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Canine distemper virus strain, bivalent vaccine based on canine distemper virus and canine parvovirus and application

A technology of canine distemper virus and dual vaccine, applied in the field of canine distemper virus strain and dual vaccine based on canine distemper virus and canine parvovirus, to achieve good immunogenicity, ensure immunogenicity and good safety Effect

Pending Publication Date: 2022-04-12
CHANGCHUN SR BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of the biological safety of the double vaccine virus and the immune protection effect of young dogs

Method used

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  • Canine distemper virus strain, bivalent vaccine based on canine distemper virus and canine parvovirus and application
  • Canine distemper virus strain, bivalent vaccine based on canine distemper virus and canine parvovirus and application
  • Canine distemper virus strain, bivalent vaccine based on canine distemper virus and canine parvovirus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation and Identification of Recombinant Baculovirus CPV-2b-VP2 Strain

[0052] 1. PCR amplification of VP2 gene

[0053] Based on the VP2 sequence (SEQID NO: 1) of the canine parvovirus (CPV, canine parvovirus) CPV-SD15 strain isolated by our company, without changing the amino acid sequence, the CPV-SD15 The original VP2 gene was optimized, and the optimized sequence CPV-2b-VP2-opti (SEQ ID NO: 2) was synthesized into the vector pUC57, named pUC-CPV-2b-VP2-opti. Using the synthesized pUC-CPV-2b-VP2-opti plasmid as a template, PCR amplification was performed with VP2-P10F primers and VP2-P10R primers to obtain a PCR product VP2-2b-opti-p10 fragment (SEQ ID NO: 11). The PCR product VP2-2b-opti-pH fragment (SEQ ID NO:12) was obtained by performing PCR amplification with VP2-PHF primers and VP2-PHR primers. See Table 1 for the primers. The reaction system was 50 μl, composed of: 10 μl of 5×HF buffer, 4 μl of 2.5 mmol / L dNTP, 1.25 μl of upstream primer (20...

Embodiment 2

[0090] Example 2. The method and identification of isolating canine distemper virus CDV-SN

[0091] 1. Isolation method of canine distemper virus CDV-SN

[0092] (1) Treatment and inspection of disease materials: freeze and thaw the blood of dogs diagnosed with canine distemper directly for 1 to 2 times, centrifuge at 4°C at a speed of 10,000r / min for 20 minutes, take the supernatant and add double antibodies (1000 units / ml), and stored in an ultra-low temperature refrigerator for later use. The sterility test is qualified, and the canine distemper antigen test is positive. The disease material is used for virus isolation.

[0093] (2) Virus isolation and culture Vero cells were revived according to conventional methods, cultured in T25 cell culture flasks, and used for inoculation after proper subculture and multiplication. Inoculate the virus when the cells are in good condition and the confluence reaches 70%, and inoculate Vero cells with qualified disease materials, 0.5m...

Embodiment 3

[0103] Example 3. Preparation of a dual vaccine based on canine distemper virus and canine parvovirus

[0104] 1. Vaccine manufacturing and inspection of semi-finished products

[0105] CDV-SN strain of canine distemper virus: Dilute the virus seeds with MEM culture medium, and inoculate the Vero cells that have grown into a monolayer with an MOI of 0.1-0.3. After culturing at 37°C for 24 hours, change to the cell maintenance solution containing 2% newborn bovine serum, and continue culturing at 33-35°C. When the cytopathy reaches more than 80%, freeze and thaw once, and store below -20°C.

[0106] Recombinant baculovirus CPV-2b-VP2 strain: culture Sf9 cells in insect cell culture medium, 27°C, 120r / min until the number of cells is about 2.0×10 6 ~2.5×10 6 Inject the recombinant baculovirus CPV-2b-VP2 strain at an MOI of 0.1 to 1.0 per ml, and continue culturing at 27°C. After 96-120 hours, the virus liquid was harvested and stored below -20°C.

[0107] Virus species ident...

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Abstract

The invention provides a canine distemper virus strain, a bivalent vaccine based on a canine distemper virus and a canine parvovirus and application of the bivalent vaccine, and belongs to the technical field of vaccines. The biosafety problem of bivalent vaccine virus seed enhancement and the immune protection effect problem of young dogs are solved. The invention provides a canine distemper virus which is named as a canine distemper virus CDV-SN strain and has a preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.23205. The invention also provides a bivalent vaccine based on the canine distemper virus and the canine parvovirus disease, and the bivalent vaccine comprises a stock solution of the canine distemper virus CDV-SN and a stock solution of recombinant baculovirus CPV-2b-VP2, and the mixing ratio of the stock solution to the stock solution of the recombinant baculovirus CPV-2b-VP2 is 1: 1. Compared with the existing product canine distemper virus and parvovirus disease bivalent live vaccine, the product has the advantages of good safety, high antigen concentration and high purity, and can be used as a preferred vaccine for basic immunity of puppies to reduce the influence of maternal antibodies on the immune effect.

Description

technical field [0001] The invention belongs to the technical field of vaccines, and in particular relates to a canine distemper virus strain, a dual vaccine based on canine distemper virus and canine parvovirus and its application. Background technique [0002] At present, the main vaccine type of canine distemper vaccine is the attenuated vaccine. Its main advantage is that it can not only activate the body's humoral immunity, but also activate the cellular immunity and induce the body to produce immune memory. It is currently the main product used by dogs and economic animals. At present, the virus strain of CDV attenuated vaccine has no reports of virulence reversion in dogs, but it has been confirmed in dogs and ferrets or primary macrophage tissue tests; genetic engineering vaccine is the current canine distemper One of the research directions of vaccines, researchers have obtained new vaccines such as canine distemper subunit vaccines, recombinant vector vaccines, nuc...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/866A61K39/175A61P31/14A61K39/23A61P31/20C12R1/93
CPCY02A50/30
Inventor 夏振强张馨月石晶金宏丽
Owner CHANGCHUN SR BIOLOGICAL TECH
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