VP2 fusion gene, recombinant rabies virus rHEP-rVP2 strain and construction method and application of strain

A rabies virus and fusion gene technology, applied in the field of microbial immunology, can solve the problems of mutation of weak virus strains and strong virulence, and achieve good immunogenicity and cost reduction

Active Publication Date: 2017-03-15
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the effect of the attenuated vaccine is ideal, the attenuated strain may mutate and become stronger

Method used

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  • VP2 fusion gene, recombinant rabies virus rHEP-rVP2 strain and construction method and application of strain
  • VP2 fusion gene, recombinant rabies virus rHEP-rVP2 strain and construction method and application of strain
  • VP2 fusion gene, recombinant rabies virus rHEP-rVP2 strain and construction method and application of strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of VP2 fusion gene

[0029] Use the coding sequence of the linear neutralizing epitope (PPDQLVNLHDFRSDEIEHLVVEE, SEQ ID NO: 2) between the 253-275 positions of the rabies virus (RV) G protein to replace the VP2 gene between the 684-686 positions of the loop2 Sequence, construct VP2 fusion gene, such as figure 1 shown. The sequence of the VP2 fusion gene is shown in SEQ ID NO: 1.

[0030] 1. Primer Design

[0031] According to the upstream and downstream sequences of the canine parvovirus VP2 gene registered in Genbank, the forward primer VP2-P1 and the reverse primer VP2-P4 were designed to amplify the complete sequence of the VP2 gene, and the forward primer introduced the BsiWI restriction site, and the reverse primer introduced NheI restriction site, the primer sequence is as follows:

[0032] VP2-P1:5-AGTCGTACGATGAGTGATGGA-3;

[0033] VP2-P4: 5-CCTGCTAGCTTAATATAATTT-3.

[0034] Primers 23aa-P2 and 23aa-P3 were designed according to the u...

Embodiment 2

[0052] Example 2 Cloning of VP2 fusion gene to full-length cDNA of HEP-Flury strain

[0053] Insert the VP2 fusion gene between the NheI and BsiWI restriction sites in the pseudogene region between the G gene and the L gene in the full-length cDNA of the HEP-Flury strain to construct the recombinant plasmid pHEP-rVP2. For the strategy, see figure 2 shown. The specific method is as follows:

[0054] The VP2 fusion gene amplified by PCR was recovered and purified by gel cutting, and the purified VP2 gene and pHEP-3.0 plasmid (recombinant plasmid carrying the full-length cDNA of HEP-Flury strain, see Ito N., Takayama I. M., YamadaK for the construction method) ., et al. Improved recovery of rabies virus from cloned cDNA using avaccinia virus-free reverse genetics system [J]. Microbiology and Immunology, 2003, 47 (8): 613-617.) NheI and Bsi WI nucleic acid restriction Dicer performs double enzyme digestion, and the enzyme digestion system is shown in Table 5:

[0055] table 5 ...

Embodiment 3

[0065] Example 3 Rescue and Screening of Recombinant Virus rHEP-rVP2 Using Recombinant Plasmid pHEP-rVP2

[0066] 1. Transfection and virus screening methods refer to the method of Ito (2003) (Ito N., Takayama I. M., YamadaK., et al. Improved recovery of rabies virus from cloned cDNA using avaccinia virus-free reverse genetics system [J]. Microbiology and Immunology, 2003, 47 (8):613-617.).

[0067] Transfection:

[0068] (1) Prepare BHK-21 cells: Add BHK-21 cells to a 12-well cell culture plate at a cell density of 1×10 5 Cells / well were cultured at 37°C for 12-16 hours in culture medium containing 10% fetal bovine serum until the cells were 50%-60% confluent. Before transfection, the cells were washed once with 800 μL PBS buffer.

[0069] (2) Mix 1.25µg recombinant plasmid pHEP-rVP2, 0.625µg pH-N, 0.3125µg pH-P, 0.125µg pH-L and 0.1875µg pH-G in a 1.5mL centrifuge tube, add sterilized deionized water to 75 μL and centrifuge briefly so that the droplet at the top of the t...

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Abstract

The invention provides a VP2 fusion gene, a recombinant rabies virus rHEP-rVP2 strain and a construction method and application of the strain, and belongs to the field of microbial immunology. The sequence of the VP2 fusion gene is as shown in SEQ ID NO:1. The recombinant rabies virus rHEP-rVP2 strain is obtained by inserting the VP2 fusion gene between a G gene and an L gene of a genome of a HEP-Flury strain. The construction method of the recombinant rabies virus strain rHEP-rVP2 includes the steps that the VP2 fusion gene is inserted in full-length cDNA of the HEP-Flury strain to construct recombinant plasmids, a host cell is subjected to transfection, a virus is rescued, and the rHEP-rVP2 strain is obtained. The recombinant rabies virus rHEP-rVP2 strain has virus titer similar to that of a parent strain, has good immunogenicity, can generate a high-level antibody for the rabies virus and the canine parvovirus after immunizing, and can serve as a bivalent vaccine candidate strain.

Description

technical field [0001] The invention belongs to the field of microbial immunology, and specifically relates to a VP2 fusion gene, a recombinant rabies virus rHEP-rVP2 strain, a construction method and an application thereof. Background technique [0002] Rabies caused by Rabies virus (RV) has a fatality rate of almost 100%. my country is a high-incidence area of ​​rabies, and the number of cases is second only to India. At present, for rabies, effective vaccines are mainly used for prevention and control. Once a human or animal becomes ill, there is no specific medicine to treat it. Dogs have become the main host and source of infection of rabies in our country. To eliminate human rabies, it is necessary to reduce the number of stray dogs, popularize the immunity of domestic dogs, and make the immunity rate reach more than 70%. Eliminate rabies in animals first. Veterinary inactivated rabies vaccines in the prior art have high cost and unsatisfactory effects after immunizat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N7/01A61K39/205A61P31/14A61P31/20
Inventor 谭业平郭霄峰赵静何孔旺
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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