 g of disodium edetate, glycerin 250g, add purified water to fully dissolve and mix, adjust the pH value to 7.4, and dilute to 5L.

[0079] Collect 0.2 g of recombinant Hansenula yeast cells, add 1 mL of ...

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
crush indicatorsaaaaaaaaaa
crush indicatorsaaaaaaaaaa
Login to View More

Abstract

Embodiments of the invention disclose a cell disruption method for recombinant hansenula polymorpha for expressing an enterovirus type 71 antigen and an application of the method in preparation of a hand-foot-mouth disease vaccine. The method includes the following steps: collecting recombinant hansenula polymorpha, adding a cell washing buffer solution, performing washing and resuspension, performing centrifugation, and discarding the supernatant; adding a cell disruption buffer solution to fully resuspend microbes; adding a phenylmethylsulfonyl fluoride solution, polysorbate 80 and acidifiedglass beads to prepare a microbial suspension; and placing the microbial suspension in a cell disrupter, performing shaking disruption, after the disruption is completed, performing centrifugation, and taking the supernatant to obtain a cell disruption solution. The method provided by the invention can effectively maintain the activity of the EV71 antigen, has simple operation and a high disruption rate, and can simultaneously disrupt multiple samples, thereby effectively saving sample processing time.

Description

technical field [0001] The invention relates to the preparation technology of genetically engineered recombinant enterovirus 71 vaccine, in particular to a method for disrupting cells of recombinant Hansenula expressing enterovirus 71 antigen and its application in preparing hand, foot and mouth disease vaccine. Background technique [0002] Enterovirus 71 (EV71) is one of the important pathogens causing HFMD. Our company has constructed a recombinant Hansenula expressing EV71 antigen. In order to measure the EV71 antigen content in the recombinant Hansenula spp. to monitor the stability of the fermentation process, it is necessary to disrupt the cells of the recombinant Hansenula spp. to release the EV71 antigen in the cells for its determination. [0003] The commonly used cell crushing method in the prior art is crushing with a high-pressure homogenizer. This method consumes a lot of energy, is cumbersome to operate, has a low crushing rate, and can only crush one sample...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/06A61K39/125A61P31/14C12R1/78
CPCC12N1/063C12N1/066A61K39/12A61P31/14C12N2770/32322C12N2770/32334C12N2770/32351
Inventor 钟子辉刘萌萌王笑天蓝小凤雷国民骆应宏甘建辉
Owner SHENZHEN KANGTAI BIOLOGICAL PROD
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