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Production method of porcine foot-and-mouth disease type O genetically engineered composite epitope protein vaccine

A genetic engineering, swine foot-and-mouth disease technology, applied in the field of immunization, can solve the problems of small unit output, high production cost, unsuitable for industrial production, etc., and achieve the effects of short production cycle, fewer personnel, and less reagent consumption.

Pending Publication Date: 2019-01-25
天津威特生物医药有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] This technology is a method of laboratory preparation, with high production cost and small unit output, and is not suitable for industrial production.

Method used

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  • Production method of porcine foot-and-mouth disease type O genetically engineered composite epitope protein vaccine
  • Production method of porcine foot-and-mouth disease type O genetically engineered composite epitope protein vaccine
  • Production method of porcine foot-and-mouth disease type O genetically engineered composite epitope protein vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Engineering bacteria culture and induced expression:

[0034] Take 500 mL of engineered bacteria seed liquid cultivated overnight and inoculate it into 50 L of LB culture liquid (containing 75 μg / mL kanamycin), ferment at 37 ° C, pH 7.2, dissolved oxygen 40%, and add it by variable index flow feeding method; after 5 hours of fermentation, IPTG was added to a final concentration of 1.0 mmol / L, and the expression was induced for 5 hours to obtain the recombinant protein after induction.

[0035] Bacteria collection and crushing:

[0036] The fermented broth collected was concentrated using a 750KD hollow fiber column, and stopped when it reached 5L. Using a high-pressure homogenizer, the concentrated solution is crushed under the conditions of 900 bar and two cycles.

[0037] Inclusion body washing and denaturation:

[0038] Use a 750KD hollow fiber column to wash the broken fermentation broth. The buffer is 1×IBWashBuffer (EDTA 10mmol / L; Tris-HCl 20mmol / L, pH7.5; Trit...

Embodiment 2

[0044] Engineering bacteria culture and induced expression:

[0045] Take 400mL of engineered bacteria seed solution cultivated overnight and inoculate it into 50L of LB culture solution (containing 75μg / mL kanamycin), ferment at 37°C, pH7. feeding method; after 5 hours of fermentation, IPTG was added to a final concentration of 1.0 mmol / L, and the expression was induced for 5 hours to obtain the recombinant protein after induction.

[0046] Bacteria collection and crushing:

[0047] The fermented broth collected was concentrated using a 750KD hollow fiber column, and stopped when it reached 5L. Using a high-pressure homogenizer, the concentrated solution is crushed under the conditions of 900 bar and two cycles.

[0048] Inclusion body washing and denaturation:

[0049] Use a 750KD hollow fiber column to wash the broken fermentation broth. The buffer is 1×IBWashBuffer (EDTA 10mmol / L; Tris-HCl 20mmol / L, pH7.5; TritonX-1001%) in an ice bath, and equal volume replacement 5 t...

Embodiment 3

[0055] Engineering bacteria culture and induced expression:

[0056] Take 300mL of engineered bacteria seed solution cultivated overnight and inoculate it into 50L of LB culture solution (containing 75μg / mL kanamycin), ferment at 37°C, pH7. feeding method; after 5 hours of fermentation, IPTG was added to a final concentration of 1.0 mmol / L, and the expression was induced for 5 hours to obtain the recombinant protein after induction.

[0057] Bacteria collection and crushing:

[0058] The fermented broth collected was concentrated using a 750KD hollow fiber column, and stopped when it reached 5L. Using a high-pressure homogenizer, the concentrated solution is crushed under the conditions of 900 bar and two cycles.

[0059] Inclusion body washing and denaturation:

[0060] Use a 750KD hollow fiber column to wash the broken fermentation broth. The buffer is 1×IBWashBuffer (EDTA 10mmol / L; Tris-HCl 20mmol / L, pH7.5; TritonX-1001%) in an ice bath, and equal volume replacement 5 t...

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Abstract

The invention relates to an industrial production method of porcine foot-and-mouth disease O-type genetic engineering composite epitope protein vaccine, comprising the following steps in sequence: (1)engineering bacteria fermentation and induced expression; (2) collection and fragmentation of bacteria; (3) inclusion body washing and denaturing dissolution; (4) protein purification; (5) Renaturation of purified protein; and (6) vaccine emulsification. The invention has the advantages that: the product quality: the vaccine is sterile, the purity of the target protein is above 95%, the effectiveantigen content is above 1.0 mg / mL, the indirect hemagglutination inhibition titer is above 1:211, the endotoxin is below 31.25 EU / mL. And for the production cost and efficiency, less people are used, the consumption of reagents is less, and the production cycle is shorter.

Description

technical field [0001] The invention belongs to the technical field of immunization, and in particular relates to a method for producing a porcine foot-and-mouth disease O-type genetic engineering composite epitope protein. Background technique [0002] Foot-and-Mouth Disease (FMD) is a severe infectious disease of cloven-hoofed animals caused by Foot-and-Mouth Disease Virus (FMDV), and is a severe infectious disease that must be reported by the World Organization for Animal Health (OIE). Rank it at the top of a class of infectious diseases. There are seven serotypes of foot-and-mouth disease virus, which are O, A, C, AsiaⅠ, SAT1, SAT2, and SAT3. It has been found that O, A and AsiaⅠ are prevalent in my country, and O-type foot-and-mouth disease is currently the most serious serotype. The epidemic has continued year after year, causing serious economic losses. At present, there are three lineages of O-type foot-and-mouth disease epidemic virus in my country: Myanmar 98 (Mya...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K14/09C07K1/14A61K39/135A61P31/14
CPCA61K39/12A61P31/14C07K14/005C12N2770/32122C12N2770/32134C12N2770/32151
Inventor 宗惠刘斌刘杰康文娟邹慧芳岳晓蓉秦天达陈晓宇李金杰牟克斌黄银君温建波魏徵邢向茹
Owner 天津威特生物医药有限责任公司
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