Mink enteritis virus recombinant subunit vaccine and preparation method thereof

A mink enteritis virus and subunit vaccine technology, applied in biochemical equipment and methods, antiviral agents, viruses/bacteriophages, etc., can solve the problem of low expression level of foreign proteins

Active Publication Date: 2014-05-28
QILU ANIMAL HEALTH PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression level of the foreign protein is usually still much lower than that of the polyhedrin protein expressed by the wild-type virus

Method used

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  • Mink enteritis virus recombinant subunit vaccine and preparation method thereof
  • Mink enteritis virus recombinant subunit vaccine and preparation method thereof
  • Mink enteritis virus recombinant subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] ——Batch preparation of recombinant baculovirus seed virus

[0107] Preparation of rBac-HBM-HS4-VP2P1 seed virus library (200 tubes, 1ml / tube): Infect 250 mL of Sf9 cells in the logarithmic growth phase with the purified P0 generation recombinant virus, MOI=0.1-0.01, and harvest 96 hours after infection Clear, obtain P1 generation seed virus, concrete operation is the same as embodiment 3. Take an appropriate amount of the collected P1 seed virus to detect the virus titer, and divide the rest into 1.8ml cell cryopreservation tubes, and add 1ml P1 virus and 0.11ml fetal bovine serum (product of Invitrogen Company) to each tube. Store the above-mentioned aliquoted cryopreservation tubes in an ultra-low temperature freezer at -80°C.

Embodiment 2

[0109] ——Preparation of batch poisonous seeds for production

[0110] The Sf9 cells cryopreserved in liquid nitrogen were resuscitated and inoculated into 250ml shaker flasks, and cultured on a constant temperature shaker at 27°C with a rotation speed of 110r / min. When the cell density reaches 3.0×10 6 When the cells / ml is above, scale up to a 1L shake flask at a ratio of 1:2, and finally scale up to a 3L shake flask. 3L shake flask cells up to 2.0 x 10 6 cells / ml, inoculate rBac-HBM-HS4-VP2 recombinant virus according to MOI=0.05~0.1, culture in suspension at 27°C for about 96 hours, harvest the culture medium, take samples to determine the titer of HA, quantitatively aliquot and store in freezer, which is for production Poison batch. Indicate the date of harvest, generation of poisonous seeds, etc.

Embodiment 3

[0112] ——Preparation of vaccine virus liquid (taking 1311001 batches, 1311002 batches and 1311003 batches of vaccines trial-produced according to the technical scheme of the present invention as examples)

[0113] (1) Throw 1ml of seeded cells of cryopreserved Sf9 cells in 100ml of serum-free medium and place them in a 500ml screw-top glass Erlenmeyer shaker flask for culture. The temperature of the shaker is 27°C and the rotation speed is 110r / min. After culturing for 48 hours, 200ml of serum-free medium was added, diluted and passaged in a 3000ml screw-top glass Erlenmeyer flask. After culturing for 48 hours, 400ml of serum-free medium was added. According to every 48h, the 1:2 dilution ratio was passed to 3000ml seed cells, and the cell density reached 3.0×10 6 cells / ml;

[0114] (2) The above 3000ml seed cells were inoculated into a 30L stirred bioreactor (working volume 20L) and the culture conditions were set as follows: inoculation density: 1.5×10 6 cells / ml, tempera...

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Abstract

The invention relates to a mink enteritis virus recombinant subunit vaccine and a preparation method thereof. The strain that produces the vaccine is recombinant autographa California nuclear polyhedrosis virus (CGMCC No. 8051), the expression vector of the strain is expressed through secretion, comprises six histidine labels, and is prone to be used in protein detection and analysis; mink enteritis virus recombinant subunit vaccine produced by the strain does not contain any viral genome and thus does not have any animal safety hazard or potential infectivity; and moreover the vaccine has a strong immunity specificity, can activate the immunized animals to generate enough immunity antibodies, and generates a long-term protection. An MEV virus-like particle is produced according to the preparation method, the titer of virus liquid generated by a bio-reactor with a volume of 1000 L can reach a hemagglutination titer of 1:4096 to 1:32768; the expression quantity of granule protein is 65 mg / L, and reach liter of virus liquid semi-finished product can produce 3000 to 8000 doses of standard mink vaccine. The recombinant subunit vaccine is a novel vaccine having a prominently better effect than that of conventional inactivated tissue vaccines.

Description

technical field [0001] The invention relates to a mink enteritis virus recombinant subunit vaccine and a preparation method thereof. The vaccine is a recombinant subunit vaccine produced by using an insect baculovirus expression system, has the effect of resisting mink viral enteritis, and belongs to the field of veterinary biological products and biotechnology. Background technique [0002] Mink Viral Enteritis (MVE) is an acute, highly contagious disease caused by Mink Enteritis Virus (MEV). The disease is mainly characterized by diarrhea and has a high incidence and mortality rate. [0003] MEV belongs to the family Parvoviridae and the genus Parvovirus in classification. After MEV infects mink, it can cause inflammation and necrosis of the intestinal mucosa of the mink, and then cause severe diarrhea in the mink. and Veterinary Medicine. 2007,39(3):52-53.). MEV can infect minks of all ages, and is highly infective to young minks. In 1949, Schofield first reported th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C07K14/015A61K39/23A61P31/20
Inventor 闵庆如易小萍范志永滕小锘王秀梅冀海明宋晓飞徐龙涛张连秀王蕾禚宝山鲍海忠
Owner QILU ANIMAL HEALTH PROD
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