WO2011067446A1 - Procedimiento de identificación de animales vacunados frente a brucella - Google Patents
Procedimiento de identificación de animales vacunados frente a brucella Download PDFInfo
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- WO2011067446A1 WO2011067446A1 PCT/ES2010/070784 ES2010070784W WO2011067446A1 WO 2011067446 A1 WO2011067446 A1 WO 2011067446A1 ES 2010070784 W ES2010070784 W ES 2010070784W WO 2011067446 A1 WO2011067446 A1 WO 2011067446A1
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- strain
- gfp
- identification
- brucella
- mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
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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
- A61K39/098—Brucella
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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
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/23—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Brucella (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
- G01N2333/43595—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from coelenteratae, e.g. medusae
Definitions
- the present invention is within the field of Preventive Medicine and Public Health, and refers both to the development and use of live vaccines of Brucella spp. genetically modified, such as the development and use of direct and indirect diagnostic techniques that allow the identification of animals that have been vaccinated from those suffering from infection by virulent strains of Brucella spp.
- Brucellosis is a disease that is transmitted from animals to man.
- Brucella infection causes abortions, infertility, decreased production and limitations in the trade of animals and derived products, which constitutes an Animal Health problem with economic repercussions.
- the bacteria are transmitted from infected animals to humans, causing them a debilitating and often disabling disease, against which there are no vaccines and whose treatment requires high doses of antibiotics for prolonged periods, with frequent recurrences. Therefore, brucellosis constitutes a relevant Public Health problem. It has been shown that the prevalence of human brucellosis is directly related to the prevalence of animal brucellosis.
- Walsh et al. (Walsh et al., 2000. Journal of General Virology, 81, 709-718) proposed the possible use of a GFP protein as a marker of a veterinary vaccine. However, they failed to express it conveniently in the Bovine Plague virus.
- the authors of the present invention describe how a strain derived from B. abortus S19 (the prototype S19-GFPp vaccine), is able to express GFP stably, without altering the microbiological or biological characteristics (attenuation and efficacy). against infection in experimental animals) of the reference strain S19 and, in addition, it is capable of inducing the response of anti-GFP antibodies detectable by specific serological tests, which allow differentiating hosts that have been vaccinated from those infected by strains virulent of Brucella spp. They also describe a indirect ELISA method of serological diagnosis capable of specifically identifying animals that have been immunized with the new vaccine that expresses the GFP protein.
- a first aspect of the invention relates to the use of a strain of Brucella spp. which stably expresses the Green Flourescent Protein (GFP) protein and is capable of provoking an immune response in the host comparable to that induced by the reference vaccine strains and also generates anti-GFP antibodies detectable by specific serological methods, in the preparation of a medicine or, alternatively, a strain of Brucella sp. which stably expresses the Green Flourescent Protein (GFP) protein and is capable of eliciting an immune response in the host comparable to that induced by the reference vaccine strains and also generates anti-GFP antibodies detectable by specific serological methods, to Its use as a medicine.
- the medicament is a vaccine.
- Another aspect of the invention relates to the use of a strain of Brucella spp. which stably expresses the Green Flourescent Protein (GFP) protein and is capable of provoking an immune response in the host comparable to that induced by the reference vaccine strains and also generates anti-GFP antibodies detectable by specific serological methods, in the preparation of a medicament for the prevention or treatment of brucellosis in a mammal or, alternatively, a strain of Brucella spp.
- GFP Green Flourescent Protein
- GFP Green Flourescent Protein
- Brucella spp. any cellular organism that can be defined as taxonomically belonging to the super kingdom Bacteria, phylum Proteobacteria, Alphaproteobacteria class, Rhizobiales order, Brucellaceae family and Brucella genus.
- "Mammalian” means any organism of the Eukaryota super kingdom, Metazoa kingdom, Chordata phylum, Craniata subphylum, Gnathostomata superclass and Mammalia class. Ruminant means any mammal belonging to the superorder Laurasiatheria, suborder Ruminantia. And bovine, sheep and goats are understood as any mammal that can be classified as belonging to the Bovidae family.
