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Attenuated African swine fever virus with gene deletion and its application as a vaccine

An African swine fever virus, gene deletion technology, applied in the direction of viruses, vaccines, viral peptides, etc., can solve the problem of unsatisfactory vaccine effectiveness and other problems

Active Publication Date: 2020-02-18
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In view of the lack of African swine fever vaccine and the unsatisfactory effectiveness of known vaccines, especially the blank of domestic vaccines, the preparation method of African swine fever gene deletion attenuated vaccine was developed, and two safe and effective vaccine strains were successfully obtained

Method used

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  • Attenuated African swine fever virus with gene deletion and its application as a vaccine
  • Attenuated African swine fever virus with gene deletion and its application as a vaccine
  • Attenuated African swine fever virus with gene deletion and its application as a vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, homologous recombination vector construction

[0040] The genome of about 1000bp around the CD2V gene ORF (as the left and right homology arms, the sequence position is at position 73394-74476 relative to the full-length sequence) and the PCR fragment of eGFP were cloned using a one-step cloning kit (purchased from China Novozyme Biotechnology Co., Ltd. Company) was cloned into the pBluescriptII KS (+) vector, in which the eGFP gene was expressed using the promoter of the CD2V gene itself, and the plasmid pB-LRΔCD2V-eGFP ( figure 1 ).

[0041]MGF360-505R (about 1000 bp on the left side of the 5'-aagccctgagaacagcagca-3' sequence of the ASF genome and about 1000 bp on the right side of the 5'-gcgagacgtttcaataaaag-3' sequence) left and right homology arms (relative to the full-length sequence position at 27942-35500 position), p72 promoter [Reis, A.L., et al., Deletion of the African Swine FeverVirus Gene DP148R Does Not Reduce Virus Replication in Culture ...

Embodiment 2

[0042] Example 2. Construction, purification and identification of gene-deleted African swine fever virus

[0043] Plasmids pB-LRΔ360-eGFP and pB-LRΔ360-mCherry were transfected into ASFV-infected PAM cells with TranslT-LT1 transfection reagent (purchased from Mirus Bio, USA), and CD2V and MGF360-505R genes were purified by plaque cloning method, respectively. Deletion of the African swine fever gene deletion virus that expresses green fluorescent protein [Reis, A.L., et al., Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigsand Induces High Levels of Protection against Challenge.J Virol, 2017.91(24).; Chen, W., et al., A goat poxvirus-vectored peste-des-petits-ruminants vaccine induces long-lasting neutralization antibody to high levels in goats and sheep. Vaccine, 2010.28(30) : p.4742-50.; Chen Weiye, et al., Research on recombinant goat pox virus vaccine expressing Peste des petits ruminants...

Embodiment 3

[0047] Embodiment three, the titration of virus titer

[0048] The titration of African swine fever virus adopts half cell infection dose (50%Tissue Culturelnfectious Dose, TCID respectively) 50 ) and half hematosorption (50% haemadsorption, HAD 50 ) in two ways.

[0049] TCID 50 Titration was carried out according to the following steps: ASFV was serially diluted 10 times with serum-free 1640 culture medium, inoculated and cultured in PAM cells with a density of about 90-100% in a 96-well culture plate, and each dilution was inoculated into 8 wells, each well 0.02mL, under the conditions of 37°C and 5% CO2 for 1 hour, then add 0.1mL of 1640 complete culture solution containing 10% fetal bovine serum to each well, and culture under the conditions of 37°C and 5% CO2, observe 3-7 days, according to cytopathic or green fluorescence and Reed and Muench method [4] to calculate the half of the infected cells (TCID 50 ).

[0050] HAD 50 The experiment was carried out according ...

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Abstract

The invention relates to a gene deletion attenuated African swine fever virus as a vaccine and the vaccine, and a construction method thereof. An African swine fever Chinese epidemic strain Pig / CN / HLJ / 2018 is adopted, a virulence gene of the African swine fever virus is deleted by a genetic engineering technology, and the gene deletion virus of MGF360-505R deletion and joint deletion of CD2V and MGF360-505R is obtained. Experiments show that the two virus strains can provide 100% immune protection against the African swine fever Chinese epidemic virulent strains, can be used as vaccines for safe and effective prevention and control of African swine fever in China, and have great social value.

Description

technical field [0001] The invention relates to the field of veterinary medicine, in particular to the prevention and treatment of animal diseases, and more specifically to attenuated African swine fever virus and vaccines. Background technique [0002] African swine fever virus is the only member of the Asfarviridae family and replicates primarily in the cytoplasm of cells. African swine fever is an acute, virulent, and highly contagious disease of domestic pigs and wild boars. The virulent strain can kill domestic pigs within about 5-14 days of infection, and the mortality rate is close to 100%. There is no effective preventive vaccine. There is no specific treatment drug. Domestic pigs, wild boars and soft ticks are the natural hosts of African swine fever at various stages. They can be transmitted directly between domestic pigs and wild boars, or through tick bites, or through virus-contaminated swill, feed, and cured dried ham and other pork products spread across cou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01A61K39/12A61P31/20C12R1/93
CPCA61K39/12A61K2039/552A61P31/20C07K14/005C12N7/00C12N2710/12021C12N2710/12022C12N2710/12034
Inventor 步志高陈伟业赵东明何希君刘任强柳金雄
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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