Three recombinant lactic acid bacteria for expressing avian influenza virus proteins as well as construction method and application thereof

A technology of avian influenza virus and recombinant lactic acid bacteria, applied in the biological field, can solve the problems of low human health, and achieve the effect of great harm and rapid spread

Inactive Publication Date: 2020-08-07
张大生
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

H9 virus is ubiquitous in Asia, and due to its low toxicity, it poses a low threat to human health, but it is necessary to beware of the "shuffling" effect of H9 and other influenza viruses, and the risk of creating new influenza viruses

Method used

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  • Three recombinant lactic acid bacteria for expressing avian influenza virus proteins as well as construction method and application thereof
  • Three recombinant lactic acid bacteria for expressing avian influenza virus proteins as well as construction method and application thereof
  • Three recombinant lactic acid bacteria for expressing avian influenza virus proteins as well as construction method and application thereof

Examples

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Effect test

Embodiment 1

[0037] This embodiment provides a method for constructing recombinant lactic acid bacteria expressing H5N6 avian influenza virus NP protein, comprising the following steps:

[0038] (1) extract H5N6 avian influenza virus RNA, carry out reverse transcription, obtain cDNA; The gene sequence of coding H5N6 avian influenza virus NP protein is shown in SEQ ID NO: 1, and the primer group H5N6-NP-1077F and H5N6 of synthetic NP gene -NP-1077R, the sequence is shown in SEQ ID NO:7 and SEQ ID NO:8;

[0039] (2) carry out PCR amplification with described cDNA and described primer set, obtain amplified product, carry out agarose gel recovery with described amplified product, obtain target gene;

[0040] (3) Perform homologous recombination of the target gene and the pVE5523 vector to obtain a homologous recombination product; clone the homologous recombination product, pick positive clones for sequencing verification, and obtain the recombinant plasmid pVE5523-H5N6-NP, map Such as figur...

Embodiment 2

[0046] This embodiment provides a method for constructing recombinant lactic acid bacteria expressing H7N9 avian influenza virus NP protein, comprising the following steps:

[0047] (1) extract H7N9 avian influenza virus RNA, carry out reverse transcription, obtain cDNA; The gene sequence of coding H7N9 avian influenza virus NP protein is shown in SEQ ID NO: 3, the primer group H7N9-NP-1515F and H7N9 of synthetic NP gene -N9-1515R, the sequence is shown in SEQ ID NO:9 and SEQ ID NO:10;

[0048] (2) carry out PCR amplification with described cDNA and described primer set, obtain amplified product, carry out agarose gel recovery with described amplified product, obtain target gene;

[0049] (3) Perform homologous recombination of the target gene and the pVE5523 vector to obtain a homologous recombination product; clone the homologous recombination product, pick positive clones for sequencing verification, and obtain the recombinant plasmid pVE5523-H7N9-NP, map Such as figure ...

Embodiment 3

[0055] This embodiment provides a method for constructing recombinant lactic acid bacteria expressing H9N2 avian influenza virus HA protein, comprising the following steps:

[0056] (1) extract H9N2 avian influenza virus RNA, carry out reverse transcription, obtain cDNA; The gene sequence of encoding H9N2 avian influenza virus HA protein is shown in SEQ ID NO: 5, and the primer set H9N2-HA-579F and H9N2 of synthetic NP gene -HA-579R, the sequence is shown in SEQ ID NO:11 and SEQ ID NO:12;

[0057] (2) carry out PCR amplification with described cDNA and described primer set, obtain amplified product, carry out agarose gel recovery with described amplified product, obtain target gene;

[0058] (3) Combine the target gene with the pVE5523 vector (the map is as follows: Figure 4 shown) for homologous recombination to obtain homologous recombination products; clone the homologous recombination products, pick positive clones for sequencing verification, and obtain recombinant plas...

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Abstract

The invention relates to three recombinant lactic acid bacteria for expressing avian influenza virus proteins as well as a construction method and application thereof. The recombinant lactobacillus strain for expressing avian influenza virus protein contains any one of a gene sequence for encoding H5N6 avian influenza virus NP protein, a gene sequence for encoding H7N9 avian influenza virus NP protein or a gene sequence for encoding H9N2 avian influenza virus HA protein. The construction method mainly comprises the following steps: bonding a target gene with a lactic acid bacteria expression vector; and introducing recombinant plasmid into the competence of ATCC393 lactobacillus casei to obtain a recombinant lactic acid bacteria strain. The recombinant lactic acid bacteria can express specific protein in an animal body, stimulate the body to generate an antibody with a protective effect, and have important significance for preventing and treating H5, H7 and H9 avian influenza. The invention also provides an oral vaccine or medicine for preventing and treating avian influenza, which can effectively prevent and treat H5, H7 and H9 avian influenza.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to three recombinant lactic acid bacteria expressing avian influenza virus protein and its construction method and application. Background technique [0002] Avian influenza virus, which belongs to type A influenza virus, is divided into three levels of high, medium and low / non-pathogenicity according to the pathogenicity of avian influenza virus to chickens and turkeys. Due to the characteristics of the hemagglutinin structure of the avian influenza virus, it generally infects poultry. When the virus undergoes gene reassortment during the replication process, the structure changes and the ability to infect humans may result, which may cause human infection with avian influenza. [0003] There are many types of clinical manifestations of this disease, some are asymptomatic latent infection, some are respiratory infection with low lethality, and some are acute hemorrhagic inf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/44C12N15/74A61K39/145A61P31/16C12R1/245
CPCC07K14/005C12N15/74A61K39/12A61P31/16A61K2039/523A61K2039/542A61K2039/552
Inventor 张大生
Owner 张大生
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