- the green fluorescent protein GFP produced by the jellyfish Aequorea sp. is a protein that emits bioluminescence in the green zone of the visible spectrum.
- GFP is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the GFP protein, and which would comprise various variants from:
- nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,
- nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a) are nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),
- nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code
- nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 1,
- polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the GFP protein.
- the strain of Brucella spp. It belongs to the species B. abortus.
- the Brucella strain is a strain derived from the reference strain B. abortus S19 (OIE Terrestrial Manual, 2009 -chapter 2.4.3.-).
- the Brucella strain belongs to species B. melitensis
- the Brucella strain is a strain derived from the reference strain B. melitensis Rev 1 (OIE Terrestrial Manual, 2009 -chapter 2.7.2.-). "Brucella abortus S19" or "Brucella abortus bv. 1 str.
- S19 is a spontaneously attenuated strain discovered by Dr. John Buck in 1923, which has been used worldwide since the early 1930s, as an effective vaccine to prevent Brucellosis in animals, and is considered internationally the reference vaccine for the control of bovine brucellosis (OIE Terrestrial Manual, 2009 -chapter 2.4.3.-).
- the original planting lot can be obtained from the United States Department of Agriculture (USDA, National Veterinary Services Laboratories -NVSL-, 1800 Dayton Road, Ames, lowa 50010, United States of America) and the Reference Laboratory for OIE Brucellosis of the Veterinary Laboratories Agency (VLA; Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom).
- the strain is also known as NCTC 8038 (http://www.broadinstitute.org/annotation/genome/brucella_group/GenomeDesc riptions.html) "Brucella melitensis Rev 1", “Brucella melitensis bv. 1 str. Rev.1” is a strain also known as BCCN V4a,
- the Rev 1 strain has been used worldwide since the 1950s as the only effective vaccine to prevent brucellosis in small ruminants, and is considered internationally the reference vaccine for the control of ovine and caprine brucellosis (OIE Terrestrial Manual, 2009 -chapter 2.7.2.-). Revi's original planting lots can be obtained at the Laboratory of Reference for Brucellosis of the OIE of the AFSSA (94706 Maisons-Alfort, France) or in the European Pharmacopoeia (BP 907, 67029 France Cedex 1, France).
- the mammal is a ruminant.
- the mammal is a bovine, and even more preferably, belonging to the subfamily Bovinae or Caprinae.
- composition of the invention comprising a strain of Brucella spp. which expresses the GFP protein and, preferably, a pharmaceutically acceptable carrier.
- the Brucella strain belongs to the species B. abortus.
- the Brucella strain is a strain derived from B. abortus S19.
- the Brucella strain belongs to the species B. melitensis.
- the Brucella strain is a strain derived from B. melitensis Rev 1.
- the composition is a vaccine.
- the composition of the invention further comprises an adjuvant.
- the composition of the invention, comprising a strain of Brucella spp. which expresses the GFP protein also comprises another active ingredient.
- composition of the invention can be formulated for administration to an animal, and more preferably to a mammal, including ruminants, in a variety of ways known in the state of the art, for use as an immunogen. These immunogens can also be used as vaccines in animals, and more particularly in mammals, or produce a response in the production of antibodies therein.
- an immunologically effective amount of the strain of Brucella spp. it is mixed with a suitable physiologically acceptable transporter for administration to mammals including humans.
- the composition of the invention can be found, but not limited to, in sterile aqueous solution or in biological fluids, such as serum. Aqueous solutions may be buffered or unbuffered and have additional active or inactive components.
- Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelants and the like, and nutrients including, but not limited to, glucose, dextrose, vitamins and minerals.
- the active ingredient can be prepared for solid administration.
- the active substance can be combined with various inert vehicles or excipients, including, but not limited to, binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch; lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint or methyl salicylate.
