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Canine adenovirus genetic engineering subunit vaccine, its preparation method and application

A technology of encoding genes and recombinant genes, applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., can solve problems such as inability to resist viruses, weakening viruses and returning to strong ones, and dispersing viruses

Active Publication Date: 2021-05-11
苏州沃美生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the use of CAV-1 attenuated vaccine has greatly reduced the incidence of canine infectious hepatitis, but the vaccine will cause blue eye disease in dogs after immunization, and the excreta of dogs is still infected. Strong possibility, so there are some limitations
Although the CAV-2 attenuated vaccine can provide cross-protection to CAV-1, the virus strains contained in the vaccine are all attenuated strains, and there is a risk of shedding the virus after immunization of animals, and it takes a period of time to produce antibodies after immunization, so it cannot be quickly established resistance to virus

Method used

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  • Canine adenovirus genetic engineering subunit vaccine, its preparation method and application
  • Canine adenovirus genetic engineering subunit vaccine, its preparation method and application
  • Canine adenovirus genetic engineering subunit vaccine, its preparation method and application

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preparation example Construction

[0052] For example, in a specific embodiment of the embodiments of the present invention, a method for preparing a canine adenovirus genetically engineered subunit vaccine may specifically include:

[0053] (1) preparing a nucleic acid molecule for encoding the fusion protein (CAV-Fu protein);

[0054] (2) Cloning the nucleic acid molecule encoding the fusion protein prepared in step (1) into a shuttle vector to obtain a recombinant shuttle vector containing the gene of interest;

[0055] (3) Transform DH10Bac bacteria with the recombinant shuttle vector obtained in step (2), select the recombinant bacteria, extract the genome and transfect Sf9 cells (or other aforementioned insect cells) to obtain recombinant baculovirus;

[0056] (4) Cultivate the Sf9 cells (or other aforementioned insect cells) and then recombinantly express and produce the fusion protein;

[0057] (5) Mixing the fusion protein into an adjuvant to obtain the vaccine.

[0058] In this specific embodiment o...

Embodiment 1

[0062] Example 1 Construction and Identification of Transfer Vector pF-CAV-Fu

[0063] 1. Amplification and purification of CAV-Fu gene

[0064] The codon-optimized CAV-Fu gene (SEQ ID NO: 1) was synthesized in Nanjing GenScript Biotechnology Co., Ltd. and cloned into the pUC17 vector to obtain the pUC-CAV-Fu plasmid vector. With pUC-CAV-Fu plasmid as template, CAV-Fu-F, CAV-Fu-R carry out PCR amplification (gene sequence of CAV-Fu-F, CAV-Fu-R as SEQ ID NO: 3 and 4), the amplification system is shown in Table 1.

[0065] Table 1 CAV-Fu gene amplification system

[0066]

[0067] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0068] Perform gel electrophoresis on the PCR product to verify the size of the target gene, such as figure 1 As shown, the target band appears at the position near 1.6kbp, indicating that the...

Embodiment 2

[0084] Example 2 Construction of recombinant baculovirus genome Bac-CAV-Fu

[0085] 1. DH10Bac transformation

[0086] Take 1 μl of the pF-CAV-Fu plasmid in Example 1 and add it to 100 μl of DH10Bac competent cells, mix well, put it in an ice bath for 30 minutes, heat shock in a 42°C water bath for 90 seconds, and then add 900 μl of LB liquid without Amp Medium, cultured at 37°C for 5 hours. After 100 μl of bacterial solution was diluted 81 times, 100 μl of diluted bacterial solution was spread on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37°C for 48 hours.

[0087] 2. single clone

[0088] Use an inoculation needle to pick large white colonies, then streak on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal, and IPTG, culture at 37°C for 48 hours, and then pick single colonies Inoculate the LB liquid medium containing gentamicin, kanamycin, and tetracycline for culture, preserve the strain, and ex...

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Abstract

The invention discloses a canine adenovirus genetic engineering subunit vaccine, its preparation method and application. The vaccine comprises a fusion protein and a pharmaceutically acceptable carrier, and the fusion protein has the sequence shown in SEQ ID NO:2. The vaccine provided by the present invention has high safety and good immunogenicity, and can generate strong humoral immunity in animals, and the immunized animals can resist the attack of strong viruses, and can also be suspended in a large scale without serum by using a bioreactor Culture preparation has the advantages of easy quality control, batch-to-batch stability, and low production cost.

Description

technical field [0001] The invention relates to a genetic engineering vaccine, in particular to a canine adenovirus genetic engineering subunit vaccine, its preparation method and application, and belongs to the technical field of animal immune medicines. Background technique [0002] The infectious disease caused by canine adenovirus (Canine Adenovirus, CAV) infection is called canine adenovirus disease (Canine Adeno, CA). CA is a highly pathogenic, wide-ranging and highly contagious infectious disease to many mammals including dogs. CAV is often co-infected with canine distemper virus (CDV), canine parvovirus (CPV), canine parainfluenza virus (CPIV), etc., which increases the complexity of clinical symptoms. According to the different effects of hemagglutination inhibition and neutralization, CAV is divided into two serotypes: canine adenovirus type 1 (CAV-1) and canine adenovirus type 2 (CAV-2). . CAV-1 can cause systemic infection characterized by infectious canine he...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/866C12N5/10G01N33/569A61K39/235A61P31/20
CPCA61K39/12A61K2039/552A61P31/20C07K14/005C07K2319/00C07K2319/30C12N5/0601C12N15/86C12N2510/02C12N2710/10034C12N2710/10322C12N2710/10334C12N2710/10351C12N2710/14043C12N2800/105G01N33/56983G01N2333/075
Inventor 曹文龙孔迪滕小锘
Owner 苏州沃美生物有限公司
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