Porcine pseudorabies virus gene deleted strain, as well as construction method and application thereof
A technology for porcine pseudorabies virus and porcine pseudorabies disease, which is applied in the fields of genetic engineering technology and virology to achieve the effect of high safety and good immune protection effect
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Embodiment 1
[0037] Example 1. Isolation and identification of a variant strain of porcine pseudorabies virus PRV HNxy
[0038] A brain tissue sample was taken from a diseased pig suspected of PRV infection from a pig farm in Xinyang, Henan, my country, which was immunized with Bartha-K61 vaccine. Part of the tissue was taken to extract genomic DNA, and PCR amplification was performed on P1F / P1R and P2F / P2R using specific primers. Increased PRV gE and gB genes, the result was positive ( figure 1 ), the amplified band sizes were 1740bp and 2893bp, which were in line with expectations; at the same time, the PCR product of the gE gene was sequenced, and the sequencing results were compared with the gE genes of multiple PRV strains published by NCBI. The results showed that the gene sequence was consistent with The current PRV variant strains are highly homologous, with an aspartic acid mutation inserted at amino acid 48 and 494 respectively, and a mutation at amino acid 316 of gE gene to threonine...
Embodiment 2
[0041] Example 2. Construction of PRV HNxy strain gene deletion virus strain
[0042] The swine pseudorabies virus variant strain gene deletion strain of the present invention uses the swine pseudorabies virus variant strain PRV HNxy isolated in Example 1 as the parent strain, by partially deleting the gI and US2 genes of the swine pseudorabies virus variant strain HNxy , GE, US9 genes are all deleted (the DNA sequence between the 268th base in the CDS region of the gl gene in the genome of the PRV HNxy variant to the 284th base in the CDS region of the US2 gene), so that gI, gE, US9 and An attenuated strain obtained by inactivating the US2 protein. The sequences of the gl, gE, US9 and US2 proteins are sequence 1, sequence 2, sequence 3 and sequence 4, respectively.
[0043] 1. Construction and identification of homologous recombination transfer plasmid containing GFP gene
[0044] 1. According to the PRV sequence published by NCBI, design and synthesize the primer pairs P3F / P3R an...
Embodiment 3
[0057] Example 3. Evaluation of the safety and immune efficacy of a live vaccine prepared by gene deletion virus PRV HNxy-ΔgIgE CGMCC No. 16900 in pigs
[0058] 1. Safety test on piglets
[0059] Eight 30-day-old healthy and susceptible piglets that were negative for PRV antibody and antigen were selected and randomly divided into 2 groups. Among them, the experimental group had 5 heads / group, each of which was injected into the neck intramuscularly with a live vaccine PRV HNxy-ΔgIgE 1.0mL (10 5 TCID 50 ); At the same time, 3 piglets in the control group were injected with 1.0 mL DMEM medium into the neck muscle. Observation continued for 14 days after immunization. The body temperature of all vaccinated pigs was measured before and after immunization, and clinical symptoms were observed and recorded. After being vaccinated with the PRV HNxy-ΔgIgE gene deletion strain vaccine, all piglets in the experimental group survived, their body temperature was normal, and did not show clini...
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