- binders such as microcrystalline cellulose, gum tragacanth or gelatin
- excipients such as starch or lactose
- dispersing agents such as alginic acid or corn starch
- lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide
- sweetening agents such as sucrose or saccharin
- flavoring agents such as peppermint or methyl salicylate.
- composition of the invention can be administered to an animal, including a mammal, preferably a ruminant, and even more preferably belonging to the Bovinae or Caprinae subfamilies, in a variety of ways, including, but not limited to, intraperitoneal, intravenous routes, intramuscular, subcutaneous, conjunctival, intracecal, intraventricular, oral, enteral, parenteral, intranasal or dermal.
- the dosage to obtain a therapeutically effective amount depends on a variety of factors (such as age, weight, sex, physiological status - such as pregnancy or lactation -, tolerance, immune system status) of the animal, preferably mammal.
- the term “therapeutically effective amount” refers to the amount of Brucella spp. which expresses the GFP protein stably, producing the desired effect (the generation of immunity and anti-GFP antibodies).
- the "adjuvants” and “pharmaceutically acceptable vehicles” that They can be used in such compositions are the vehicles known to those skilled in the art.
- the term “medication” refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in humans and animals.
- it refers to the composition comprising the strains of Brucella spp. that express the GFP protein stably, and that are capable of generating immunity against brucellosis and anti-GFP antibodies, or the composition comprising Brucella spp. that expresses the GFP protein stably, and that is capable of generating immunity against brucellosis and anti-GFP antibodies and a pharmaceutically acceptable carrier and / or additionally, an adjuvant.
- the term medication therefore includes vaccines.
- vaccine refers to an antigenic composition or preparation used to establish the immune system's response to a disease. They are prepared of antigens that once inside the organism provoke the response of the immune system, through the production of antibodies, and generate immunological memory producing permanent or transient immunity.
- adjuvant refers to an agent, as long as it does not have an antigenic effect in itself, which can stimulate the immune system by increasing its response to the vaccine.
- aluminum salts “aluminum phosphate” and “aluminum hydroxide” are the two adjuvants most commonly used in vaccines.
- Other substances such as squalene, can also be used as adjuvants.
- active ingredient means any component that potentially provide a pharmacological activity or other effect different in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.
- the term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.
- method of the invention relates to a method for the identification of mammals vaccinated with the strain or composition of the invention, hereafter "method of the invention", comprising:
- the mammal is a ruminant.
- the mammal is a bovine, sheep or goat, and even more preferably, belonging to the Bovinae or Caprinae subfamilies.
- an "isolated biological sample” includes, but is not limited to, cells, tissues and / or biological fluids of a mammal, obtained by any method known to a person skilled in the art.
- the isolated biological sample is a biological fluid, such as, without limitation, milk, semen, seminal fluid, vaginal exudates, conjunctival secretions, blood, plasma or blood serum. More preferably, the biological fluid is blood serum.
- the isolated biological sample is cells found in blood, milk, semen, vaginal exudates or conjunctival secretions of the mammal. In another preferred embodiment, they are cells or tissues.
- the detection of the presence of the gfp gene or the products of its expression, in the isolated mammalian biological sample is performed by the detection of antibodies against the GFP protein ( anti-GFP antibodies).
- the detection of anti-GFP antibodies can be carried out by any method known in the state of the art for example, without being limited to, by immunoassay or immunohistochemistry.
- the immunoassay is an enzyme-linked immunosorbent assay or ELISA.
- An antigen or immunogen is a substance capable of producing an adaptive immune system response through lymphocyte activation.
- the antigens are usually proteins or polysaccharides. Lipids and nucleic acids are antigenic only when combined with proteins and polysaccharides.
- the term "antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts) with the protein. (antigen) GFP.
- IgM immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts) with the protein. (antigen) GFP.
- IgM immunoglobulin molecules
- IgD immunoglobulin molecules
- IgG immunoglobulin glycoprotein
- IgA immunoglobulins
- anti-GFP antibody refers to an antibody capable of reacting with the GFP protein, with a variant of the GFP protein or with a fragment thereof, as long as said variant or said fragment is functionally equivalent.
