A kind of live vaccine against African swine fever recombinant pseudorabies virus and preparation method thereof

A technique for recombining pseudorabies and African swine fever, applied in botany equipment and methods, biochemical equipment and methods, viruses, etc., can solve problems such as slow transmission, achieve the effect of reducing injection stress response and stabilizing expression

Active Publication Date: 2022-04-26
北京中海生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although ASFV has a high fatality rate, it is not as infectious as severe diseases such as foot-and-mouth disease, and may spread slowly among pigs

Method used

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  • A kind of live vaccine against African swine fever recombinant pseudorabies virus and preparation method thereof
  • A kind of live vaccine against African swine fever recombinant pseudorabies virus and preparation method thereof
  • A kind of live vaccine against African swine fever recombinant pseudorabies virus and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 - Obtaining the coding genes of recombinant CD2V / P72 / B602L and sacas9SgRNA. In order to increase the recombination rate, the CD2V / P72 / B602L and sacas9SgRNA expression cassettes were placed in the recombinant homology arms gl and US2 genes. The specific gene position in the recombination arm is shown in Table 2.

[0078] Table 2 Recombinant vector design

[0079] name 1 Gene iGO 1-1121 promoter 1 1122-1671 Cd22 gene 1672-2754 promoter 2 2755-3044 P72 gene 3045-4730 promoter 3 4731-4279 B601L 4280-6872 WPRE terminator 6873-7463 promoter 4 7464-8077 Sacag gene 8079-11248 polya terminator 11249-11459 U6 promoter P72PAMgRNA 11460-11872 US2 homology arm 11873-12881

[0080] To clone the optimized antigen gene shown in SEQ ID No.1, the expression cassette containing CD2V / P72 / B602L and sacas9SgRNA is cloned into the recombinant sequence containing gl and US2. Shown, wherein ...

Embodiment 2

[0081] Embodiment 2——recombinant virus construction

[0082] 1 Construction of recombinant CD2V / P72 / B602L and sacas9SgRNA gene recombination vector

[0083] 1.1 Use PCR to amplify the gI gene US2 gene of the laboratory PRV virus, use the 3-terminal gI gene as the homology arm and the 5-terminal US2 gene as the homology arm gene, and introduce the Mlu1 / Ase1 restriction site at the same time, the gI gene and the US2 gene Introduce Ase1 and Mlu1 enzyme digestion sites in the middle, use gI and US2: to splice into recombinant arm genes, connect pEGFP vector, sequence and store correctly, shake the bacteria to extract the vector and recover it with Mlu1 and Ase1 enzyme digestion gel for recombinant CD2V / P72 / B602L expression box and sacas9SgRNAPAM gene expression box.

[0084] Primer sequence GIF: ggaggcgcgc cggct attaa t 21 (Sequence 5)

[0085] GIR: cgagccgggg gagatacgcg t 21 (sequence 6).

[0086] 1.2 Expression CD2V / P72 / B602L gene expression frame expansion (gene synthe...

Embodiment 3

[0121] Example 3—Simulation determination of rPRV TIE1872V2Sag72 strain virus to pseudovirus interferon effect

[0122] 1. Constructed with fluorescent P72

[0123] The wild-type P72 gene (sequence 19) was cloned into PDC315 to construct the MINIP72egfp gene vector to ensure no frame shift, and the adenovirus was packaged after correct sequencing.

[0124] sequence 19

[0125]

[0126] 2. BHK suspension cells were co-transfected with rPRV and MINIP72egfp-carrying adenovirus as the experimental group, and common PRV was used as the negative control to ensure that the sample volume of adenovirus in the two groups was the same, and the fluorescence values ​​of diluted detection and undiluted were weaker than wild PRV Virus experimental group, the results are as follows Figure 6 ; It shows that the sacas9SgP72PAMRNA original of the recombinant virus group works normally and can cut the P72 gene. If the wild virus encounters this working original, it will interfere with virus...

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Abstract

The invention relates to a live recombinant pseudorabies vaccine for preventing African swine fever and its preparation. The African swine fever virus CD2V / P72 / B602L gene constructed by the invention and the sacas9SgRNA sequence recombinant pseudorabies virus TIE1872V2Sag72 strain capable of knocking out the wild-type P72 gene are The BHK cell line obtained can express the recombinant CD2V / P72 / B602L protein antigen, and its expression level is stable. At the same time, the in vitro cell simulation experiment shows that this method can interfere with virus replication. The recombinant pseudorabia virus strain is used as the production strain, and the recombinant CD2V / P72 / B602L protein antigen obtained by suspension microcarrier culture is prepared into a live vaccine, which can prevent the recently popular African swine fever virus and can also be made into a spray Drinking water form of the vaccine reduces injection stress.

Description

technical field [0001] The invention relates to a live vaccine for preventing African swine fever recombinant pseudorabies virus and a preparation method thereof, belonging to the field of veterinary biological products. [0002] technical background [0003] African swine fever (ASF) is an acute and severe infectious disease of pigs caused by African swine fever virus (ASFV). ASFV can infect domestic pigs, wild boars and ticks, and there is no obvious difference in species, sex and age. It is the only DNA virus transmitted by insects. Widely distributed ticks are important storage hosts and vectors. Contact transmission, food transmission and blood-sucking transmission of soft ticks are the main routes of transmission of the disease. The virus circulates among wild boars, between wild boars and domestic pigs, and between domestic pigs, making it difficult to eradicate the disease. The disease has a short course of disease and a high mortality rate. The mortality rate of do...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/85C12N15/34C12N15/38A61K39/12A61P31/20C12R1/93
CPCC12N7/00C12N15/85C07K14/005A61K39/12A61P31/20C12N2710/16721C12N2710/12022C12N2710/12034C12N2710/16752C12N2800/107A61K2039/5256A61K2039/552A61K2039/542
Inventor 陈晴周远成王彬高洁伏显华冯倩倩杜继文李淑萍
Owner 北京中海生物科技有限公司
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