Coding hemaagglutinin gene of poultry influenza virus, plant expressing carrier and application thereof

A plant expression vector and avian influenza technology, applied in the field of genetic engineering, can solve the problems of reduced poultry productivity, a large amount of manpower, and a long time, and achieve the effects of good immunogenicity, increased expression, and enhanced applicability

Inactive Publication Date: 2006-11-15
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI +2
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  • Abstract
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AI Technical Summary

Problems solved by technology

The inactivated whole virus vaccine mainly used at present must be injected with a poisonous amount many times higher than the live vaccine, and an adjuvant must be added in addition, which increases the cost of the inactivated vaccine, and must be vaccinated one by one, requiring a lot of manpower, and During the process of catching chickens and injecting vaccines, a stress response occurs, resulting in a decline in poultry productivity. At the same time, during the process of immunization, the flow of epidemic prevention personnel between poultry houses and direct contact with poultry may easily cause the spread of the virus and the loss of inactivated vaccines. Another disadvantage is that the serological test of inoculated animals is positive, so it cannot be distinguished from natural infection, which affects the monitoring of epidemic diseases and cannot be used in routine immunization. In addition, inactivated vaccines cannot induce effective mucosal immune sIgA antibody production and cellular immune responses Therefore, it cannot effectively inhibit the replication of influenza virus in the respiratory tract; the attenuated vaccine has the risk of mutation into a highly pathogenic virus strain and the risk of contamination by the same strong strain, and at the same time, the attenuated vaccine also affects the monitoring of the AIV epidemic situation; A long time is not suitable for the needs of emergency prevention. With the in-depth research of DNA vaccines, people are worried that DNA will be integrated into the host cell genome, causing cancer risks. In addition, it is difficult for DNA to pass through the nuclear membrane, which limits its role. To a certain extent, it hinders the promotion and application of DNA vaccines, and it is still in the research stage, and there is still a long way to go to clinical application; recombinant live vector vaccines should be avoided in chickens immunized with vector virus vaccines or chickens naturally infected with wild vector viruses Otherwise, it will not be able to induce a good immune response to avian influenza; the conventional subunit vaccine is made by extracting the immunogenic protein of AIV. This vaccine has good safety and can also stimulate sufficient immunity. It will not interfere with the serological investigation of avian influenza virus, and it does not have the problems of strong virulence, loose poison and environmental pollution, so it is a relatively safe vaccine

Method used

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  • Coding hemaagglutinin gene of poultry influenza virus, plant expressing carrier and application thereof
  • Coding hemaagglutinin gene of poultry influenza virus, plant expressing carrier and application thereof
  • Coding hemaagglutinin gene of poultry influenza virus, plant expressing carrier and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] [Example 1] Preparation of avian influenza hemagglutinin gene NAIVHA of the present invention

[0066] According to the coden usage database published on the Internet, it is calculated that plants prefer to use codons, and then use the biological software DNASTAR to maintain the amino acid sequence of the hemagglutinin of the highly pathogenic strain of avian influenza A / Goose / Guangdong / 3 / 96 (H5N1) Under the condition of no change, the plant preferred codons are used for reverse translation, and at the same time, the plant polyadenylation signal sequence (PPSS) (AATAAA, AATAAT) and the sequence (ATTTA) in the gene sequence are shielded, and the mRNA degradation-promoting sequence (ATTTA) is improved. For the G+C% of the gene, avoid A+T%>80% in 15-30 base pairs, avoid 4 or more consecutive A or T in 10 base pairs, and avoid continuous A+T or The number of G+C is more than 5, some restriction sites are avoided, and at the same time, complex secondary structures are not fo...

Embodiment 2

[0068] [Example 2] Construction of efficient plant expression vector pC234ANAIVHA and control wild-type expression vector pC234ACKHA

[0069] 2.1 Preparation of Escherichia coli DH5a competent cells

[0070] 1) Take a single colony of the recipient bacteria DH5α grown on the LB solid medium, inoculate it in 5ml LB liquid medium, and cultivate it on a shaking table at 37°C overnight (200r / min);

[0071] 2) Transfer 1% of the inoculum to 100ml LB liquid medium, shake at 37°C and 300r / min for about 3hrs, and measure the OD 600 value (0.4-0.6) to detect the growth status of the culture;

[0072] 3) Under sterile conditions, transfer the bacteria to a sterile, ice-precooled 50ml centrifuge tube, place on ice for 10 minutes, and cool the culture to 0°C;

[0073] 4) 4°C, 4000rpm, centrifuge for 10 minutes, recover the bacterial cells, and remove the culture medium;

[0074] 5) Use 10ml of ice-cold 0.1M CaCl 2 Resuspend each cell pellet and place on ice for 30 minutes;

[0075] 6) ...

Embodiment 3

[0087] [Example 3] Agrobacterium dipping method or biolistic transformation of japonicus japonicus and identification

[0088] 3.1 Preparation of Agrobacterium Competent Cells

[0089] 1) Pick a single colony EHA105 and inoculate it in 5ml of YEB (galifampicin 100mg / l) liquid medium, culture overnight at 28°C and 200r / min shaking;

[0090] 2) Transfer 2ml of bacterial liquid into 50ml of YEB liquid medium and continue culturing to OD 600 ≈0.5;

[0091] 3) Transfer to a sterile centrifuge tube, ice bath for 30min, centrifuge at 5000r / min for 5min, and remove the supernatant;

[0092] 4) Add 2ml 20mM CaCl 2 resuspended bacteria;

[0093] 5) Dispense 200 μl per tube into sterile Eppendorf tubes and store at 4°C.

[0094] 3.2 Transformation of Agrobacterium Competent Cells

[0095] 1) Take 20 μg of extracted and purified recombinant pC234ANAIVHA and pC234ACKHA plasmid DNA, add them to 200 μl competent cells respectively, and mix well;

[0096] 2) Ice bath for 5 minutes, tra...

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Abstract

This invention discloses a new coding fowl influenza hemagglutinin protein gene VAIVHA and the construction, conversion and expression of the plant carrier. This gene can be highly expressed in the plant, and the expression value can be improved more than 40 times than the wild fowl influenza hemagglutinin gene. The gene transfer plant can be directly feed on the animal, or the protein extraction and purified protein immunize the animal, and the protection result of it is good. This shows that the gene plant has the ability to prevent the fowl influenza.

Description

technical field [0001] The present invention relates to a new avian influenza gene, in particular to a new gene encoding avian influenza hemagglutinin, the construction, transformation and expression of the gene plant expression vector and the application of the obtained transgenic plant in the prevention and control of avian influenza , belonging to the field of genetic engineering. Background technique [0002] Avian influenza (Avian influenza, AI) is a poultry disease caused by type A avian influenza virus (Avian influenza virus, AIV) of Orthomyxoviridae. Highly pathogenic avian influenza (HPAI) can cause 100% death of chicken flocks, and is identified as a class A infectious disease by the International Office of Epizootics. AIV is an RNA virus, and its single-stranded negative-strand RNA has a total length of 13.6Kb, consisting of 8 independent RNA segments, encoding PB2, PB1, PA, HA, NP, NA, M, and NS, and the RNA nucleic acid fragments are helical Aligned capsomers,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/33A61K39/12C12N15/82
Inventor 吴燕民刘建利唐益雄卢运明陈化兰
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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