Recombinant phage peculiar smell removal vaccine for boars

A phage and odor-removing technology, applied in the direction of virus/phage, recombinant DNA technology, drug combination, etc., can solve problems affecting pig growth performance, etc., and achieve the effect of being convenient for large-scale production, easy to cultivate, and satisfying feeding management

Active Publication Date: 2013-04-24
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hormone treatment requires repeated injections, and chemical castration can sterilize but affect the growth performance of pigs

Method used

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  • Recombinant phage peculiar smell removal vaccine for boars
  • Recombinant phage peculiar smell removal vaccine for boars
  • Recombinant phage peculiar smell removal vaccine for boars

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of recombinant phage

[0042] 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

[0043] 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.

[0044] Recombinant pUC-19 vector double enzyme digestion system:

[0045] 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

[0046] Mix the above reaction system and place it in a 37 °C water bath for 4 h. After the digested products are also identified by 1% agarose gel electrophoresis, the gel recovery kit is used for gel...

Embodiment 2

[0058] Example 2 Large scale amplification of recombinant phage

[0059] 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

[0060] Example 3 Preparation of phage deodorant oil emulsion vaccine

[0061] The preparation method of recombinant phage deodorizing oil emulsion vaccine is as follows:

[0062] 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 peculiar smellremoval vaccine for boars. In the vaccine, recombinant T7 phage is used as an antigen; gonadotropin-releasing hormone (GnRH) protein shown as SEQ ID No:2 in amino acid sequences is expressed on the surface of the recombinant T7 phage; and the GnRH protein is expressed by inserting three copied GnRH genes into the downstream of a P10B gene of the T7 phage. The recombinant phage peculiar smell removal vaccine has the advantages that: (1) the recombinant phage vaccine prepared from a T7 phage carrier can overcome the defect of poor immunogenicity of a GnRH peptide hormone, improve immunogenicity effectively, stimulate organisms to generate an antibody with a higher level and inhibit the development of testicles of the boars; (2) the T7phage is easy to cultivate and purify, and has multiple advantages when used for producing genetic engineering vaccines, so the recombinant phage peculiar smell removal vaccine is convenient to produce in large scale and low in cost; and (3) an immunological method is more humanistic, so the conventional castration method is replaced, and the requirements of modern large-scale feeding and management are met.

Description

technical field [0001] The invention relates to a boar odor-removing recombinant phage vaccine which removes boar endogenous odor substances and improves meat quality by immunological methods, and belongs to the field of molecular biology technology. Background technique [0002] GnRH is synthesized by GnRH neurons in the hypothalamus and stored in the cells in the form of granules or vesicles. Thirteen different GnRHs have been isolated from the nerve tissues of vertebrates and primordial animals. The GnRH structures of all mammals including humans It is the same, its amino acid sequence is: PYROGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, that is, pyroglutamic acid-histidine-tryptophan-serine-tyrosine -Glycine-Leucine-Arginine-Proline-Glycyl Peptide. After active immunization of animals with GnRH vaccine, the animal body can be induced to produce specific anti-GnRH antibodies, which bind to the endogenous GnRH secreted by the animal itself and make it lose its biological ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/12C12N7/01C12N15/70A61P43/00C12R1/19C12R1/92
Inventor 徐海王义伟侯继波
Owner JIANGSU ACAD OF AGRI SCI
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