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
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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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