Genetic engineering subunit oral vaccine for African swine fever virus

An African swine fever virus and genetic engineering technology, applied in the field of vaccine preparation, can solve the problems of large losses in pig farming, inability to provide immune protection, and hidden dangers in the biological safety of attenuated live vaccines

Active Publication Date: 2022-05-10
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the preventive measures for African swine fever are mainly to strengthen supervision, strengthen disinfection and extinction, and take good biosafety protection measures. Once an outbreak occurs, the whole group can only be culled in order to be completely eradicated, which will cause great losses to the pig industry.
At present, there is still a lack of effective vaccines on the market. Among them, inactivated vaccines cannot resist the attack of virulent ASF strains and cannot provide effective immune protection, so they cannot be used as the focus of future research; while live attenuated vaccines still have biological safety issues. Hidden dangers, the safety and effectiveness of vaccination need to be further evaluated

Method used

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  • Genetic engineering subunit oral vaccine for African swine fever virus
  • Genetic engineering subunit oral vaccine for African swine fever virus
  • Genetic engineering subunit oral vaccine for African swine fever virus

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preparation example Construction

[0040] 4. Vaccine preparation.

[0041] The cell concentration is about 1.0×10 8 The CFU / mL bacterial liquid was prepared into freeze-dried powder by freeze-drying technology for preservation. Take the freeze-dried powder and add 1ml of normal saline, mix well, and take directly orally.

Embodiment 1

[0043] Embodiment 1: Preparation of African swine fever virus genetically engineered subunit oral vaccine

[0044] 1. Optimize gene fragments:

[0045] ①According to the amino acid sequences of ASFV p30 (GenBank accession number: MN270980.1), p54 (GenBank accession number: MN393476.1) and p72 (GenBank accession number: MN886930.1), the amino acid sequences obtained by transformation are SEQ ID NO : 1, the antigenic protein of ASFV p30, p54, p72 of SEQ ID NO: 2 and SEQ ID NO: 3, as the target protein expressed by host bacteria.

[0046] ② In order to enhance its immune effect, the LTB gene, which has the function of eliminating toxicity and retaining adjuvant activity, and has good antigenicity, is linked to ASFV p30, p54, and p72.

[0047] ③ For easy identification of protein expression, the nucleotide sequence of 6×His Tag was linked to the target gene, and the target protein was expressed by indirect expression of His tag.

[0048] (2) Expression vector construction

[00...

Embodiment 2

[0061] Embodiment 2: The immune effect test of combination oral vaccine to non-target animals

[0062] 1 Materials and methods

[0063] 1.1 Materials

[0064] 1.1.1 Experimental animals 40 female New Zealand white rabbits weighing about 2kg were purchased from a company in Qingdao.

[0065] 1.1.2 Two batches of recombinant vaccine products prepared by the experimental drug laboratory, the number of viable bacteria after dilution was 1.0×10 8 / ml.

[0066] 1.2 Test method

[0067] 1.2.1 Immunization test

[0068] 40 female New Zealand white rabbits weighing about 2kg were divided into 4 groups, 10 rabbits in each group, Vaccination group without LTB: combined immunization with MG1363 / pMG36e-p30-His, MG1363 / pMG36e-p54-His, MG1363 / pMG36e-p72- His group; Vaccination group with LTB: combined immunization with MG1363 / pMG36e-p30-LTB-His, MG1363 / pMG36e-p54-LTB-His, MG1363 / pMG36e-p72-LTB-His group; empty vector control group and PBS control group, Each rabbit in the recombinant b...

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Abstract

The invention provides an African swine fever virus genetic engineering subunit oral vaccine, which comprises genetic engineering lactococcus lactis for recombinant expression of African swine fever virus antigen protein, and the antigen protein is ASFV p30 protein with an amino acid sequence of SEQ ID NO: 1, ASFV p54 protein with an amino acid sequence of SEQ ID NO: 3 or ASFV p72 protein with an amino acid sequence of SEQ ID NO: 5. The antigen protein is connected with an Escherichia coli heat-labile enterotoxin B subunit LTB gene protein. The African swine fever virus genetic engineering subunit oral vaccine can effectively stimulate local immune cells of intestinal tracts to generate secretory IgA, stimulate an organism to generate mucosal reaction, and further stimulate the organism to generate cellular immunity and humoral immunity through blood circulation.

Description

technical field [0001] The invention belongs to the technical field of vaccine preparation, in particular to a genetically engineered subunit oral vaccine of African swine fever virus. Background technique [0002] African swine fever (ASF) is an acute, febrile, highly contact and fatal zoonotic disease caused by African swine fever virus (ASFV). ASFV is the only member of the African swine fever virus genus (Asfivirus) in the African swine fever virus family (Asfarviridae), and is the only known arbovirus with a DNA genome. The main manifestations of the diseased pigs are elevated body temperature, extensive bleeding of the skin and mucous membranes, and abortion of pregnant sows. The fatality rate of African swine fever is as high as 100%, which has brought serious harm to the pig industry. [0003] ASF is different from other animal diseases. It has been more than 100 years since the first outbreak in 1921. At present, the preventive measures for African swine fever ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C12N15/34C07K19/00C12N15/62C12N15/74C12N1/21A61K39/12A61P31/20C12R1/01
CPCC07K14/005C07K14/245C12N15/746A61K39/12A61P31/20A61K2039/542A61K2039/60A61K2039/552C12N2710/12034C12N2710/12022C07K2319/55Y02A50/30
Inventor 蔡秀磊赵赛赛张洪亮单虎秦志华张皓杰
Owner QINGDAO AGRI UNIV
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