- anti-GFP antibody refers to an immunoglobulin G (IgG).
- immunoassay refers to any analytical technique that is based on the reaction of conjugation of an antibody with an antigen.
- Examples of immunoassays known in the state of the art are, but are not limited to: immunoblot, enzyme-linked immunosorbent assay (ELISA), linear immunoassay (LIA), radioimmunoassay (RIA), immunofluorescence, x-map or protein or lipopolysaccharide chips (LPS)
- the immunoassay is an enzyme-linked immunosorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay).
- the ELISA is based on the premise that an immunoreactive (antigen or antibody) can be immobilized on a solid support, then bringing that system into contact with a fluid phase containing the complementary reagent that can bind to a marker compound.
- an immunoreactive antigen or antibody
- the authors of the present invention describe a serological diagnostic method capable of identifying animals that have been vaccinated with the prototype of the new S19-GFPp vaccine and differentiating them from those infected by virulent strains of Brucella spp.
- the ELISA is an indirect ELISA, and even more preferably comprises the following steps:
- marker compound refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection of the anti-GFP antibody.
- the marker compound is selected from the list comprising radioisotopes, enzymes, fluorophores or any molecule capable of being conjugated with another molecule or detected and / or quantified directly. This marker compound may bind directly to the antibody or, indirectly, through another compound.
- directly binding marker compounds are, without limitation, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as 32 P or 35 S, fluorochromes such as fluorescein, rhodamine or its derivatives, or metal particles, for direct detection. by colorimetry, autoradiography, fluorimetry, or metallography respectively.
- enzymes such as alkaline phosphatase or peroxidase
- radioactive isotopes such as 32 P or 35 S
- fluorochromes such as fluorescein, rhodamine or its derivatives, or metal particles
- the detection of the presence of the gfp gene or the products of its expression, in the isolated biological sample of the mammal is performed by ultraviolet illumination of said sample.
- the detection of the presence of the gfp gene or the products of its expression, in the isolated biological sample of the mammal is performed by fluorescence microscopy.
- the detection of the presence of the gfp gene or the products of its expression, in the isolated mammalian biological sample is performed by polymerase chain reaction (PCR).
- kits for identification of mammals vaccinated with the strain or composition of the invention, which comprises the appropriate means for carrying out the method of the invention.
- Said kit may contain all those reagents necessary to analyze the presence of the gfp gene, or the products of its expression, in the isolated mammalian biological sample by means of any of the methods described hereinabove as, for example, without being limited a, specific antibodies of the GFP protein, secondary antibodies or positive and / or negative controls.
- the kit can also include, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc.
- the kit can also include, without any limitation, the use of buffers, polymerases, cofactors to obtain optimum activity from these, agents to prevent contamination, etc.
- the kit can include all the supports and containers necessary for commissioning and optimization.
- the kit further comprises instructions for carrying out the methods of the invention.
- polynucleotide and “nucleic acid” are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).
- amino acid sequence amino acid sequence
- peptide oligopeptide
- polypeptide a polypeptide
- protein a polymeric form of amino acids of any chemical or biochemically modified length.
- Fig. 1 Growth of the new S19-GFPp vaccine and the parental strain S19 in triplicase soy agar plates illuminated with ultraviolet light in a "Rad Doc Gel” apparatus of Bio Rad and visualized with the appropriate filter (520DF30 nm, Bio Rad ) (A) or fluorescence microscope (B).
- Fig. 2 PCR amplification of the ery region in the new S19-GFPp vaccine (lower molecular weight), compared to that of the virulent strain B. abortus 2308.
- Fig. 3 Proportion of intracellular and extracellular bacteria in HeLa cells infected with the new S19-GFPp vaccine or with the S19 parental strain, one hour after infection. HeLa cells were infected with 200 CFU / cell and, 1 hour later, were fixed and labeled by double immunofluorescence with rhodamine and FITC, following previously described protocols (Chaves-Olarte et al., 2002. Cellular Microbiology 4 (10): 663 -675).
