A kind of mink enteritis virus recombinant subunit vaccine and its preparation method

A technology of recombinant baculovirus and enzyme cutting sites, applied in biochemical equipment and methods, antiviral agents, viruses/phages, etc., can solve the problem of low expression level of foreign proteins

Active Publication Date: 2017-10-03
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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  • A kind of mink enteritis virus recombinant subunit vaccine and its preparation method
  • A kind of mink enteritis virus recombinant subunit vaccine and its preparation method
  • A kind of mink enteritis virus recombinant subunit vaccine and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0107] Preparation of rBac-HBM-HS4-VP2P1 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. An appropriate amount of the collected P1 generation seed virus was taken to detect the virus titer, and the rest were divided into 1.8ml cell cryopreservation tubes, and 1ml P1 virus and 0.11ml fetal bovine serum (product of Invitrogen Company) were added 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 In 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 produced as an example by the technical scheme of the present invention)

[0113] (1) Thaw 1 ml of seed cells of cryopreserved Sf9 cells in 100 ml of serum-free medium, place them in a 500 ml screw-top glass Erlenmeyer shaker flask for culture, the temperature of the shaker is 27° C., and the rotation speed is 110 r / 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-mentioned 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, temperatu...

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Abstract

The invention relates to a mink enteritis virus recombinant subunit vaccine and a preparation method thereof. The production strain of the vaccine related to the present invention is the recombinant Autographa californica nuclear polyhedrosis virus CGMCC No.8051, the expression vector adopted by the virus strain is secreted expression, and contains 6 histidine tags, which is easy to use for protein detection and analysis ; use it to produce mink enteritis virus recombinant subunit vaccine, the vaccine does not contain virus genome, no animal safety hazards and potential dissemination; immunity is highly targeted, can stimulate immune animals to produce sufficient immune antibodies, and produce long-term protection. According to the production of MEV virus-like particles according to the present invention, the titer of the virus liquid harvested in the 1000L bioreactor reaches the hemagglutination value of 1:4096~1:32768, the expression of the granule protein is about 65 mg / L, and the semi-finished product of the virus liquid per liter can be made The standard mink vaccine is 3000-8000 doses; the recombinant subunit vaccine is a new vaccine obviously superior to the existing inactivated tissue vaccine.

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), characterized by diarrhea and high morbidity 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 mink, and then cause severe diarrhea in 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 the disease in Canada. Wills isolated...

Claims

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Application Information

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Patent Type & Authority Patents(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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