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African swine fever virus antigen protein recombinant expression vector, recombinant plant lactobacillus as well as preparation method and application of recombinant plant lactobacillus

A technology of African swine fever virus and expression vector, which is applied in the field of recombinant plant lactic acid bacteria and its preparation, and recombinant expression vector of African swine fever virus antigen protein, which can solve problems such as chronic or persistent infection, lack of production cell lines, and inability to be used as a vaccine

Active Publication Date: 2022-05-03
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is generally only protective against homologous strains of the same genotype, and is ineffective against infection by heterologous viruses
In addition, live attenuated strains often have associated side effects, such as skin lesions and joint swelling, making them unavailable as safe vaccines
Live virus vaccines may also cause chronic or persistent infection with risk of regressive virulence
Another problem with live attenuated vaccines is the lack of stable production cell lines, since ASFV replicates preferentially in primary monocytes or macrophages

Method used

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  • African swine fever virus antigen protein recombinant expression vector, recombinant plant lactobacillus as well as preparation method and application of recombinant plant lactobacillus
  • African swine fever virus antigen protein recombinant expression vector, recombinant plant lactobacillus as well as preparation method and application of recombinant plant lactobacillus
  • African swine fever virus antigen protein recombinant expression vector, recombinant plant lactobacillus as well as preparation method and application of recombinant plant lactobacillus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Construction and verification of recombinant plant lactic acid bacteria expressing African swine fever virus fusion antigen

[0112] 1.1 Materials and methods

[0113] The strains and plasmids used in this experiment are listed in Table 1.

[0114] Table 1 Strains and plasmids

[0115]

[0116] 1.1.1.2 Enzymes and main test reagents

[0117] Restriction enzymes Xba I and HindⅢ, PrimeSTAR Max Premix (2×), DNA Marker (DL5000, DL10000, DL15000), 10xLoading Buffer, and nucleic acid electrophoresis gel agarose were purchased from Yubao Bioengineering (Dalian) Co., Ltd. ( Takara); Rapid DNA Ligation Kit (Rapid DNA Ligation Kit), DNA Blunting Kit (DNA Blunting Kit), BCA Protein Concentration Assay Kit (Enhanced BCA Protein Assay Kit) were purchased from Beyond Biotech Co., Ltd.; SppIP was preserved by the Animal Microecological Preparation Engineering Research Center of Jilin Province; beef powder was purchased from OXOID company in the UK; DAP and D-alanine were purchas...

Embodiment 2

[0343] The immune effect of the recombinant plant lactic acid bacteria constructed in embodiment 1:

[0344] Plant lactic acid bacteria vaccine has the advantages of low cost and convenient large-scale production, and can colonize the intestinal tract of the body for a long time and continue to play a role. Moreover, plant lactic acid bacteria themselves are probiotics, which can improve the body's immunity and promote mucosal immunity. Oral immunization makes it more convenient. Therefore, the recombinant plant lactic acid bacteria vaccine has broad development prospects. The recombinant plant lactic acid bacteria constructed in this experiment are non-antibiotic plant lactic acid bacteria, which are more friendly to the environment and avoid antibiotic tolerance for animals.

[0345] Example 2 The experiment set up the initial immunization and booster immunization procedures, induced the expression of recombinant plant lactic acid bacteria in two groups, and set up a contro...

Embodiment 3

[0411] Construction of recombinant Lactobacillus plantarum and protein expression verification and purification

[0412] 1.1 Materials

[0413] 1.1.1 Plasmids

[0414] Taking the African swine fever virus strain BA71V as the reference sequence, the sequence of the P14.5 protein gene (E120R) in the strain was selected and synthesized by Nanjing GenScript Biotechnology Co., Ltd. according to the codon expression of Lactobacillus plantarum. At the same time, the two adjuvant sequences were optimized and placed at the 5'3' end (CTA1-DD) and 3' end (IL-33) of the E120R gene, P14.5, CTA1-P14.5-D-D, P14.5- After the IL-33-Mus sequence was synthesized, it was inserted into the single-anchor expression vector pLP-S with erm as the selection marker, and three plasmids pLP-S-p14.5, pLP-S-p14.5-IL-33 and pLP- S-CTA1-p14.5-D-D.

[0415] 1.1.2 Strains and plasmid vectors

[0416] NC8 lactic acid bacteria are preserved by the laboratory, and the lactic acid bacteria surface display syste...

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Abstract

The invention relates to the technical field of microorganisms, in particular to an African swine fever virus antigen protein recombinant expression vector, recombinant plant lactic acid bacteria and a preparation method and application thereof, and the African swine fever virus antigen protein recombinant expression vector comprises P54, Fc, P30 and EGP gene segments; wherein the P54 and the P30 are cell membrane structure protein gene sequences related to host adsorption and internalization effects, which are coded in an African swine fever virus gene type II SY-18 strain; the Fc is a porcine IgG3Fc gene sequence, and the EGP is an intestinal cell HSPGs receptor targeting ligand polypeptide sequence. The recombinant lactobacillus plantarum obtained by the invention can efficiently express P54 / P30 antigen protein, and has good immunogenicity. Meanwhile, compared with the original strain L.Plantarum NC8, the strain can induce mucosal immunity, cellular immunity and humoral immunity response, and has good practical application value.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a recombinant expression vector of African swine fever virus antigenic protein, recombinant plant lactic acid bacteria and a preparation method and application thereof. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] African swine fever (ASF) is an acute, highly contagious, severe infectious disease caused by African swine fever virus (ASFV), with high mortality. As there are still no effective commercialized vaccines and specific drugs to prevent and treat the disease, African swine fever virus has mutated during the transmission process between hosts, resulting in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/62C12N1/21A61K39/12A61K39/39A61P31/20A23K10/18C12R1/25
CPCC12N15/746C07K14/005C07K14/54A61K39/12A61K39/39A61P31/20A23K10/18C12N2800/22C12N2800/101C07K2319/30C07K2319/035C07K2319/55C07K2319/705C12N2710/12022C12N2710/12034A61K2039/523A61K2039/552A61K2039/55527A61K2039/55544A61K2039/55516C12R2001/25Y02A50/30
Inventor 王春凤杨桂连邹博识牛天明杨文涛石春卫
Owner JILIN AGRICULTURAL UNIV
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