- Fig. 4 Replication of the new S19-GFPp vaccine and the parental strain S19 in HeLa cells and in Raw macrophages 264.7, at 48 hours post-infection.
- the cells were infected with 200 CFU / cell and the number of CFU / mL was determined, following previously described protocols (Chaves-Olarte et al., 2002. Cellular Microbiology 4 (10): 663-675; Celli et al., 2003 Journal of Experimental Medicine, 198 (4): 545-556).
- Fig. 5 Splenic kinetics of the new S19-GFPp vaccine and the parental strain S19 in the murine model.
- Groups of 30 mice were inoculated intraperitoneally with 1 x 10 5 CFU of the new S19-GFPp vaccine or the parental strain S19.
- At 7, 14, 25, 40 and 60 days after infection 6 mice from each group were sacrificed to determine the number of CFU / spleen of each vaccine strain and construct the corresponding curves of splenic multiplication, following previously described protocols (Sangari et al., 1998. Vaccine, 1 6 (17): 1640-1 645).
- Fig. 6 Protective assay in BALB / c mice immunized with the new S19-GFPp vaccine.
- Groups of 6 mice were immunized, subcutaneously, with 1 x 10 5 CFU of the new S19-GFPp vaccine or the parental strain S19.
- As a control a group of mice was inoculated with sterile PBS. After 60 days, all mice were experimentally infected, intraperitoneally, with 5x10 4 CFU of the virulent reference strain B. abortus 2308 and, 2 weeks later, were sacrificed to count the CFU number of 2308 per spleen. Animals vaccinated with S19-GFPp showed similar levels of protection to those shown by the S19 reference vaccine.
- Fig. 8 Determination of the degree of purity and immunogenicity in mice of the recombinant GST-GFP protein obtained.
- Fig. 9 Standard curves in the ELISA-GFP developed, using sera from mice (a) or sheep (b) previously immunized with the recombinant GST-GFP protein).
- Fig. 10 Intensity of the antibody response against GFP in sera of mice inoculated with the new S19-GFPp vaccine or with the reference vaccine S19. The intensity of the reaction of each individual serum was calculated according to the percentage of positivity of the sample with respect to the control (D.O. of the positive serum / D.O. of the serum sample X 100). Each point represents the average of 10 sera from different mice.
- the reference vaccine B. abortus S19 (obtained from the OIE Reference Laboratory for Brucellosis of the AFSSA, France) was genetically modified by electroporation with a plasmid encoding GFP.
- the plasmid used was pBBR-2-gfp, derived from plasmid pBBRIMCS-2, which contains a kanamycin resistance insert (Kovach et al., 1995. Gene. Vol 1 66: 175-176) and an insert with the gene that encodes the GFP, under the control of the lac promoter. It is known that this plasmid generates GFP constitutively, without integrating into the Brucella chromosome (Celli et al., 2003. Journal heard Experimental Medicine, 198 (4): 545-556). 2.- Genetic and microbiological characterization of S19-GFPp.
- plasmid pBBR-2-gfp allows the expression of GFP in Brucella in adequate proportions so that the new S19-GFPp vaccine can be identified with the naked eye after its isolation.
- the expression of the GFP protein in Brucella does not alter the colonial size and phase of S19 in bacteriological cultures, nor its classical bacteriological characteristics.
- the colonial size was determined by measuring the diameter of the colonies obtained on agar plates, after 3 days of incubation.
- the colonial phase was determined by observing bacterial growths in an oblique illumination magnifying glass and staining by flood technique with a violet-oxalate crystal solution (Alton et al. 1988. Techniques for the brucellosis laboratories. In. INRA (Ed.), Paris, France; 1988, 190 pp).
- the new strain obtained was analyzed microbiologically by standard techniques for the identification and typing of Brucella spp. Briefly, identification at the gender level was carried out by testing catalase, oxidase, urease and agglutination with acriflavin. The bacteriophage sensitivity test Tb, Wb, Iz and R / C was used to identify the species level.
