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

Method for cultivating recombinant avian influenza subtype virus through full-suspension cell

A cell culture, avian influenza technology, applied in the methods of using microorganisms, viruses, animal cells, etc., can solve problems such as the inability to meet the production process, and achieve the effect of ensuring stability and increasing performance stability.

Inactive Publication Date: 2018-06-29
吉林冠界生物技术有限公司 +1
View PDF8 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expanded seed virus is suitable for the subculture of chicken embryo virus, and cannot meet the production process of avian influenza inactivated vaccine (H5 subtype) full-suspension cell culture. The seed virus for preparing the vaccine needs to be adaptively domesticated to a certain generation of MDCK cells. Can be used for scale-up production

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 cultivating recombinant avian influenza subtype virus through full-suspension cell
  • Method for cultivating recombinant avian influenza subtype virus through full-suspension cell
  • Method for cultivating recombinant avian influenza subtype virus through full-suspension cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Preparation of suspended MDCK cells, the steps are as follows:

[0035] Adherent cultured MDCK cells were introduced from ATCC by the Gansu Animal Cell Engineering Technology Research Center (GsACC) of Northwest University for Nationalities. The introduction time: February 2011, ATCC number: CCL-34, generation: P56, preservation number: 58860056 . A cell bank was established after GsACC expansion and culture, and the number of the cell bank is: GsACC2B0000090.

[0036] The MDCK cells were revived with DMEM / F12 medium containing 10% FBS, grown densely and then passaged.

[0037] Select well-grown cells and gradually domesticate and cultivate them, as follows:

[0038] Subcultured with DMEM / F12 medium containing 8% fetal bovine serum for 3 times;

[0039] Subcultured 5 times with DMEM / F12 medium containing 5% fetal bovine serum;

[0040] Subcultured 5 times with DMEM / F12 medium containing 2% fetal bovine serum;

[0041] DMEM / F12 medium and low-serum medium were used ...

Embodiment 2

[0054] Recombinant avian influenza subtype virus chick embryo seed virus is inoculated in the domestication of MDCK monolayer cell culture, and the steps are as follows:

[0055] 1. MDCK cell preparation: take MDCK cells grown for 72 hours, digest with 0.25% EDTA-trypsin, 25cm 2 The amount of trypsin used in monolayer cells is 0.5ml, (if it is 75cm 2 For monolayer cells, the amount of trypsin used is 1.5ml, and so on), at 37°C for 3 minutes. After about 90% of the cells shrink, the cells are dispersed by blowing and blowing with DMEM culture medium (containing 10% fetal bovine serum), and the cells are counted.

[0056] Subculture according to counting results, 25cm 2 Monolayer seeded cells were 5.0×10 5 ~7.0×10 5 each, 75cm 2 Cells were seeded in a monolayer of 1.5 x 10 6 ~2.1×10 6 one, and so on.

[0057] Set at 37°C, CO 2 Incubator (CO 2 concentration of 5%). After 72h of growth, cell count, 25cm 2 The number of monolayer cells is 5.0×10 6 ~7.0×10 6 , 75cm 2 ...

Embodiment 3

[0074] Suspension MDCK cell seed poisoning preparation:

[0075] 1. Suspension Cell Preparation

[0076] Resuscitate suspended MDCK cells frozen in liquid nitrogen according to conventional methods, add the cell solution into a 500ml Erlenmeyer shaker flask with a pipette, and add filter-sterilized serum-free cell culture medium with a pH of 7.2±0.2 to 100ml. Place shaker flask in CO 2 In the shaker, 37 ℃, 80 ~ 100 rpm for cultivation and propagation.

[0077] 2. Reactor scale-up culture

[0078] Take the suspended MDCK cells cultured in shake flasks, and use 1.0×10 6.0 ~2.0×10 6.0 The cell density of each / ml was inoculated into a 5L reactor for culture, the rotation speed was 80-100 rpm, the temperature was 37°C, the dissolved oxygen was 30%-60%, and the pH was 7.2±0.2.

[0079] When the cell density is ≥6.0×10 6.0 cells / ml, add cell culture medium to adjust the initial cell density to 1.0×10 6.0 ~2.0×10 6.0cells / ml, adjust the reactor control parameters (rotating spe...

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 relates to the preparation field of the avian influenza virus vaccine, and especially relates to a method for cultivating recombinant avian influenza subtype virus through full-suspension cell. The method comprises the following steps: inoculating the recombinant avian influenza subtype virus chick embryo virus into MDCK monolayer cell to acclimate and culture, inoculating the harvested culture, repeating the cultivating until the proliferation speed of the recombinant avian influenza subtype virus is stable, wherein the virus content is larger than or equal to 10<7.5>EID[50]; and then inoculating the suspension cell; acquiring virus liquid while cultivating until the cytopathy achieves 75% or above, namely obtaining the recombinant avian influenza subtype virus. The chick embryo virus is firstly inoculated into the MDCK monolayer cell to acclimate and cultivate, and then is inoculated to the suspension MDCK cell, thereby effectively increasing the performance stability of the suspension virus obtained through the production, wherein the obtained suspension virus HA is larger than or equal to 1 to 1024, each 0.1ml virus content is larger than or equal to 10<8.0>EID[50], and each 1ml virus content is larger than or equal to 10<8.0>TCID[50].

Description

technical field [0001] The invention relates to the field of preparation of avian influenza virus vaccines, in particular to a method for culturing recombinant avian influenza subtype viruses in full suspension cells. Background technique [0002] Avian influenza (AI) is a severe infectious disease of poultry caused by type A avian influenza virus. At present, vaccine immunization is the main measure to prevent and control avian influenza outbreaks at home and abroad. At present, most manufacturers in my country use chicken embryos as the substrate to produce avian influenza vaccines. This process has a great dependence on chicken embryos; the virus liquid titer produced Unstable; the conditions in the cultivation process are not uniform enough, and there are large differences between batches; chicken embryos need to consume a large number of healthy chicken embryos for the production of influenza vaccines, and chicken embryos have potential contamination, and the cultivation...

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): C12N5/071C12N7/00C12N1/36
CPCC12N1/36C12N5/0686C12N7/00C12N2760/16151
Inventor 李莉赵海源曾显营陈化兰田国彬陈宏冯玉强王玉红朱长动蒋晓梅张天舒杜鑫张丽娜高晓庆刘金伟曾晓敏王仁君王琳雅
Owner 吉林冠界生物技术有限公司
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