Recombinant rabies viruses in which canine distemper virus main immune genes are embedded and application of recombinant rabies viruses

A technology of rabies virus and canine distemper virus, applied in application, antiviral agent, genetic engineering, etc., can solve the problems of high production cost and limited scale of vaccine production

Inactive Publication Date: 2019-06-28
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

In recent years, my country has launched domestic rabies inactivated vaccines, but due to the limited scale of vaccine production and high production costs, domestic inactivated veterinary vaccines have not been widely used in China

Method used

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  • Recombinant rabies viruses in which canine distemper virus main immune genes are embedded and application of recombinant rabies viruses
  • Recombinant rabies viruses in which canine distemper virus main immune genes are embedded and application of recombinant rabies viruses
  • Recombinant rabies viruses in which canine distemper virus main immune genes are embedded and application of recombinant rabies viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 CDV epidemic strain screening

[0048] 1. Process and extract nucleic acid from suspected CDV samples collected clinically, and perform PCR amplification.

[0049] (1) See Table 1 for primer sequences

[0050] Table 1 Amplification primers of CDV F, H and N genes

[0051]

[0052] (2) Utilize primer CDV1 / CDV2, carry out PCR amplification according to the system shown in Table 2:

[0053] Table 2 PCR amplification system

[0054]

[0055] 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.

[0056] The amplified product was cloned into the pMD19-T Simple vector, the ligated product was transformed into JM109 competent cells, the plasmid was extracted for PCR identification, and the positive plasmid was sent to Beijing Jinweizhi Biotechnology Co., Ltd. for sequencing. According to the MegAlign software in DNAStar, the sequencing res...

Embodiment 2

[0057] Example 2 CDV strain F and H genes are cloned into pBNSP

[0058] 1. For the strategy of constructing recombinant plasmids pBNSP-CDV-F and pBNSP-CDV-H, see figure 1 shown.

[0059] (1) Utilize the upstream primer and the downstream primer (see Table 1) of primer CDV-F and H to amplify the F and H gene (sequence as shown in SEQIDNO.1 and SEQIDNO.2) of the CDV strain selected in embodiment 1 , CDV-F and H upstream primers and downstream primers are expected to amplify the lengths were 1989bp and 1815bp (amplification reaction system as shown in Table 2).

[0060] The PCR amplification program was: 95°C for 5 min; 94°C for 30 s, 56°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 10 min.

[0061] (2) After the reaction, 5 μL of PCR products were taken for electrophoresis detection on 1.0% agarose gel. Obtain a specific DNA electrophoresis band, the size is consistent with the test expectation, see figure 2 shown.

[0062] (3) The F and H genes amplified by PCR were re...

Embodiment 4

[0071] Example 4 Rescue and Screening of Recombinant Viruses Using Recombinant Plasmids pBNSP-CDV-F and BNSP-CDV-H

[0072] 1. The method of transfection and virus screening was carried out according to the method of Matthias Schnell.

[0073]Transfection: (1) Prepare Vero-E6 cells: 6-well cell culture plate with a cell density of 5×10 5 cells / well, the culture medium contains 10% fetal bovine serum; culture at 37°C for 24 hours, and transfect when the cells are about 75%.

[0074] (2) Take 24 μl of transfection reagent X-tremeGene 9, add it to 600 μl of Optimem medium in a 1.5mL centrifuge tube, mix well and let it stand at room temperature for 5 minutes.

[0075] (3) Take 5μl pBNSP-CDV-F and BNSP-CDV-H, 2.5μl pTIT-N, 1.25μl pTIT-P, 1.25μl pTIT-L, 1μl pTIT-G plasma, 1.5μl pCAGGS-T7 in another 1.5mL Mix in a centrifuge tube.

[0076] (4) Add the mixed plasmid into the tube of the transfection reagent, and let it stand at room temperature for 15 minutes after mixing.

[007...

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Abstract

The invention discloses recombinant rabies viruses BNSP-CDV-F and BNSP-CDV-H in which canine distemper virus main immune genes are embedded. The recombinant viruses take a rabies virus strain SAD-B19as a framework, CDV-F and CDV-H genes shown in SEQ ID NO.1 and SEQ ID NO.2 are separately inserted between an N gene and P gene of a recombinant plasmid SAD-19 full-length cDNA, and finally recombinant rabies virus strains BNSP-CDV-F and BNSP-CDV-H are saved through a reverse genetic operation technology. The recombinant viruses BNSP-CDV-F and BNSP-CDV-H can express fusion protein and hemagglutinin protein of canine distemper viruses; after a vaccine prepared by mixing the recombinant viruses is used for immunizing animals, a great number of anti-rabies virus antibodies and main immune gene antibodies resistant to the canine distemper viruses can be induced and generated. The cost of the vaccine can also be reduced, and the recombinant viruses BNSP-CDV-F and BNSP-CDV-H have a good application and popularization prospect.

Description

technical field [0001] The invention belongs to the field of molecular biological immunology. More specifically, it relates to a recombinant rabies virus chimeric with the main immune gene of canine distemper virus and its application. 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 increa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/45C12N7/01C12N15/63C12N15/66A61K39/295A61K39/205A61K39/175A61P31/14C12R1/93
Inventor 殷相平张志东张韵卫巧林刘翠殷娟强周亚花
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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