- abortus S19 preserving the characteristic deletion of 702 bp (base pairs) in the ery gene (low molecular weight band, Fig. 2) and that it allows to differentiate the S19 vaccine strain of virulent strains of B. abortus (higher molecular weight band, Fig. 2).
- the new S19-GFPp vaccine has a capacity for adhesion and internalization in human HeLa epithelial cells practically identical to that of the reference vaccine (Fig. 3).
- the new S19-GFPp vaccine has an attenuation in mice practically identical to that of the classic S19 vaccine (Fig. 5), indicating that the expression of the gfp gene used does not modify the splenic kinetics (multiplication capacity and persistence) of the classic parental vaccine.
- the GST-GFP fusion protein was expressed in the E. coli XL1-Blue system with the plasmid pGEX-GFP, purified by affinity chromatography and its purity was determined by acrylamide gel electrophoresis (Fig. 8A).
- mice were obtained in mice, following conventional immunization protocols. For this, an injection of 100 ⁇ g of GST-GFP protein was administered together with complete Freund's adjuvant, followed by 2 consecutive immunizations separated by a week with the GST-GFP protein together with incomplete Freund's adjuvant. Immunizations were performed for several weeks until the sera of animals immunized with GST-GFP showed precipitation bands in gel immunodiffusion against the purified GFP protein (Fig. 8B). These control antibodies showed no cross reactions with any Brucella antigen (results not shown).
- control sera against GFP were obtained in sheep, following conventional immunization protocols. For this, an injection of 100 ⁇ g of GST-GFP protein was administered together with complete Freund's adjuvant, followed by 5 consecutive immunizations separated by two weeks with the GST-GFP protein together with incomplete Freund's adjuvant. Immunizations were carried out for two months until the sera of animals immunized with GST-GFP showed precipitation bands in gel immunodiffusion against the purified GFP protein (results not shown). These control antibodies showed no cross reactions with any Brucella antigen (results not shown).
- the work carried out demonstrates that the GFP protein can be expressed in Brucella vaccine strains, without altering the biological properties of the parental strain and inducing in animals a clearly distinguishable serological response from that induced by other Brucella strains.
- Antibodies generated against the GFP protein can be identified by the indirect ELISA assay against GFP developed in this patent.
- the incorporation of the gfp gene in Brucella allows both visual identification (by ultraviolet illumination or fluorescence microscope) and molecular identification (by means of a PCR that amplifies the gfp gene; PCR-GFP) of the developed Brucella vaccine strains, a from both bacteriological cultures and tissue samples, exudates or animal fluids.
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CN2010800630275A CN102781468A (zh) | 2009-12-03 | 2010-11-29 | 用于鉴别接种对抗布鲁氏菌的疫苗的动物的方法 |
MX2012006303A MX2012006303A (es) | 2009-12-03 | 2010-11-29 | Procedimiento de identificacion de animales vacunados frente a brucella. |
EP10834263.5A EP2508201A4 (en) | 2009-12-03 | 2010-11-29 | METHOD FOR IDENTIFYING ANIMALS VACCINATED AGAINST BRUCELLA |
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CN110863031A (zh) * | 2019-09-29 | 2020-03-06 | 安徽医科大学第一附属医院 | 一种检测布鲁菌病治疗效果的标志物及其应用 |
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CN111796091A (zh) * | 2020-07-20 | 2020-10-20 | 天康生物股份有限公司 | 用于区分动物感染布氏杆菌或布氏杆菌菌影疫苗的试剂盒 |
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EP2508201A1 (en) | 2012-10-10 |
EP2508201A4 (en) | 2013-08-07 |
CN102781468A (zh) | 2012-11-14 |
ES2372187B8 (es) | 2013-04-29 |
CR20120301A (es) | 2013-02-27 |
PE20121648A1 (es) | 2012-12-24 |
ES2372187B1 (es) | 2012-11-26 |
ES2372187A1 (es) | 2012-01-17 |
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