Porcine epidemic diarrhea virus variant strain 2a, 2b bivalent inactivated vaccine and preparation method thereof

A technology of porcine epidemic diarrhea and dual inactivated vaccines, applied in multivalent vaccines, biochemical equipment and methods, viruses, etc., can solve the problems of pig side effects and high prices

Active Publication Date: 2021-08-20
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these single or combined vaccines are not effective against the currently circulating 2a subtype or 2b subtype variants
In addition, the problem of serum in vaccine production is becoming more and more serious. The high price and side effects on pigs are driving the rapid development of serum-free virus culture technology.

Method used

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  • Porcine epidemic diarrhea virus variant strain 2a, 2b bivalent inactivated vaccine and preparation method thereof
  • Porcine epidemic diarrhea virus variant strain 2a, 2b bivalent inactivated vaccine and preparation method thereof
  • Porcine epidemic diarrhea virus variant strain 2a, 2b bivalent inactivated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The preparation of embodiment 1 porcine epidemic diarrhea virus mutant strain 2a, 2b virus liquid

[0027] 1. Breeding of poisonous species

[0028] The whole suspension ST cells were mixed with 0.5×10 6 -1×10 6 Cells / ml density inoculated in shake flasks, at 37°C, 5% CO 2 , 150rpm incubator, and after 72 hours, dilute the cell density to 2×10 with ST serum-free medium 6 -3×10 6 pc / ml, add trypsin to a final concentration of 10ug / ml, inoculate porcine epidemic diarrhea virus variant 2a or 2b according to 1% of the culture volume, continue to culture at 37°C, harvest the virus liquid at 18h-24h, and test the virus content and purity , after conforming to the regulations, quantitatively aliquoted, freeze-dried, and stored below -80°C.

[0029] 2. Propagation of poisonous species for production

[0030] Including cell expansion, virus inoculation, virus liquid harvest, virus titer determination, sterility and exogenous detection.

[0031] ①Expanded cell culture in t...

Embodiment 2

[0046] Example 2 Optimum Condition Optimization of Porcine Epidemic Diarrhea Virus Mutant 2a, 2b Antigen Preparation

[0047] The present embodiment provides porcine epidemic diarrhea virus mutant strain 2a, 2b cultivation optimal condition optimization, specifically as follows:

[0048] 1. Determination of the optimal cell density: the cell density in the shake flask reaches 7×10 6 -10×10 6 cells / ml, dilute ST cells with ST serum-free medium, add trypsin at a concentration of 15 μg / ml, inoculate at 1% by volume, and set the cell density to 2×10 6 / ml and 3×10 6 samples / ml, and the virus titer was determined after 24 hours. The results showed that the optimal density of inoculated cells was 2×10 6 pieces / ml.

[0049] 2. Determination of optimal trypsin concentration: when the cell density reaches 7×10 6 -10×10 6 cells / ml, ST cells were diluted to 2×10 with ST serum-free medium 6 The cell density of each / ml was inoculated with 1% of the volume ratio, and the concentrati...

Embodiment 3

[0051] The preparation of embodiment 3 porcine epidemic diarrhea virus mutant strain 2a, 2b bivalent inactivated vaccine

[0052] According to the inactivated virus liquid prepared under the optimal conditions for culturing porcine epidemic diarrhea virus mutant strains 2a and 2b in Example 2, the dual inactivated vaccine was prepared.

[0053] 1. Preparation of PEDV-2a / 2b bivalent inactivated vaccine

[0054] ①Water phase preparation: mix the inactivated porcine epidemic diarrhea virus variant strains 2a and 2b evenly at a volume ratio of 1:1, and the titers of porcine epidemic diarrhea virus variant strains 2a and 2b after mixing are both ≥10 6 TCID 50 / 0.1mL.

[0055] ②Emulsification and sub-packaging: Mix the water phase and water adjuvant IMS1313 at a volume ratio of 3:1 and then emulsify. After passing the test, quantitative sub-package, capping and labeling are carried out.

[0056] 2. Vaccine testing method

[0057] Three batches of vaccines were prepared according...

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Abstract

The invention provides a porcine epidemic diarrhea virus variant strain 2a, 2b bivalent inactivated vaccine and a preparation method thereof. The bivalent inactivated vaccine includes two currently popular porcine epidemic diarrhea variant strains 2a and 2b subtypes, which can prevent diarrhea caused by these two different variant strains. One injection is dual-purpose, safe and effective, and reduces animal immunity. And stress times, piglets can be protected by immunizing sows. The preparation method comprises: fully suspending and culturing porcine epidemic diarrhea virus variant strains 2a and 2b without serum, mixing the inactivated porcine epidemic diarrhea virus variant strain 2a and 2b virus liquids in proportion, adding adjuvant and fully emulsifying to obtain the product. The invention adopts the full-suspension serum-free ST cell culture process to prepare the virus liquid with high antigen content, easy scale-up culture, large batch size and small batch-to-batch difference. The process reduces the risk of pollution and low cost, and provides an effective means for the prevention and control of PEDV.

Description

technical field [0001] The invention relates to the fields of biotechnology and preventive veterinary medicine, in particular to a bivalent inactivated vaccine of porcine epidemic diarrhea virus mutant strain 2a and 2b and a preparation method thereof. Background technique [0002] Porcine diarrheal diseases include diarrhea caused by bacteria and diarrhea caused by viruses, and porcine epidemic diarrhea virus plays a major role in the prevention of viral diarrhea. Most porcine diarrhea vaccines currently available on the market are inactivated porcine epidemic diarrhea virus vaccines or live vaccines, dual vaccines of porcine epidemic diarrhea virus and porcine transmissible gastroenteritis or other viruses, and porcine epidemic diarrhea virus-porcine infectious Triple vaccine of gastroenteritis virus-porcine rotavirus and other viruses. However, these single or combined vaccines are not effective against the currently circulating 2a subtype or 2b subtype variants. In add...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K39/295A61K39/215A61P31/14
CPCA61K39/12A61K2039/5252A61K2039/552A61K2039/70A61P31/14C12N7/00C12N2770/20021C12N2770/20034C12N2770/20051
Inventor 张华伟周明光余蕾徐高原倪冬冬金建云罗修鑫
Owner WUHAN KEQIAN BIOLOGY CO LTD
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