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Recombinant rabies virus of chimeric canine parvovirus VP2 gene and application of recombinant rabies virus

A technology of rabies virus and canine parvovirus, applied in the field of immunology, can solve the problems of high production cost and short duration of antibody response

Pending Publication Date: 2021-05-28
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, inactivated vaccinated animals induce CPV neutralizing antibody responses of shorter duration and are more expensive to produce

Method used

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  • Recombinant rabies virus of chimeric canine parvovirus VP2 gene and application of recombinant rabies virus
  • Recombinant rabies virus of chimeric canine parvovirus VP2 gene and application of recombinant rabies virus
  • Recombinant rabies virus of chimeric canine parvovirus VP2 gene and application of recombinant rabies virus

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preparation example Construction

[0037] The invention provides the preparation method of the recombinant rabies virus, comprising the following steps: 1) using the canine parvovirus genome as a template, PCR amplification obtains the VP2 gene; 2) connecting the VP2 gene to the pBNSP plasmid to obtain the recombinant plasmid pBNSP -CPV-VP2; the pBNSP plasmid carries the full-length cDNA of SAD-B19 strain; 3) the recombinant plasmid pBNSP-CPV-VP2, helper plasmid N, helper plasmid P, helper plasmid L, helper plasmid G and helper plasmid The recombinant rabies virus was obtained after pCAGGS-T7 was mixed and transfected into cells.

[0038] In the present invention, the VP2 gene is obtained by PCR amplification using the canine parvovirus genome as a template. In the present invention, the source of the canine parvovirus genome is not particularly limited, and the canine parvovirus genome from a conventional source in the field can be used. In the specific implementation process of the present invention, the cani...

Embodiment 1

[0055] 1. Centrifuge the clinically collected suspected CPV sample (number CPV-LZ-1) at 5000rpm for 10min, take the supernatant, and then follow the Tiangen Feces Genomic DNA Extraction Kit

[0056] According to the instruction manual, the CPV genomic DNA was extracted and then amplified by PCR.

[0057] The primer sequences are listed in Table 1.

[0058] Table 1 CPV VP2 gene amplification primers

[0059]

[0060] (2) Utilize primer CPV1 / CPV2, carry out PCR amplification according to the system shown in Table 2:

[0061] Table 2 PCR amplification system

[0062]

[0063] Amplification program: 95°C for 5min; 94°C for 30s, 56°C for 30s, 72°C for 2min, 30 cycles; 72°C for 10min. The amplified PCR product was electrophoresed on 1% agarose gel.

[0064] The amplified product was cloned into the pMD19-T Simple vector (purchased from Dalian TaKaRa Company), the ligated product was transformed into JM109 competent cells (purchased from Dalian TaKaRa Company), the plasmid ...

Embodiment 2

[0066] Cloning of VP2 gene of CPV strain into pBNSP

[0067] 1. For the strategy of constructing the recombinant plasmid pBNSP-CPV-VP2, see figure 1 shown.

[0068] (1) utilize primer CPV-VP2 / F and CPV-VP2 / R (see Table 1) to amplify the VP2 gene (sequence shown in SEQ ID No.1) of the CPV strain selected in the embodiment 1, CPV-VP2 The expected amplification length of / F and CPV-VP2 / R primers is 1755bp (the amplification reaction system is shown in Table 2).

[0069] The PCR amplification program was: 95°C for 5 minutes; 94°C for 30s, 56°C for 30s, 72°C for 2min, 30 cycles; 72°C for 10min.

[0070] (2) After the reaction, 5 μL of the PCR product was taken for electrophoresis detection on 1.0% agarose gel. results as seen figure 2 As shown, a specific DNA electrophoresis band with a size of 1755bp was obtained, which was consistent with the experimental expectation.

[0071] (3) The VP2 gene amplified by PCR is recovered and purified by gel cutting, and the purified VP2 g...

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Abstract

The invention provides a recombinant rabies virus of a chimeric canine parvovirus VP2 gene, and belongs to the technical field of immunology. The recombinant rabies virus takes a rabies vaccine candidate strain SAD-B19 as a skeleton, and the VP2 gene of a CPV-2a strain currently popular in China is inserted into an SAD-B19 genome; the recombinant rabies virus strain of the chimeric VP2 protein is obtained through rescue, after immunization, high-level anti-rabies virus antibodies and anti-canine parvovirus VP2 antibodies can be induced to be generated, and the vaccine is low in cost, safe and effective.

Description

technical field [0001] The invention belongs to the technical field of immunology, and in particular relates to a recombinant rabies virus of chimeric canine parvovirus VP2 gene and application thereof. Background technique [0002] Rabies (Rabies) is a zoonotic infectious disease caused by rabies virus (RV) infection. Once the onset occurs, the fatality rate is almost 100%. According to WHO statistics, about 60,000 people die from rabies every year in the world. Asia and Africa are high-incidence areas of rabies, and India occupies the first place. On average, about 3,000 people die of rabies every year in my country, and about 99% of the patients are bitten or scratched by dogs or cats. To reduce or eliminate the occurrence of human rabies, it is necessary to control dog rabies, so the control of dog rabies in my country should be the focus of future prevention and control. With the improvement of people's living standards and the increase in the number of pets raised, h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/35C12N15/63A61K39/295A61K39/23A61K39/205A61P31/14A61P31/20C12R1/93
CPCC12N7/00C12N15/63C07K14/005A61K39/12A61P31/14A61P31/20C12N2760/20121C12N2760/20122C12N2760/20134C12N2750/14022C12N2750/14034A61K2039/5256A61K2039/552A61K2039/70
Inventor 殷相平卫巧林毛箬青王相伟周亚花殷娟斌孙跃峰
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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