WO2017211052A1 - Vaccin en microcapsule de protéine flic - Google Patents

Vaccin en microcapsule de protéine flic Download PDF

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Publication number
WO2017211052A1
WO2017211052A1 PCT/CN2016/107373 CN2016107373W WO2017211052A1 WO 2017211052 A1 WO2017211052 A1 WO 2017211052A1 CN 2016107373 W CN2016107373 W CN 2016107373W WO 2017211052 A1 WO2017211052 A1 WO 2017211052A1
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flic
vaccine
microcapsules
microcapsule vaccine
microcapsule
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PCT/CN2016/107373
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English (en)
Chinese (zh)
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类延乐
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类延乐
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/501—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]

Definitions

  • the invention belongs to the technical field of preparation of aquaculture vaccines, and particularly relates to a floc protein microcapsule vaccine.
  • Edwardsiella tarda is an important pathogen of aquatic animals. It belongs to the family Enterobacteriaceae and belongs to the genus Edwards. This bacterium was first reported by Hoshina in blush disease and is currently a great hazard in aquaculture. Pathogens. The bacteria can infect a variety of fish including freshwater and marine aquaculture, and can cause a large number of deaths in a short period of time, which has brought huge economic losses to China's aquaculture industry.
  • vaccines can improve the specific immunity level of farmed fish, make the fish body immune to the infection of specific pathogenic microorganisms, do not pollute the environment, and have no drug residues. It is the mainstream development direction of fish disease prevention and control in the future. Therefore, screening immunogenic antigens and using them to prepare vaccines has practical application significance.
  • the object of the present invention is to provide a fliC protein microcapsule vaccine, which can effectively prevent diseases caused by E. faecalis, thereby making up for the deficiencies of the prior art.
  • microcapsule vaccine provided by the present invention, wherein the amino acid sequence of the antigen fliC protein is SEQ ID NO: 1;
  • the gene encoding the fliC protein has a nucleotide sequence of SEQ ID NO: 2;
  • the microcapsule vaccine of the invention comprises mixing the fliC protein with the astragalus polysaccharide, mixing with sodium alginate, and preparing the emulsion with distilled water, wherein the final concentration of sodium alginate is 2%, the emulsion is evenly stirred, and then sprayed.
  • Calcium alginate microcapsules were formed in a solution with a concentration of 3% CaCl 2 , and stirring was continued for 20 min after the spraying; the calcium alginate microcapsules were collected by centrifugation, and then the microcapsules were dispersed into a 0.2% chitosan solution and thoroughly stirred. Thereafter, the microcapsules are collected by centrifugation, washed with distilled water, and the collected microcapsules are dispersed in a mannitol solution, mixed, frozen, and lyophilized to prepare a microcapsule vaccine.
  • microcapsule vaccine of the present invention can be used as a feed additive.
  • the fliC protein vaccine provided by the invention has strong immunogenicity and has good immune effect. Moreover, the oral immunization has less stress on the smaller fish body, and the labor cost is low, which is convenient for large-scale promotion and application.
  • the vaccine has no peculiar smell, good palatability, and easy to eat fish.
  • Figure 1 Sequence comparison of the fliC protein of the present invention with the fliC gene in NCBI.
  • a gingival brood cultured in a gingival farm in Shandong province showed obvious symptoms of delayed E. faecium infection, but the diseased fish had been previously treated with a delayed E. faecium vaccine.
  • the fish were obtained from the dead fish under sterile conditions, and streaked on a common nutrient agar plate. After incubation at 30 ° C, the dominant colonies were picked and purified until the pure culture was obtained.
  • fliC forward primer CGGGATCCATGGCACAAGTAATCAACACC
  • fliC reverse primer CCGCTCGAGACGCAGCAGAGACAGGACGTTC;
  • the bacterial genome extraction kit was used to extract bacterial genomic DNA as a template, and the positive and negative primers of each gene were used for PCR amplification.
  • the PCR reaction system and reaction conditions were: 50 ⁇ l reaction system containing 37 ⁇ l H 2 O, 5 ⁇ l.
  • the amino acid sequence obtained by the present invention is the fliC protein of SEQ ID NO: 1 compared to the fliC of NCBI. There are differences in the presence of more than 10 amino groups in the gene (Fig. 1).
  • the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) obtained is SEQ ID NO: 3, NCBI ratio
  • GAPDH is consistent with the published sequences in NCBI.
  • the immunization effect showed that the vaccine prepared by using the fliC protein having the amino acid sequence of SEQ ID NO: 1 had a better immunological effect on the ET strain than the vaccine prepared by the published fliC protein in NCBI.
  • Example 2 Expression of recombinant protein and its coupling with lipopolysaccharide
  • fliC gene expression vector The amplified diploid E. faecalis fliC gene product was recovered and purified, and the purified product was digested with BamHI and XholI, ligated into prokaryotic expression vector pET32a, and its expression vector pET32a- fliC, and the expression vector was transformed into E. coli BL21 (DE3), cultured on LB plate containing ampicillin, and the positive clone expression bacteria were screened by PCR method, and the PCR-positive clones were further utilized. Sequencing was confirmed.
  • the recombinant protein of Escherichia coli fliC has a molecular weight of 63 kDa, the target gene encodes 416 amino acids, the theoretical molecular weight of the expressed protein fragment is 43 kDa, and the size of the plasmid pET32a is about 20 kDa. The result is consistent with the theoretical size.
  • Example 3 Preparation of an oral microcapsule vaccine of fliC subunit:
  • the coupled product of the above-prepared fliC recombinant protein and LPS is mixed with the Astragalus polysaccharide in a ratio of 2-4:1 by mass ratio, and then the antigen mixture is mixed with sodium alginate at a mass ratio of 1:3.
  • the emulsion was prepared by using distilled water to ensure that the final concentration of sodium alginate in the emulsion was 2%, and it was embedded for 30 minutes under the stirring of the mixer.
  • the emulsion was directly sprayed into a 3% CaCl 2 solution agitated by a mixer using a spray-type acute pore coagulation bath method to form a calcium alginate microcapsule, and the volume ratio of the emulsion to the CaCl 2 solution was 1: 1.
  • microcapsules were collected by centrifugation, and then the microcapsules were dispersed into a 0.2% chitosan solution, stirred well for 30-40 min, the microcapsules were collected by centrifugation, washed three times with distilled water and centrifuged, and the collected microcapsules were dispersed at 8%.
  • mannitol solution it is mixed and frozen, and after lyophilization, it is packaged and packaged to prepare a subunit oral microcapsule vaccine.
  • the apparent properties of the prepared microcapsules were observed by a microscope, and it was found that the microcapsules were uniform in size and the average diameter was 150 ⁇ 10 ⁇ m.
  • a total of 80 healthy gums were prepared, with an average body weight of 50g ⁇ 5g.
  • the gingiva was kept for 7 days using a temperature-controlled circulating culture system, and the water temperature was controlled at 21 ° C.
  • the commercial pellet feed was normally fed during the period.
  • the fish were divided into two groups, 40 mice in each group. After fasting for 24 hours, the fishes of the immunized group were fed with oral microcapsule vaccine of fliC subunit.
  • the feeding method oral microcapsules
  • the vaccine is mixed with fish feed and put into the water body for free feeding.
  • the dosage is: 0.5g of microcapsule vaccine per kg body weight of fish, and continuous feeding for 5 days.
  • the control group was fed with microcapsule granules prepared from sodium alginate solution containing no immunogen and Astragalus polysaccharide.
  • the feeding method and feeding amount were the same as those in the immunized group, and 35 hours after the first feeding, the preparation was slow with physiological saline.
  • the live bacteria suspension of Edwards was at a concentration of 1 ⁇ 10 7 CFU/ml.
  • the experimental fish body was challenged by intramuscular injection, and 100 ⁇ l was injected per tail. After the challenge, the disease incidence and mortality of the fish were continuously observed and recorded, and the relative immune protection rate of the oral microcapsule vaccine of the Edwards subunit was calculated.
  • the results of the challenge experiment with the E. faecalis ET strain showed that the fliC prepared by the present invention
  • the subunit oral microcapsule vaccine was significantly more immune (p ⁇ 0.05) than the other commercially available strains of E. sinensis, presumably due to differences in the amino acid sequence of the fliC gene.
  • the oral microcapsule vaccine of the retarded Edwards subunit produced by the invention is convenient to use, has suitable particle size, has good sustained release and antigen protection, and can effectively stimulate the intestinal immune system to produce immunity. Response, so that the fish body to obtain specific immune protection against Edwards deficient, has a good development and application prospects.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Inorganic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Polymers & Plastics (AREA)
  • Mycology (AREA)
  • Communicable Diseases (AREA)
  • Animal Husbandry (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un vaccin en microcapsule. Une séquence d'acides aminés d'une protéine fliC est représentée par SEQ ID No: 1. Le vaccin en microcapsule selon l'invention peut également être utilisé comme additif alimentaire.
PCT/CN2016/107373 2016-06-08 2016-11-26 Vaccin en microcapsule de protéine flic WO2017211052A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610411274.9A CN105920594A (zh) 2016-06-08 2016-06-08 一种fliC蛋白微胶囊疫苗
CN201610411274.9 2016-06-08

