Attenuated and live vaccine of African swine fever with deletion of E66L and I267L genes

An African swine fever virus, E66L technology, applied in the field of veterinary biological products, can solve problems such as chronic diseases of vaccines

Pending Publication Date: 2020-11-27
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, the attenuated vaccine strains that have deleted genes such as ASFV MGF360, MGF505, and CD2v, as well as the natural attenuated strains isolated in nature, have been proved to have certain immune effects in pigs. However, it was found in field experiments that these natural attenuated viruses and The use of artificial deletion strain vaccines can cause chronic lesions, such as skin ulcers, fever, joint swelling and other adverse reactions (King et al., 2011; Leitao et al., 2001; Revilla et al., 1992)

Method used

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  • Attenuated and live vaccine of African swine fever with deletion of E66L and I267L genes
  • Attenuated and live vaccine of African swine fever with deletion of E66L and I267L genes
  • Attenuated and live vaccine of African swine fever with deletion of E66L and I267L genes

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Experimental program
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Effect test

Embodiment 1

[0030] Preparation of cells, strains and plasmids

[0031] 1. Porcine primary alveolar macrophages (PAM) and primary bone marrow macrophages (BMDM) were obtained from piglets aged 2-3 months. The isolated PAM cells were cultured in 1640 complete medium (gibco) containing 10% FBS (TBD) ) culture, BMDM should be induced and cultured in 1640 complete medium containing 10ng / ml GM-CSF and 10% FBS at the final concentration;

[0032] 2. African swine fever virus SY-18 strain, Genebank accession number: MH766894.1, was isolated by the Epidemiology Laboratory of the Military Veterinary Research Institute in 2018. The strain used in this research is the fourth generation of PAM cell expansion virus. Store in separate packages at -80°C;

[0033] 3. The expression vectors pUC-EGFP and pUC-mCherry are plasmids carrying green fluorescent EGFP and red fluorescent mCherry respectively, and their fluorescent genes are expressed under the control of p72 promoter.

Embodiment 2

[0035] Construction of ASFV△E66L gene deletion strain ASFV△E66L

[0036] 1. Construction of homologous recombination vector Using the method of homologous recombination, the left and right homology arms of the gene to be deleted are the left homology arm of the E66L gene (about 1200bp on the left side of the E66L gene, E66L-Larm) and the right homology arm of E66L Arm (about 1200bp on the right side of the E66L gene, E66L-Rarm), cloned into the pUC-EGFP vector sequentially, such as figure 1 Shown; get the recombinant plasmid p△E66L-EGFP;

[0037] 2. Screening, purification and identification of recombinant virus. Transfect the recombinant plasmid p△E66L-EGFP into BMDM cells. After 4 hours, add SY-18 virus liquid according to the infection amount of 1MOI. After culturing at 37°C for 48 hours, about 50- 100 scattered green fluorescent cells, pick the cells with green fluorescence and put them into fresh BMDM cells to complete a round of purification, this is the P1 round of vir...

Embodiment 3

[0039] Construction of ASFV△I267L gene deletion strain ASFV△I267L

[0040] 1. Construction of homologous recombination vector Using the method of homologous recombination, the left and right homology arms of the gene to be deleted are the left homology arm of the I267L gene (about 1200bp on the left side of the I267L gene, I267L-Larm) and the right homology arm of I267L Arm (about 1200bp on the right side of the I267L gene, I267L-Rarm), cloned into the pUC-EGFP vector sequentially, such as figure 2 Shown; get the recombinant plasmid p△I267L-EGFP;

[0041] 2. Screening, purification and identification of recombinant virus. Transfect the recombinant plasmid p△I267L-EGFP into BMDM cells. After 4 hours, add SY-18 virus liquid according to the infection amount of 1MOI. After culturing at 37°C for 48 hours, about 50- 100 scattered green fluorescent cells, pick the cells with green fluorescence and put them into fresh BMDM cells to complete a round of purification, this is the P1 r...

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Abstract

The invention discloses an attenuated and live vaccine of African swine fever with deletion of E66L and I267L genes, and discloses a novel attenuated gene-deleted African swine fever virus. By deleting part or all of gene functions of E66L and I267L, compared with a parent strain, an obtained recombinant strain has the advantages that after pigs are infected, fever and viremia do not appear or areremarkably relieved, and test pigs are all healthy and alive; and the vaccine prepared from the gene-deleted live virus can protect susceptible pigs from ASFV virulent infection or artificial attack,and can be used for preventing the African swine fever.

Description

[0001] field of invention [0002] The present invention relates to an attenuated African swine fever gene deletion, in particular discloses an attenuated African swine fever gene that lacks part or all of the functions of E66L and I267L genes. The present invention further provides an African swine fever gene prepared by using the attenuated gene A missing live vaccine belongs to the technical field of veterinary biological products. Background technique [0003] African swine fever (ASF) is a highly contagious and fatal disease of pigs caused by African swine fever virus (ASFV), with a mortality rate close to 100%, which brings great losses to the pig industry. For the prevention and control of African swine fever, the methods of culling and removal were mainly adopted in the past. So far, no vaccine has been successfully developed. The African swine fever virus genome is 170-190kb in length and encodes 150-180 ORFs. So far, a large number of genes related to virus virulenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N7/04C12N15/85A61K39/12A61P31/20C12R1/93
CPCC12N7/00C12N15/85A61K39/12A61P31/20C12N2710/12021C12N2710/12062C12N2800/107C12N2710/12034A61K2039/552
Inventor 张静远陈腾张艳艳岳慧贤扈荣良
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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