O-type foot-and-mouth disease recombined phage vaccine of pig and construction method thereof

A construction method and phage technology, which can be applied to viruses/phages, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of overlapping single topological structures, and achieve the effects of improving immunogenicity, easy cultivation and low cost.

Inactive Publication Date: 2012-04-18
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

They are both functionally independent, but the

Method used

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  • O-type foot-and-mouth disease recombined phage vaccine of pig and construction method thereof
  • O-type foot-and-mouth disease recombined phage vaccine of pig and construction method thereof
  • O-type foot-and-mouth disease recombined phage vaccine of pig and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of recombinant phage

[0041] 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

[0042] 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.

[0043] Recombinant pUC-19 vector double enzyme digestion system:

[0044] 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

[0045] 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

[0057] Example 2 Large scale amplification of recombinant phage

[0058] 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 a 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

[0059] Example 3 Preparation of porcine O-type foot-and-mouth disease recombinant phage oil emulsion vaccine

[0060] The preparation method of porcine O type foot-and-mouth disease recombinant phage oil emulsion vaccine is as follows:

[0061] 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 an O-type foot-and-mouth disease recombined phage vaccine of a pig and a construction method thereof. The vaccine adopts recombined T7 phage as an antigen, and the amino acid sequence such as FMDVVP1(129-169) polypeptide of Mya98 pedigree shown in SEQIDNO:2 is expressed on the surface of the recombined T7 phage; and the VP1 (129-169) polypeptide is obtained by inserting single-copy VP1(129-169) genes into the downstream of the P10B gene of the T7 phage to carry out fusion. The invention has the following technical effects that (1) the recombined phage vaccine prepared by the T7 phage carrier can overcome the shortcomings of weaker self immunogenicity of the FMDVVP1 polypeptide and the defect of lack of T auxiliary cell epitope, effectively improve the immunogenicity and stimulate the organism to generate the higher-level antibody; and (2) since the T7 phage is easy to culture, the purification is convenient, and the invention for producing the gene engineering vaccine has multiple advantages. In the O-type foot-and-mouth disease recombined phage vaccine of the pig, the on-scale production is convenient and the cost is low.

Description

Technical field [0001] The invention involves T7 phage display, pig -Oh -type hoof -oral virus VP1 protein's main antigen surface restructuring and application, which belongs to the field of molecular biotechnology. Background technique [0002] Skill disease (FMD) is an acute and severe infectious disease of occasional hoof animals, which seriously threaten domestic and foreign trade at home and abroad and even human health of animals and products.Once FMD outbreaks, compulsory measures such as blocking, isolation, and killing livestock adopted will bring major economic losses to the country or region.Therefore, FMD has been listed by the World Animal Health Organization (OIE) in the first infectious disease of Class A.The pathogen of the foot -and -mouth disease is a faire -and -mouth vaccine virus, which is a member of the small RNA virus and a member of the foot -and -mouth disease virus.The virus particles are twenty -mandarin, no capsules, 20 to 30nm in diameter, and the st...

Claims

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Application Information

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IPC IPC(8): A61K39/135A61P31/14C12N7/01C12N15/63C12R1/92
Inventor 徐海王义伟侯继波
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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