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Serum-free full-suspension culture method of avian metapneumovirus and application of serum-free full-suspension culture method in vaccines

An avian metapneumovirus and serum-free culture medium technology, which is applied in the biological field, can solve the problems of difficulty in preparing avian metapneumovirus vaccines, cannot fully satisfy the avian metapneumovirus, secondary infection with bacteria, etc., and achieves the goal of reducing virus spread. Risks, components are clear and controllable, and the effect of saving operating costs

Inactive Publication Date: 2020-11-20
WENS FOOD GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Avian metapneumovirus is highly contagious and spreads quickly, which can cause immunosuppressive reactions and secondary bacterial infection, causing huge losses to poultry
At present, avian metapneumovirus is mainly isolated, cultured and identified through SPF chickens or chicken embryos. There are also reports of using Vero cells to study virus in vitro cell culture, but it cannot fully meet the needs of large-scale and low-cost culture of avian metapneumovirus.
In addition, in the prior art, the titer of the virus solution obtained by culturing the avian metapneumovirus is low, which has caused great difficulties in the preparation of the avian metapneumovirus vaccine

Method used

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  • Serum-free full-suspension culture method of avian metapneumovirus and application of serum-free full-suspension culture method in vaccines
  • Serum-free full-suspension culture method of avian metapneumovirus and application of serum-free full-suspension culture method in vaccines

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Experimental program
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Effect test

Embodiment 1

[0036] A serum-free full suspension culture method of avian metapneumovirus, comprising the following steps:

[0037] Step 1. Strain adaptation: the avian metapneumovirus strain was continuously passed on to the suspension BHK-21 cell line for 15 generations, and the TCID was measured using the adherent BHK-21 cells 50 , with a poison price of 10 7.33 TCID 50 / mL, as the basic strain;

[0038] Step 2, seed cell preparation: Suspended BHK-21 cells were cultured in Erlenmeyer shaker flasks with serum-free medium after resuscitation, and the cell density was kept at 5.0×10 5 ~1.0×10 7 cells / mL; the culture conditions are shaker parameter temperature of 37°C, carbon dioxide concentration of 5%, and speed of 100r / min;

[0039] Step 3, expand host cells: add serum-free culture medium to the sterile bioreactor, set the culture conditions as temperature 37°C, pH 7.2, DO 60%, stirring speed 70r / min, and then the seed cells prepared in step 2 Inoculate into the bioreactor to keep t...

Embodiment 2

[0047] A serum-free full suspension culture method of avian metapneumovirus, comprising the following steps:

[0048] Step 1. Strain adaptation: Continuously pass the avian metapneumovirus strain in the suspension BHK-21 cell line for 16 generations, and use the adherent BHK-21 cells to measure TCID 50 , the poison price is 10 7.33 TCID 50 / mL, as the basic strain;

[0049] Step 2, seed cell preparation: Suspended BHK-21 cells were cultured in Erlenmeyer shaker flasks with serum-free medium after resuscitation, and the cell density was kept at 5.0×10 5 ~1.0×10 7 cells / mL; the culture conditions are shaker parameter temperature of 37°C, carbon dioxide concentration of 5%, and speed of 110r / min;

[0050] Step 3. Expand host cells: add serum-free culture medium into the sterile bioreactor, set the culture conditions as temperature 37°C, pH 7.0, DO 40%, stirring speed 60r / min, inoculate the seed cells prepared in step 2 to the bioreactor, maintaining a cell density of 1.0 x 1...

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Abstract

The invention relates to a serum-free full-suspension culture method of avian metapneumovirus and an application of the serum-free full-suspension culture method in vaccines. The method comprises thefollowing steps of performing continuous passage of an avian metapneumovirus strain in a suspension BHK-21 cell line to obtain virus adaptive cells, and taking an adaptive strain with the highest virus content as a basic strain; resuscitating suspension BHK-21 cells, and culturing the resuscitated suspension BHK-21 cells in a triangular shake flask by using a serum-free culture solution; adding the serum-free culture solution into a bioreactor, and inoculating seed cells into the bioreactor; continuously culturing the basic strain of the suspension BHK-21 cells; performing glucose monitoring every day after virus inoculation, and when the content of glucose in the culture solution is 1-5 mmol / L, supplementing a nutrition bag; in 72 hours after virus inoculation, performing sampling and calculating the cell viability, and harvesting a virus culture solution when the cell viability is lower than 60%; and repeatedly freezing and thawing the virus solution, performing centrifugation and performing storage. The suspension BHK-21 cells are adopted for culture, the virus adaptability is good, and the virus content is high; the operation cost is low and the product quality is stable; and the method is easy to operate and has industrial large-scale production feasibility.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a serum-free full-suspension culture method of avian metapneumovirus and its application in vaccines. Background technique [0002] Avian metapneumonitis is an acute upper respiratory tract infection caused by avian metapneumovirus. It is one of the highly contagious diseases of poultry. Decreased egg rate, etc. Avian metapneumovirus was called avian pneumovirus in the early days. Because it can infect turkeys and cause rhinotracheitis, it is also called turkey rhinotracheitis virus. It belongs to Paramyxoviridae and Pneumoviridae subfamily. According to genetic differences and serological analysis, avian metapneumovirus can be divided into four subtypes: A, B, C and D. In 1998, it was first reported that avian metapneumovirus A was isolated from chickens with swollen head syndrome. In 2012, subtype B and subtype C were successively isolated. In recent years, there have...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N5/071A61K39/155A61P31/14C12R1/93
CPCA61K39/12A61K2039/552A61P31/14C12N5/0686C12N7/00C12N2500/90C12N2760/18334C12N2760/18351
Inventor 蒋天华周庆丰严专强温燕兰靖宇
Owner WENS FOOD GRP CO LTD
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