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
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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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