Method for large-scale production of porcine pseudorabies inactivated vaccine

A technology for porcine pseudorabies and porcine pseudorabies virus, which is applied in the field of vaccines, can solve the problems of increased residual concentration of harmful substances, large side reactions in vaccine products, and increased effective antigen content, so as to reduce the degree and probability of side reactions, and improve social Benefit and economic benefits, the effect of reducing the number of production times

Active Publication Date: 2017-10-20
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional spinner bottle culture antigen combined with conventional production process, the effective antigen content in the produced porcine pseudorabies virus inactivated vaccine increases, and the residual concentration of harmful substances also increases, resulting in large side effects and poor immune effect in vaccine products and other adverse effects

Method used

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  • Method for large-scale production of porcine pseudorabies inactivated vaccine
  • Method for large-scale production of porcine pseudorabies inactivated vaccine
  • Method for large-scale production of porcine pseudorabies inactivated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 large-scale production prepares porcine pseudorabies virus (XF-1 strain)

[0041] The concentration of 300L is 4.0~10.0×10 6 Each / ml BHK-21 cell suspension was inoculated in a 650L bioreactor, and 350L of BHK-21 medium containing 0.5% to 2% (v / v) newborn bovine serum was added, and the cell culture temperature was controlled at 36°C to 37°C. ℃, pH6.5~7.5, stirring speed 80rpm~150rpm, dissolved oxygen concentration 30%~60%, reactor parameter setting four-way gas, clean air, oxygen, nitrogen and CO 2 , according to the culture time, cell density and pH value, set different gas parameter ranges. After culturing for 24 hours, the cell count and viability were observed by trypan blue staining. After 2 days of suspension culture, new BHK-21 medium should be supplemented according to 5% (v / v) of the total culture volume in the bioreactor. The cells were cultured for 3 days, and the density reached 4.0~10.0×10 6 cells / ml to obtain BHK-21 cell suspension.

[00...

Embodiment 2

[0043] The purification of embodiment 2 porcine pseudorabies virus liquid

[0044] 1. Hollow fiber column clarification process

[0045] 1 system pretreatment

[0046] 1.1 Install the 0.45 μm hollow fiber column into the hollow fiber column control equipment, connect the corresponding pipeline, and infiltrate the hollow fiber column with sterile injection water for 30 minutes after assembly.

[0047] 1.2 System Integrity Detection

[0048] The pressure hold method checks the integrity of the system.

[0049] 1.3 System processing

[0050] 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;

[0051] 1.4 Detection of water flux

[0052] 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.

[0053] 1.5 ...

Embodiment 3

[0081] The preparation of embodiment 3 porcine pseudorabies inactivated vaccine

[0082] After purification of porcine pseudorabies virus (XF-1 strain), the antigenic virulence is at 10 8.0 TCID 50 / ml or more, add formaldehyde solution with a final concentration of 0.4% (v / v) to inactivate at 37°C for 48 hours, and store it at 4°C for use after passing the relevant inspection according to the appendix of the current "Chinese Veterinary Pharmacopoeia".

[0083] After the qualified porcine pseudorabies virus (XF-1 strain) is diluted according to the requirements of the vaccine, it is mixed with MONTANIDE respectively TM ISA 201 adjuvant was emulsified according to the mass ratio of 1:1 (g / g), the stirring speed was 500rpm / min, and the emulsification time was 30min. The antigen content in the final porcine pseudorabies inactivated vaccine is 10 6.0 TCID 50 / head portion, 10 7.0 TCID 50 / head portion and 10 7.5 TCID 50 / first portion, after the emulsification is complet...

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Abstract

The invention belongs to the technical field of vaccines and relates to a method for large-scale production of a porcine pseudorabies inactivated vaccine. The method comprises preparing a virus solution of porcine pseudorabies (XF-1 strain), carrying out continuous flow centrifugation, hollow fiber column clarification filtration, hollow fiber column ultrafiltration concentration and Sepharose 4FF molecular sieve gel chromatography purification treatment to obtain purified porcine pseudorabies viruses, adding a formaldehyde solution having a final concentration of 0.4% (v / v) into the purified porcine pseudorabies viruses, carrying out inactivation at 37 DEG C for 48h, and carrying out emulsification with a 201 adjuvant to obtain the porcine pseudorabies inactivated vaccine. The porcine pseudorabies inactivated vaccine can well prevent highly pathogenic mutant pseudorabies prevailing in the market.

Description

technical field [0001] The invention belongs to the technical field of vaccines, in particular to a method for large-scale production of porcine pseudorabies inactivated vaccines. Background technique [0002] Pseudorabies is an acute infectious disease caused by pseudorabies virus (Pseudorabies virus, PRV), including a variety of livestock and wild animals. Pigs should be the natural host and reservoir of the disease, which is characterized by fever, severe itching, encephalomyelitis and severe reproductive impairment. Since 2011, large-scale pig farms that have been immunized with gE gene-deficient live vaccines in many provinces across the country have experienced weak piglets, stillbirths, mummies, and abortions in sows; gilts and empty pregnant sows often return to oestrus, and repeated infertility or no estrus; boars often have testicular atrophy after infection, sexual function decline and loss of breeding ability; new piglets have neurological symptoms and death and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02C12N7/06A61K39/245A61P31/22C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552C12N7/00C12N2710/16721C12N2710/16734C12N2710/16751C12N2710/16763
Inventor 徐高原陈波周明光洪灯方玉林陈关平
Owner WUHAN KEQIAN BIOLOGY CO LTD
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