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Preparation method of avian infectious bursal disease virus live vaccine

A chicken infectious bursal virus technology, applied in the field of preparation of chicken infectious bursal virus live vaccine, can solve the problem of increasing foreign virus, mycoplasma pollution, increasing the difficulty of downstream separation and purification, and difficult to control product quality, etc. problems, achieve good immune protection effect, improve equipment utilization, and expand production scale

Active Publication Date: 2019-06-21
哈药集团生物疫苗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Most of the traditional DF-1 culture adopts the method of serum adherence, which is complicated in process, large in area, requires a lot of manual operation and needs to add serum during the culture process, which increases the possibility of contamination by exogenous viruses, mycoplasma, etc. In addition, the difference in serum between different batches will also make it difficult to control the quality of the product, and also increase the difficulty of downstream separation and purification

Method used

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  • Preparation method of avian infectious bursal disease virus live vaccine
  • Preparation method of avian infectious bursal disease virus live vaccine
  • Preparation method of avian infectious bursal disease virus live vaccine

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Acclimatization of DF-1 cells adapted to the serum-free medium and growing in a suspension state The DF-1 cells were acclimatized by gradually lowering the serum to adapt to the culture at low concentration of serum. When DF-1 cells growing stably in MEM medium containing 10% newborn bovine serum grow to the mid-logarithmic growth phase, replace them with MEM medium containing 5% newborn bovine serum until the cells grow to 80%-90% At confluence, trypsinize the cells to a cell density of 2.0 x 10 5 The cells / ml were subcultured in MEM medium containing 5% newborn bovine serum. After several generations, the survival rate of DF-1 cells in the MEM medium containing 5% newborn bovine serum was maintained at more than 90%, and maintained a relatively fast growth rate, which was used to further reduce serum acclimation culture. In the same way, DF-1 cells were gradually adapted to the culture condition of MEM containing 1% newborn bovine serum. The DF-1 cells ada...

Embodiment 2

[0026] The preparation of embodiment 2 chicken infectious bursal virus live vaccines

[0027] (1) The DF-1-XF cells obtained in Example 1 were domesticated and cultured according to 0.5×10 6 cells / ml is the initial inoculation density of the cells and inoculated them into a 7L bioreactor for cell suspension culture, and the culture time is 36 hours; other suspension culture conditions are as follows: pH control is 7.2, dissolved oxygen (DO) is controlled at 50%, and temperature is controlled at 37°C, the stirring speed is controlled at 100r / min;

[0028] (2) Inoculate chicken infectious bursal virus H11 strain (preservation number is CGMCC NO.6910) into DF-1-XF cells cultured in suspension for 36 hours according to the inoculation dose of 5% (v / V); carry out virus propagation Cultivate; harvest virus liquid after inoculation 36 hours; other suspension culture conditions are as follows: pH control is 7.2, dissolved oxygen (DO) control is 50%, temperature control is 37 ℃, stirr...

Embodiment 3

[0030] The preparation of embodiment 3 chicken infectious bursal virus live vaccines

[0031] (1) The DF-1-XF cells obtained in Example 1 were domesticated and cultured according to 0.3×10 6 cells / ml is the initial inoculation density of the cells and inoculate them into a 7L bioreactor for cell suspension proliferation culture; other suspension culture conditions are as follows: pH control is 7.2, dissolved oxygen (DO) is controlled at 50%, temperature is controlled at 37°C, stirring speed The control is 60r / min;

[0032] (2) Inoculate chicken infectious bursal virus H11 strain (preservation number is CGMCC NO.6910) to the DF-1-XF cells that have been cultured in suspension for 48 hours according to the inoculation dose of 1% to carry out virus propagation and culture; inoculate for 12 hours After harvesting the virus liquid; after testing, the virus price is 10 5.45 TCID 50 / ml;

[0033] (3) Mix the lyoprotectant and the virus liquid according to the volume ratio of 1:1,...

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Abstract

The invention discloses a preparation method of an avian infectious bursal disease virus live vaccine. Primary chicken embryonic fibroblasts are domesticated to obtain continuous chicken embryonic fibroblasts adaptive to serum-free culture media and in single-cell suspension growth state. The invention further provides a method of preparing the avian infectious bursal disease virus live vaccine. The method is characterized by comprising (1), inoculating the continuous chicken embryonic fibroblasts adaptive to serum-free culture media and in single-cell suspension growth state to a bioreactor so as to carry out suspension enrichment culture of cells; (2) inoculating IBDV (infectious bursal disease virus) to the cells that are subjected to suspension enrichment culture, carrying out viral enrichment culture, and colleting a viral liquid; (3) mixing the viral liquid and a lyophilization protectant, and lyophilizing. The preparation method has the advantages that pollution risk and production cost are decreased, vaccine production time is shortened effectively, and production scale can be enlarged; the prepared avian infectious bursal disease virus live vaccine has good safety and goodimmunoprotection effect.

Description

technical field [0001] The invention relates to a preparation method of a chicken infectious bursal virus live vaccine, in particular to a method for preparing a chicken infectious bursal virus live vaccine by using a serum-free suspension culture technique, and belongs to the field of preparation of a chicken infectious bursal virus live vaccine . Background technique [0002] Infectious bursal disease (IBD) is an acute, highly contagious, lympholytic infectious disease of chickens and turkeys caused by chicken infectious bursal virus (IBDV). , mainly infects young chickens aged 3 to 6 weeks. The virus mainly infects immature B lymphocytes or B lymphoid precursor cells in the chick's bursa, causing severe and long-term immunosuppression in chickens, reducing the body's ability to respond to vaccines, causing immune failure, or causing other viruses Secondary infectivity of sexual or bacterial diseases, thereby increasing the death rate of chicken flocks. There is still n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/02C12N7/00C12N7/02A61K39/12A61P31/14C12R1/91
CPCY02A50/30
Inventor 刘鑫莹杨末赵晓春柴华赵刚李建华李应鹤陈琳王宁曲海波
Owner 哈药集团生物疫苗有限公司
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