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112679616A (zh) * 2021-01-08 2021-04-20 青岛农业大学 一种牙鲆弹状病毒基因工程亚单位疫苗

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105920594A (zh) * 2016-06-08 2016-09-07 类延乐 一种fliC蛋白微胶囊疫苗
CN108285904B (zh) * 2018-02-09 2021-07-06 河北科技师范学院 一种具有免疫保护作用的迟缓爱德华菌鞭毛蛋白FlgJ

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102206257A (zh) * 2011-04-15 2011-10-05 华东理工大学 迟钝爱德华氏菌免疫保护性抗原、相关表达载体、疫苗和应用
CN105920594A (zh) * 2016-06-08 2016-09-07 类延乐 一种fliC蛋白微胶囊疫苗
CN106075418A (zh) * 2016-06-08 2016-11-09 类延乐 一种fliC蛋白和GAPDH蛋白微胶囊疫苗

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102206257A (zh) * 2011-04-15 2011-10-05 华东理工大学 迟钝爱德华氏菌免疫保护性抗原、相关表达载体、疫苗和应用
CN105920594A (zh) * 2016-06-08 2016-09-07 类延乐 一种fliC蛋白微胶囊疫苗
CN106075418A (zh) * 2016-06-08 2016-11-09 类延乐 一种fliC蛋白和GAPDH蛋白微胶囊疫苗

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OKUDA J.: "Base changes in the fliC gene of Edwardsiella tarda: possible effects on flagellation and motility", DISEASES OF AQUATIC ORGANISMS, 29 June 2007 (2007-06-29), XP055599406 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112679616A (zh) * 2021-01-08 2021-04-20 青岛农业大学 一种牙鲆弹状病毒基因工程亚单位疫苗

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