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Infectious bursal virus vero cell-adapted strain of chicken and its application

A technology of chicken infectivity and bursa, which is applied in the direction of antiviral agents, virus/bacteriophage, virus antigen components, etc., can solve the problems of inability to carry out large-scale preparation, low immune protection efficiency, low virus titer, etc., and achieve protection Effects of inoculated animals, long duration, high virus titer

Inactive Publication Date: 2016-05-04
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the virus titer of IBDV vaccine produced in China is low, and the immune protection efficiency is low; in the production method, the production method of inoculating IBDV in a spinner bottle is still used, and the culture volume is about 0.5L, which cannot be prepared on a large scale

Method used

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  • Infectious bursal virus vero cell-adapted strain of chicken and its application
  • Infectious bursal virus vero cell-adapted strain of chicken and its application
  • Infectious bursal virus vero cell-adapted strain of chicken and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1I

[0026] Example 1 Obtaining and Characterization of IBDV Virus

[0027] Using the suspected chicken infectious bursal disease feed isolated from the diseased chicken flocks in Jiangsu, my country, inoculating the specific Pathogen Free (SPF) chicken embryo isolated virus, the virus strain is propagated on the SPF chicken embryo, and the chicken embryo Plaque purification is performed on fibroblasts, and the virus NJ strain is finally obtained through purification and screening of different plaques.

[0028] The virus NJ strain was identified as follows:

[0029] (1) Amplification of virus NJ strain: Dilute the virus NJ strain with sterile PBS buffer 10 3 After doubling, 10-11-day-old SPF chicken embryos were inoculated by the chorioallantoic membrane route, the inoculation volume was 0.2mL / embryo, cultured at 37℃, and the allantoic fluid of chicken embryos was harvested within 36-48h.

[0030] (2) EID of the virus 50 :Dilute the chicken embryo allantoic fluid obtained in step (1) with s...

Embodiment 2

[0034] Example 2 Screening of Vero Cell Adapted Strains of Chicken Infectious Bursal Bursal Virus

[0035] In this implementation, the chicken infectious bursal bursal virus NJ strain was propagated on Vero cells cultured without serum, and a strain of IBDV virus NJ-23 with high virus titer and stable genetic characteristics was screened by the limiting dilution method.

[0036] specific method:

[0037] (1) Vero cells were cultured with IVT medium (serum-free medium, purchased from Gibico) to 95% confluence, the Vero cells were digested with 0.1% trypsin and centrifuged at 1000 rpm, and the cells were resuspended in fresh IVT medium . Count the cells according to 8×10 5 Cell / well spreads 96-well plates.

[0038] (2) Screening: Dilute the NJ strain virus liquid with IVT medium supplemented with a final concentration of 0.25~2.5% (mass percentage concentration) whey protein hydrolysate (Sigma), and use 5 dilutions (10 -4 ~10 -8 ) Virus infection step (1) Spread a single layer of Vero ...

Embodiment 3

[0049] Example 3 Cultivation of IBDV virus NJ-23 strain virus liquid on Vero cells cultured in serum-free microcarrier suspension

[0050] The method of culturing the virus solution of IBDV virus NJ-23 strain on Vero cells cultured in serum-free microcarrier suspension is as follows:

[0051] (1) Serum-free culture of Vero cells: Cultivate Vero cells in IVT medium. When the cells reach 95% confluence, digest with 0.1% trypsin and centrifuge at 1000 rpm. Resuspend the cells in fresh IVT medium to remove the cells. Transfer to a new culture flask according to a certain proportion, and continue to culture until the cells reach 95% confluence to obtain Vero cells.

[0052] (2) Initial Vero cell suspension culture in serum-free microcarriers: Add whey protein hydrolysate at a final concentration of 0.25% (mass percentage) to the IVT medium to obtain a virus maintenance solution with a pH of 7.1. The Vero cells obtained in step (1) were digested with 0.1% trypsin, centrifuged, and resuspe...

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Abstract

The invention provides an infectious bursal disease virus Vero cell-adapted strain and belongs to the field of bioengineering. The related infectious bursal disease virus Vero cell-adapted strain is named Ck / Jiangsu / NJ-23 / 2008 and has a collection No. of CGMCCNO.8852. The infectious bursal disease virus Vero cell-adapted strain which can efficiently multiply on serum-free cultured Vero cell is finally obtained through wild strain separation, chick embryo passage, Vero cell passage adaption; the infectious bursal disease virus Vero cell-adapted strain is subjected to continuous passage culture on the serum-free cultured Vero cell and TCID50 can be kept to be higher than 108.5 / mL. Virus culture solution is inactivated and prepared into oil emulsion; after the prepared oil emulsion is used to immunize chicken, detection proves that the prepared oil emulsion has good immunogenicity. The infectious bursal disease virus (IBDV) strain and the production process thereof are simple, safe and efficient and suitable for industrial culture.

Description

Technical field [0001] The invention relates to the field of bioengineering, in particular to a Vero cell adapted strain of chicken infectious bursal bursal virus and its application. Background technique [0002] Infectious Bursal Disease (IBD) is a highly contagious infectious disease of chickens caused by infectious bursal virus (IBDV). It first occurred in the Gamboro area of ​​Delaware, USA, and mainly infected 3~ In 6-week-old young chickens, the virus multiplies rapidly in the lymphocytes of the bursa of fabric, and damages the B lymphocytes of the bursa of fabric, causing severe immunosuppression. After 1980, the disease was introduced to our country, and a large-scale outbreak and continued epidemic have seriously affected the development of my country's chicken industry. Currently, vaccination is commonly used to prevent the disease. [0003] At present, the domestically produced IBDV vaccine usually uses chicken embryo fibroblasts to grow the virus. Chicken embryo fibr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K39/12A61P31/14C12R1/93
Inventor 吴培培冯磊禇轩唐应华侯继波
Owner JIANGSU ACAD OF AGRI SCI