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Recombinant lactic acid bacillus efficiently expressing foot and mouth disease virus antigen genes and preparation method and application thereof

A foot-and-mouth disease virus and lactobacillus technology, applied in the biological field, can solve the problems of large vaccination dose, limited broad-spectrum, short duration of immunity, etc., and achieve the effect of human and animal safety, simple process and good immune effect

Active Publication Date: 2015-10-21
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the inactivated vaccine overcomes some shortcomings of the attenuated vaccine, the vaccination dose is large and the duration of immunity is short; the inactivated virus in the vaccine may cause a new outbreak of the disease; in addition, the antiviral immune mechanism induced by the inactivated vaccine is limited. The broad-spectrum is limited, and its limitations are becoming more and more prominent. Therefore, relevant research laboratories around the world are devoting themselves to the development of safe and effective new-type foot-and-mouth disease vaccines

Method used

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  • Recombinant lactic acid bacillus efficiently expressing foot and mouth disease virus antigen genes and preparation method and application thereof
  • Recombinant lactic acid bacillus efficiently expressing foot and mouth disease virus antigen genes and preparation method and application thereof
  • Recombinant lactic acid bacillus efficiently expressing foot and mouth disease virus antigen genes and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The acquisition of embodiment 1 opti-FMDV-A / VP1 gene

[0048] 1 Materials and methods

[0049] 1.1 Materials and sources

[0050] Foot-and-mouth disease type A virus is kept in our laboratory. Escherichia coli-lactic acid bacteria shuttle plasmid expression vector pSIP411 and Lactobacillus NC8 were purchased from Beijing Bitbo Biotechnology Co., Ltd., and the cloning vector pUC57 was provided by Nanjing GenScript Biotechnology Co., Ltd.

[0051] 1.2 Test method

[0052] 1.2.1 Gene modification and synthesis

[0053] The original sequence of the Lactobacillus signal peptide SP310 is the gene sequence retrieved by the online tool of the EMBL (smart.embl-heidelberg.de) database, the sequence number is AJ238086, using the method of increasing the positive charge of the N-terminal of the SP310 signal peptide and the hydrophobicity of the H region, and the C-terminal The deletion of amino acid residues made the H region closer to the cleavage site and other methods, and f...

Embodiment 2

[0057] Example 2 Obtaining of recombinant expression vector pSIP / opti-FMDV-A / VP1

[0058] 1 Materials and methods

[0059] 1.1 Materials and sources

[0060] Restriction enzymes NcoI, Xhol and T4 ligase were all purchased from NEB Company, and Taq enzyme, dNTP, DNA Marker DL2,000, DL15,000, Agarose Gel DNA Purification Kit, MiniBEST Plasmid Purification Kit were purchased from Dalian Bao Biological Company. The cloning vector pUC57 was provided by Nanjing GenScript Biotechnology Co., Ltd.

[0061] 1.2 Test method

[0062] The small fragment of the plasmid pUC / opti-FMDV-A / VP1 of Example 1 was digested with NcoI / Xhol, and was connected with the large fragment of the Escherichia coli-lactic acid bacteria shuttle expression vector pSIP411 through the same double digestion ( figure 1 ), transformation, and alkaline lysis to extract the plasmid. The transformant was identified by electrophoresis and enzyme digestion, and the positive recombinant plasmid was named pSIP / opti-FMDV-A...

Embodiment 3

[0065]Preparation and detection of embodiment 3 expressing type A foot-and-mouth disease virus VP1 gene

[0066] 1 Materials and methods

[0067] 1.1 Materials and sources

[0068] (1) Inducer: Bacitracin (SppIP) (20mg / mL) was synthesized by Shanghai Sangon Bioengineering Co., Ltd.;

[0069] (2) Antibiotics: Ampicillin (Amr) and erythromycin (Emr) were purchased from Baird Biotechnology Co., Ltd.

[0070] 1.2 Test method

[0071] 1.2.1 Electrotransformation of the target gene in Lactobacillus NC8 and screening of resistant strains: The electrotransformed Lactobacillus NC8 was spread on the MRS medium, cultured in a 37°C incubator for 24h-72h, and the After the colonies were picked, they were cultured overnight in MRS liquid medium;

[0072] 1.2.2 PCR detection of recombinant Lactobacillus NC8: Take 5mL of overnight culture solution to extract the plasmid, PCR amplify and detect the target gene, and use the non-transgenic carrier pSIP411 as a negative control;

[0073] 1.2...

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Abstract

The invention discloses a recombinant lactic acid bacillus efficiently expressing foot and mouth disease virus antigen genes and a preparation method and application thereof, and belongs to the technical field of biology. The recombinant lactic acid bacillus contains a foot and mouth disease virus VP1 gene sequence, wherein modified lactic acid bacillus signal peptide genes are introduced into the end N of the foot and mouth disease virus VP1 gene sequence, and codon optimization is performed on the foot and mouth disease virus VP1 gene sequence, and the nucleotide sequence is shown in the SEQ ID NO:1. The structure of lactic acid bacillus signal peptide is modified, the modified lactic acid bacillus signal peptide genes are integrated with the foot and mouth disease virus VP1 gene obtained after codon optimization is performed by means of a connection short peptide being GSGGSGG, the integrated gene sequence is inserted into an escherichia coli-lactic acid bacillus shuttle expression vector to construct a recombinant plasmid pSIP / opti-FMDV-A / VP1, and the recombinant plasmid pSIP / opti-FMDV-A / VP1 is converted into lactic acid bacillus. As for a recombinant lactic acid bacillus expression system, the new thought and new method for properly modifying the signal peptide structure to improve the foreign protein secretion efficiency are built, and theoretical basis and technical support are provided for further researching and developing novel mucosal immune vaccines.

Description

technical field [0001] The invention relates to a recombinant lactobacillus and its preparation method and application, in particular to a recombinant lactobacillus expressing a foot-and-mouth disease virus antigen gene with high efficiency, its preparation method and application, and belongs to the field of biotechnology. Background technique [0002] Foot and mouth disease (FMD) is an acute, febrile, highly contagious infectious disease of artiodactyls caused by foot and mouth disease virus (FMDV), known for its rapid spread and high incidence . The World Organization for Animal Health (OIE) lists it as one of 93 legally notifiable animal diseases. Foot-and-mouth disease virus belongs to the genus Foot-and-Mouth Disease Virus of the Picornaviridae family. The virus genome is a single-stranded positive-strand RNA, about 8.5kb. There are 7 serotypes, and there is no cross-protection between types. Its open reading frame consists of L gene, P1 structural protein gene (inclu...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74A61K39/39A61K39/135A61P31/14C12R1/225
Inventor 潘丽张永光王淼张中旺王永录方玉珍吕建亮周鹏刘新生
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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