Canine distemper attenuated vaccine strain and medical purpose
An attenuated vaccine and canine distemper virus technology, applied in the biological field, can solve the problems of decreased expression level, long attenuation period of passage, and impact on the immunogenicity of canine distemper vaccine, so as to achieve genetic stability and solve the effect of variation
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Embodiment 1
[0034] 1. Screening of main popular strains:
[0035] 1) Extraction and reverse transcription of total RNA
[0036] The lung tissues of dogs clinically diagnosed as canine distemper were collected, ground with sterile PBS to make a 10% lung tissue suspension, and stored at -80°C. Take 100 μL of suspension from the stock solution, extract total RNA according to conventional methods, and use PrimeScript RT reagent kit for reverse transcription reaction. The reverse transcription system is as follows:
[0037] Table 1, RT reaction system
[0038]
[0039] Reverse transcription was carried out in a PCR machine, and the reaction conditions were reverse transcription at 37°C for 15 min, followed by heating at 85°C for 5 s to inactivate reverse transcriptase. After the reaction, the cDNA solution was stored at -20°C.
[0040] 2) PCR amplification of H gene
[0041] Using cDNA as a template, apply primers targeting the H gene
[0042] QH1: TTCTG AGGCA GATGA GTTCT TC,
[0043...
Embodiment 2
[0069] The N gene and NP gene of the total RNA of the clinical epidemic strains CC12 and CC12-30 screened in Example 1 were amplified and sequenced respectively. Wherein QN1 and QN2 are N gene amplification primers, and QNP1 and QNP2 are NP gene amplification primers. Electrophoresis of N, H and NP gene PCR products of CC12 and CC12-30 see figure 1 , figure 2 .
[0070] Primer name Primer sequence QN1 TTCTG AGGCA GATGA GTTCT TC QN2 CTTGG ATGCT ATTTC TGACA CT QNP1 ACAGGATTGCTGAGGACCTAT QNP2 CAAGATAACCATGTACGGTGC
[0071] The N gene sequence of the clinical epidemic strain is shown in SEQ ID NO: 3, and the fragment length is 829bp.
[0072] The NP gene sequence of the clinical epidemic strain is SEQ ID NO: 4, and the fragment length is 289bp.
[0073] The N gene sequence of CC12-30 is SEQ ID NO: 5, and the fragment length is 829bp
[0074] The sequence of the NP gene of CC12-30 is SEQ ID NO: 6, and the fragment length is 289bp. ...
Embodiment 3
[0077] Genetic Stability Verification of the Vaccine Strain of the Present Invention
[0078] 1) Cell passage verification
[0079] The CC12-30 of the present invention was continuously passed on for 10 generations on vero cells, and the total RNA was extracted from the vaccine virus of every 5 generations, and the method of Example 1 was used to amplify the H gene and determine the sequence, and the obtained sequence was found by sequence comparison. , its H gene is exactly the same as CC12-30.
[0080] 2) Passage verification of susceptible dogs
[0081] Take the FK81 cell culture of CC12-30, and inoculate 5 1-month-old beagle puppies intramuscularly, 2ml each, and kill the dogs 5 days later, collect lung tissue separately, prepare 10% lung tissue suspension, and freeze-thaw at -80°C Twice, collect 5000g of centrifuged supernatant, then get 40000g of precipitate and suspend it with PBS to 2ml, inoculate the puppies of the same month age again, collect and prepare PBS suspe...
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