Recombinant phage vaccine for avian influenza A and construction method for recombinant phage vaccine

A construction method and bacteriophage technology, which can be used in pharmaceutical formulations, antibody medical components, virus antigen components, etc., can solve problems such as difficulties in the production and supply of influenza vaccines and implementation of immunization, antigenic variation, and antigenic transformation, so as to facilitate large-scale production, Easy to purify, easy to culture effect

Active Publication Date: 2012-04-04
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the application of these vaccines has played a very good role in the prevention and control of influenza, due to the large number of HA and NA subtypes of influenza virus, antigenic shift and antigenic drift often occur, resulting in great variation in antigenicity
Although the HA and NA amino acid sequences and even the three-dimensional structure of influenza virus surface antigens have been determined in recent years, the variation rules of influenza virus surface antigens and the appearance of antigenic determinants are still unpredictable. The development of new vaccines from different strains has brought great difficulties to the production and supply of influenza vaccines and the implementation of immunization

Method used

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  • Recombinant phage vaccine for avian influenza A and construction method for recombinant phage vaccine
  • Recombinant phage vaccine for avian influenza A and construction method for recombinant phage vaccine
  • Recombinant phage vaccine for avian influenza A and construction method for recombinant phage vaccine

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Construction of recombinant phage

[0040] A DNA sequence was synthesized by a commercial company. The DNA sequence has been cloned into the multiple cloning site of the pUC-19 vector. The 5' end of the DNA sequence has a restriction site EcoRI, and the 3' end contains a restriction site HindⅢ. The DNA sequence As shown in SEQ ID NO: 3

[0041] The above DNA sequence was excised from the pUC-19 vector with restriction endonucleases EcoRI and HindIII and inserted between EcoRI and HindIII of the T7 phage multiple cloning site to construct a recombinant phage.

[0042] Recombinant pUC-19 vector double enzyme digestion system:

[0043] wxya 2 o 16.0 μL 10×H Buffer 5.0 μL pUC-19 vector 25.0 μL EcoRI 2.0 μL HindⅢ 2.0 μL

[0044] Mix the above reaction system and place it in a 37°C water bath for 4 hours. After the digested product is also identified by 1% agarose gel electrophoresis, the gel recovery kit is used for ...

Embodiment 2

[0056] Example 2 Large scale amplification of recombinant phage

[0057] Glycerol frozen E. coli Strain BL21 was inoculated on the plane of LB medium, cultured overnight at 37 °C; a single colony was picked from the plate, inoculated with 5 mL of LB culture medium, and cultured overnight at 37 °C with shaking at 200 rpm. Take 3mL overnight culture to inoculate 300mL LB culture medium, cultivate to OD 600 =0.8 or so. Pick a single phage plaque on the plate, inoculate it into the cultured BL21 host bacteria, and incubate with shaking at 37°C and 100 rpm for 2-3 hours until the bacterial solution changes from turbid to clear. The phage was recovered by PEG-NaCl precipitation, and the phage titer was determined according to the conventional method. Adjust the recovered phage concentration to 2×10 13 pfu / mL, and add formaldehyde solution at a ratio of 4‰, and inactivate overnight at 37°C and 100 rpm with shaking.

Embodiment 3

[0058] Example 3 Preparation of Type A Avian Influenza Recombinant Phage Oil Emulsion Vaccine

[0059] The preparation method of recombinant phage deodorizing oil emulsion vaccine is as follows:

[0060] Preparation of the oil phase of the vaccine: 4mL Siben 80, 2mL Siben 85, 94mL No. 10 mineral oil, 2g aluminum stearate, mix well, and press at 121°C for 20 minutes under high pressure. Preparation of vaccine aqueous phase: 94mL titer is 2×10 13 Pfu / mL of inactivated phage, 4 mL of autoclaved Tween-80, 3000 rpm and stir well. According to the ratio of water phase: oil ratio of 1:3, add 150mL oil phase to the tissue masher, slowly add 50mL water phase while stirring slowly, after mixing well, stir at 10000 rpm / quickly for 2 minutes until a stable The water-in-oil structure is the prepared recombinant phage oil emulsion vaccine, which is stored at 4°C for later use.

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Abstract

The invention provides a recombinant phage vaccine for avian influenza A and a construction method for the recombinant phage vaccine. In the vaccine, a recombinant T7 phage is taken as an antigen; peptide 23 (M2e), of which an amino acid sequence is shown as SEQ ID NO:2, of extramembrane fragments of M2 protein of subtype H5 avian influenza viruses is expressed on the surface of the recombinant T7 phage; and the M2e is obtained by inserting two copied M2e genes into a P10B gene of a T7 phage and performing downstream expression. The invention has the advantages that: (1) the recombinant phagevaccine prepared by using a T7 phage carrier can overcome the disadvantage that the autoimmunogenicity of the M2e is weaker, effectively improve the immunogenicity, and stimulate organisms to generate an antibody with higher level; (2) the M2 protein is conservative among different subtypes of avian influenza viruses, so the recombinant phage vaccine is expected to become a 'universal' avian influenza vaccine; and (3) the T7 phage is easy to culture and convenient to purify, and has various advantages when used for producing genetic engineering vaccines, so the prepared recombinant phage vaccine for the avian influenza A can be conveniently produced on a large scale and is low in cost.

Description

technical field [0001] The invention relates to the construction and application of a recombinant phage displaying the 23 peptide of the outer region of the M2 protein membrane of the T7 phage, which belongs to the field of molecular biology technology. Background technique [0002] Avian Influenza (Avian Inlfuenza, AI) is a variety of disease syndromes in poultry (poultry and wild birds) from respiratory system to severe systemic sepsis caused by type A influenza virus. The disease is defined as a Class A infectious disease by the World Health Organization, and is also listed as a Class I animal disease in my country. Generally speaking, different influenza viruses have different host specificities, that is, it is difficult for avian influenza viruses to infect humans, and it is also difficult for human influenza viruses to infect poultry. However, in Hong Kong in 1997, highly pathogenic H5N1 avian influenza was isolated from 18 patients, and it was confirmed for the first...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61K39/12A61P31/16
Inventor 徐海王义伟侯继波
Owner JIANGSU ACAD OF AGRI SCI
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