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Demulsification method of recombinant protein oil emulsion subunit vaccine

A technology of subunit vaccines and recombinant antigenic proteins, which is applied in the direction of vaccines, veterinary vaccines, biochemical equipment and methods, etc., can solve the problems that the positive control of the test product cannot be established, the range value of the endotoxin content cannot be detected, etc., and achieve The method is simple, the effect is good, and the antigen recovery rate is high

Inactive Publication Date: 2020-06-05
SHANDONG SINDER TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the addition of organic solvent chloroform to the oil emulsion vaccine in the process of demulsification, this method causes residual organic solvent chloroform in the water phase separated out after demulsification, which will lead to the detection of endotoxin content in the water phase. The positive control of the test product was not established, and the range value of the endotoxin content in the water phase after demulsification could not be detected

Method used

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  • Demulsification method of recombinant protein oil emulsion subunit vaccine
  • Demulsification method of recombinant protein oil emulsion subunit vaccine
  • Demulsification method of recombinant protein oil emulsion subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Establishment of Demulsification Parameters of Ultrasonic Demulsification Demulsification Method

[0017] 1.1 Establishment of Ultrasonic Demulsification Parameters Take 10ml of genetically engineered subunit vaccine for chicken infectious bursal disease, and use the following three conditions for demulsification: 1000 joules, ultrasound for 4 seconds, stop for 4 seconds, a total of 40 cycles; 1300 Joule, ultrasonic for 4 seconds, stop for 4 seconds, a total of 40 cycles; 1500 joules, ultrasonic for 4 seconds, stop for 4 seconds, a total of 40 cycles; after the ultrasonic, centrifuge at 8000 rpm / min for 10 min at room temperature to obtain the aqueous phase.

[0018] 1.2 Theoretical value of the water phase of the vaccine after demulsification The ratio of the water phase to the oil phase is 1:2 when the chicken infectious bursal disease genetically engineered subunit vaccine is emulsified, so the theoretical value of the water phase after demulsification shoul...

Embodiment 2

[0022] Example 2 Preparation of Chicken Infectious Bursal Disease Genetic Engineering Subunit Vaccine and Its Emulsification Method

[0023] 2.1 Preparation of Chicken Infectious Bursal Disease Virus VP2 Protein The recombinant Escherichia coli was cultured and induced to express in a fermenter. After the fermentation, the collected wet bacteria were resuspended with an appropriate amount of PBS solution, and used at 4°C Ultrasonic crushing. The crushed bacterial liquid was centrifuged at 12000r / min for 15 minutes, and the supernatant was collected. Protein purification by neutral salt salting out method. Triton X-114 and allantoin were used to remove endotoxin to obtain VP2 protein solution.

[0024] 2.2 Detection of VP2 protein content The agar diffusion test was performed on the supernatant after breaking the bacteria, and the AGP titer of the expression product VP2 in the supernatant was recorded. It is 1:32.

[0025] 2.3 Inactivation Put the VP2 protein solution in a ...

Embodiment 3

[0053] Example 3: Using two demulsification methods to detect water phase endotoxin content after oil emulsion vaccine demulsification

[0054] 3.1 Chloroform demulsification method Mix the tested IBD genetically engineered subunit oil emulsion vaccine with chloroform in equal amounts, place in a micro-vortex shaker for 6 minutes, then centrifuge at 8000rpm for 5 minutes, collect the precipitated The aqueous phase was tested for endotoxin. Prepare solutions A, B, C, and D according to Table 2, and use the gel limit test method for detection.

[0055] Table 3: Preparation table of gel semi-quantitative test solution

[0056]

[0057]

[0058] Judgment of results: When the dilution times of the positive control solution of the test product of B are 10 and 20, the result of the positive control solution of the test product is negative, indicating that the organic solvent has an impact on the detection of endotoxin, and the test is not established.

[0059] 3.2 Ultrasonic ...

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Abstract

The invention provides a demulsification method of a recombinant antigen protein subunit vaccine. The demulsification method comprises the following steps: carrying out ultrasonic demulsification on asubunit vaccine using an oil emulsion as an adjuvant in an ice bath; wherein one specific condition of ultrasonic demulsification is as follows: 1000-2500 joules, ultrasonic treatment is performed for 2-5 seconds, and is stopped for 2-5 seconds, 30-45 cycles are performed in total, and after ultrasonic treatment is finished, and performing centrifugation at room temperature to obtain a water phase. The method disclosed by the invention is simple, convenient and rapid, and can be completed within 10 minutes; the antigen separation effect is good, and meanwhile, the stability of the bursal disease virus VP2 protein can be ensured; the content of the VP2 antigen in a water phase separated after demulsification can be detected by adopting an agar diffusion test method; and the recovery rate of the antigen in the demulsified water phase is high and reaches 40%. The method is suitable for demulsifying chicken infectious bursal disease genetic engineering subunit vaccines and other genetic engineering subunit vaccines emulsified by a marcol52 adjuvant, and then the endotoxin content of a water phase is detected, so that the endotoxin content of a finished vaccine product is evaluated.

Description

technical field [0001] The invention belongs to the technical field of vaccine quality detection, in particular to a demulsification method of genetically engineered subunit oil emulsion inactivated vaccines. Background technique [0002] Infectious bursal disease has a high incidence and a short course of disease, and spreads rapidly. Sick chickens suffer from severe diarrhea, extreme weakness and death in varying degrees. Infection of chicks can also lead to immunosuppression, and can induce various diseases or lead to vaccine immunity failure. All breeds of chickens can be infected, and the incubation period is 2 to 3 days. It mainly occurs in chickens aged 2 to 15 weeks, with the peak period of onset at 3 to 6 weeks of age. In recent years, the age of onset of the disease has a tendency to widen, and this disease occurs in chickens from 10 days of age to laying eggs. disease reports. At the beginning of the typical onset flock disease, individual chickens suddenly beco...

Claims

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Application Information

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IPC IPC(8): B01D17/04A61K39/12A61P31/14
CPCA61K39/12A61K2039/5252A61K2039/552A61P31/14B01D17/04C12N2720/10034
Inventor 单学强张伦于泽坤栾志舫只勇郝光恩杨海明段笑笑
Owner SHANDONG SINDER TECH
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