Preparation method and application of attenuated strain of contagious pustular dermatitis virus
A impetigo virus, infectious technology, applied in the field of preparation of sheep infectious impetigo virus gene-deleted strains, can solve the problems of re-virus, etc., and achieve the effect of reducing pathogenicity and good safety
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Embodiment 1
[0051] Example material sources:
[0052] Cells and viruses: The primary fetal sheep turbinate bone cells (OFTu) used in the present invention were isolated from healthy pregnant sheep at 3-5 months of pregnancy, the pregnant sheep were anesthetized, the fetal sheep were aseptically removed, the turbinate bone tissue was collected and cut into pieces, using D-Hanks The liquid was washed 2 to 3 times, and 0.25% trypsin was added for digestion for 30 min, and then the digestion was repeated 1 to 2 times. Collect the cell suspension, centrifuge at low speed, discard the supernatant, resuspend the cells in MEM medium containing 10% FBS, set at 37°C, 5% CO 2 Cultivated in an incubator. OFTu cells were used for plasmid transfection and viral recombination experiments. The ORFV-SY17 virus strain is a preserved virus strain of the Veterinary Pathology Laboratory of Jilin University, which has been published in Zhong J#, Guan J#, Zhou Y, Cui S, Wang Z, Zhou S, Xu M, Wei X, Gao Y, Zha...
Embodiment 2
[0072] Example 2 is similar to Example 1, the difference is that the 20th to 480th nucleotides in the nucleotide sequence shown in SEQ NO: 1 are knocked out, and the YFP gene is inserted at the gene deletion site, and YFP The gene is the screening gene, and the obtained cells are screened and identified. The results showed that the aphthous lesions of the lambs inoculated with the virus attenuated strain obtained by the method provided in Example 2 were relatively mild.
Embodiment 3
[0074] The preparation method, detection method and application of the attenuated ORFV strain provided in Example 3 are similar to those in Example 1 or 2, except that the mutation method is to change the 41st base of the nucleotide sequence shown in SEQ NO: 1. The base to the 500th base is knocked out, that is, the nucleotide sequence shown in SEQ NO: 19 is knocked out from the genome.
[0075] Its left and right homology arms are the nucleotide sequence of the 119960th to 120733rd position of the genome and the nucleotide sequence of the 121194th to 122021st position of the genome, respectively.
[0076] In Example 3, the late-stage 454 promoter (SL454) was synthesized using poxvirus as the promoter, and the related sequences of the promoter were recorded in the published literature, and will not be repeated here.
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