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A method for large-scale production of high-purity porcine pseudorabies virus

A porcine pseudorabies virus and high-purity technology, which is applied in the field of large-scale production of high-purity porcine pseudorabies virus, can solve the problems of increased residual concentration of harmful substances, increased effective antigen content of vaccines, safety of vaccine products, etc. Effects of vaccine side reactions, reduction of antigen loss, and good promotion prospects

Active Publication Date: 2020-07-28
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purification process using the traditional inactivated whole virus as the antigen is mainly based on natural sedimentation, centrifugation, membrane bag concentration and other processes. The effective antigen content in the produced vaccine increases, and the concentration of harmful substance residues also increases, resulting in the safety of vaccine products. Sexual hazards

Method used

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  • A method for large-scale production of high-purity porcine pseudorabies virus
  • A method for large-scale production of high-purity porcine pseudorabies virus
  • A method for large-scale production of high-purity porcine pseudorabies virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Hollow fiber column clarification process

[0044] 1 system pretreatment

[0045] 1.1 Install the 0.2μm, 0.45μm, and 0.65μm hollow fiber columns into the hollow fiber column control equipment respectively, connect the corresponding pipelines, and soak the hollow fiber columns with sterile injection water for 10 minutes after assembly.

[0046] 1.2 System Integrity Detection

[0047] The pressure hold method checks the integrity of the system.

[0048] 1.3 System processing

[0049] Cleaning and sterilization: Use sterile 0.5mol / L NaOH solution to sterilize the system for 30 minutes, then clean the system with sterile water for injection to remove residual alkali solution until the pH is 7.0;

[0050] 1.4 Detection of water flux

[0051] Use sterile water for injection to pass through at the specified pump speed, and calculate the water flux of the hollow fiber column at the corresponding temperature.

[0052] 1.5 hollow fiber column balance

[0053] Equilibrate...

Embodiment 2

[0061] 1. Hollow fiber column ultrafiltration concentration process

[0062] 1 system pretreatment

[0063] 1.1 Install the 300KD, 500KD, and 750KD hollow fiber columns into the hollow fiber column control equipment respectively, and connect the phase

[0064] Appropriate pipelines, after assembly, soak the hollow fiber column with sterile water for injection circulation for 10 minutes.

[0065] 1.2 System Integrity Detection

[0066] The pressure hold method checks the integrity of the system.

[0067] 1.3 System processing

[0068] Cleaning and sterilization: Use sterile 0.5mol / L NaOH solution to sterilize the system for 30 minutes. Then clean the system with sterile water for injection to wash away the residual alkali solution until the pH is 7.0.

[0069] 1.4 Detection of water flux

[0070] Use sterile water for injection to pass through at the specified pump speed, and calculate the water flux of the hollow fiber column at the corresponding temperature.

[0071] 1...

Embodiment 3

[0078] Embodiment 3 molecular sieve gel chromatography process

[0079] 1 system pretreatment

[0080] 1.1 Install the 4FF molecular sieve prepacked column in place and measure the column efficiency.

[0081] 1.2 Treat 2 column volumes (CV) of the molecular sieve gel chromatography column with sterile 0.5mol / L NaOH, then wash with sterile injection water to pH 7.0, and then equilibrate the ion exchange column with 0.01mol / L PBS solution to reach the conductivity The column rate is consistent before and after the column (the conductivity before and after the column is 15.69ms / cm), the pH is stable at 7.0, and the UV 280 The baseline is stable.

[0082] 2 Purification process

[0083] Load the concentrated virus sample, set the linear flow rate of the sample to 30cm / h, and control the pressure to less than 2.0bar. The loading volume is 5%, 10%, and 15% of the column volume. After loading the sample, start to elute the protein with 0.01mol / L PBS solution, and wait until the U...

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Abstract

The invention belongs to the technical field of vaccines and in particular relates to a method for large-scale production of a high-purity porcine pseudorabies virus. The method comprises processes of continuous flow centrifugation, hollow fiber clarification filtering, ultrafiltration and concentration, and molecular sieve purification. The virus recycling rate is increased to the maximum extent, and the content of impurity proteins is reduced. A porcine pseudorabies virus concentrated solution and purified antigen produced by using the method are particularly applicable to vaccine preparation, and compared with a porcine pseudorabies virus inactivated vaccine produced according to the prior art, the high-purity porcine pseudorabies virus is relatively high in safety, relatively high in uniformity and relatively good in immune effect, and side reactions of vaccines are fundamentally reduced.

Description

technical field [0001] The invention belongs to the technical field of vaccines, in particular to a method for large-scale production of high-purity porcine pseudorabies virus. Background technique [0002] Pseudorabies (Porcine Pseudorabies) is an acute infectious disease caused by Pseudorabies Virus (PRV) and occurs in a variety of domestic animals and wild animals. The main symptoms are fever, itching, and encephalomyelitis. Pig is the only natural host of pseudorabies, which is very harmful to it. It can cause abortion in pregnant sows, stillbirth and fetal mummification. For newborn piglets, it causes neurological symptoms, ataxia, paralysis, exhaustion and death, and the case fatality rate is 100%. Adult pigs are mostly recessively infected. Sick pigs, infected pigs and infected rodents are the main sources of infection for this disease. Pigs are not only the primary infected host of the disease, but also the long-term storage and detoxification of the virus, which...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00
CPCC12N7/00C12N2710/16751
Inventor 范金秀周明光徐高原陈波陈关平
Owner WUHAN KEQIAN BIOLOGY CO LTD
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