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High-expression fermentation process of avian bursa virus VP2 protein

A fermentation process and high expression technology, applied in fermentation, virus, viral peptides and other directions, can solve the problems of difficult to achieve industrial production application, unsatisfactory VP2 protein expression, and purification that consumes a lot of manpower, material and financial resources, and achieves good commercial development. Value, improve market competitiveness, shorten the effect of production cycle

Inactive Publication Date: 2019-01-08
HENAN ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the VP2 protein expression of engineering bacteria in the existing research is not satisfactory, and purification and concentration require a lot of manpower, material resources, and financial resources, making it difficult to achieve industrial production applications

Method used

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  • High-expression fermentation process of avian bursa virus VP2 protein
  • High-expression fermentation process of avian bursa virus VP2 protein
  • High-expression fermentation process of avian bursa virus VP2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Construction test of genetically engineered bacteria

[0045] Referring to the Chinese patent document CN 106148358 A, the genetically engineered bacteria E.coli BL21(DE3)PlysS / pET-28a-IBDV-VP2 was constructed and stored in a glycerol tube at -80°C.

Embodiment 2

[0046] Embodiment 2: 10L fermentation test of genetically engineered bacteria

[0047] (1) Activation of strains

[0048] The genetically engineered bacteria preserved in the -80°C glycerol tube in Example 1 with an inoculation loop E. coli The BL21(DE3)PlysS / pET-28a-IBDV-VP2 strain was inoculated by streaking on the surface of LB solid medium containing kanamycin, and then the plate was cultured upside down in a constant temperature incubator at 37°C overnight.

[0049] (2) Seed cultivation

[0050] 1. Inoculate the activated bacterial classification into 50mL of primary seed medium, shake and cultivate at 37°C for 14 hours to obtain primary seed liquid;

[0051] Among them, the primary seed medium is LB medium, and its components are: 10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, sodium hydroxide solution or dilute hydrochloric acid to adjust the pH value to 7.0. 121 Sterilize at ℃ for 20 minutes, and add kanamycin at a concentration of 100ug / mL before inocula...

Embodiment 3

[0067] Embodiment 3: 100L fermentation test of genetically engineered bacteria

[0068] (1) Activation of strains

[0069] The genetically engineered bacteria preserved in the -80°C glycerol tube in Example 1 with an inoculation loop E. coli The BL21(DE3)PlysS / pET-28a-IBDV-VP2 strain was inoculated by streaking on the surface of LB solid medium containing kanamycin, and then the plate was cultured upside down in a constant temperature incubator at 37°C overnight.

[0070] (2) Seed cultivation

[0071] 1. Inoculate the activated bacterial classification into 500mL of primary seed culture medium, shake and cultivate at 37°C for 14 hours to obtain primary seed liquid;

[0072] Among them, the primary seed medium is LB medium, and its components are: 10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, and sodium hydroxide solution to adjust the pH value to 7.0. Sterilize at 121°C 20min, add kanamycin at a concentration of 100ug / mL before inoculation.

[0073] II. Transfe...

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Abstract

The invention discloses a high-expression fermentation process of avian bursa virus VP2 protein and aims to solve the technical problem that the expression quantity of the engineering bacterium avianbursa virus VP2 protein is low. The fermentation process comprises the following steps: (1) activating culture; (2) performing seed cultivation; and (3) fermenting supplementary materials in batches:I, a cell rapid reproduction stage: preparing a basic culture medium, putting into a fermentation tank, inoculating secondary seed liquid into a fermentation tank according to the inoculation quantityof 4 to 6 percent, performing fermentation and starting to supplement materials when the dissolved oxygen quantity is increased sharply; and II, a protein expression stage: adding an inducing agent with the final concentration of 0.5 mmol / L, adding a supplementary material culture medium II and continuously performing fermentation until ending. According to the process method, the protein expression quantity is high, the production period can be shortened, the equipment investment can be reduced, the production cost can be reduced, the production efficiency can be improved, the competitiveness of products on the market can be greatly improved, and basis is laid for development and production of IBDV gene engineering subunit vaccine.

Description

technical field [0001] The invention relates to the technical field of fermentation engineering, in particular to a high-expression fermentation process of avian bursal virus VP2 protein. Background technique [0002] Poultry bursal disease (Infectious Bursal Disease, IBD) is an acute, highly contagious infectious disease that mainly infects the immune organ of chicks, the bursa, causing immunosuppression and even death. huge impact on the poultry industry. [0003] Avian bursal disease is caused by infectious bursal disease virus (IBDV). After infection, the virus quickly invades the bursal and proliferates in the bursal, making the B lymphocytes in the bursal Cells lyse, die, and eventually produce severe immunosuppression. Long-term immunosuppression makes chickens susceptible to infection by other pathogens, and the body's immune response to vaccines is reduced, and protective immune responses cannot be induced normally, resulting in immune failure. The VP2 protein of...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K14/08C12R1/19
CPCC07K14/005C12N2720/10051C12P21/02
Inventor 张改平刘运超邓瑞广邢广旭陈玉梅刘东民魏蔷冯华周景明刘艳凯赵孟孟
Owner HENAN ACAD OF AGRI SCI
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