79 results about "Brucella melitensis" patented technology

The technology is a veterinary pharmaceutical formulation of two vaccines, one from an RNA viral vector system constituted by an RNA recombinant particle that codifies for a Cu/Zn superoxide dismutase protein of Brucella abortus, and the other based on naked RNA constituted by a recombinant molecule of naked RNA that carries a sequence for the synthesis of at least one recombinant Cu/Zn superoxide dismutase protein of Brucella abortus and some Semliki Forest virus genes. An expression system based on the Semliki Forest virus and a use of this system, in addition to a method for the preparation of the pharmaceutical formulations.

The invention provides a monoclonal antibody against smooth Brucella (Brucella) lipopolysaccharides (sLPS), and preferably provides 14F4. Cross reaction tests show that the monoclonal antibody 14F4 has no cross reaction with Escherichia coli (O:157), Salmonella Dublin (C79-86) whole thallus and the LPS. On the basis, a competitive ELISA (c-ELISA) test method using the monoclonal antibody 14F4 as a competition antibody is established, and the method is suitable for testing large-scale clinical samples, and has the characteristics of being rapid, high-throughput, high-sensitivity and high-specificity.

A PCR kit for simultaneously detecting Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis as well as a preparation method and a using method thereof, and belongs to the field of gene detection of anthropozoonosis pathogens. The PCR kit comprises a PCR liquid reactant, DNA polymerase, positive quality control standard substances and negative quality control standard substances; the PCR liquid reactant comprises four pairs of primers for identifying Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis and a PCR buffer solution containing dNTPs, Mg<2+> and double distilled water. The PCR kit has high practicability, can be used for directly detecting the type of Brucella in a contaminated sample, has high detection speed, high sensitivity (10<2>cfu/ml), high specificity and good repeatability, is safe, reliable, quick, simple and convenient, can be used for qualitative detection of the type of Brucella in the sample, and can replace traditional etiological and serological methods.

The invention provides a primer, a probe and a kit for detecting brucella based on a recombinase polymerase amplification method and belongs to the technical field of microbiological diagnosis. The primer pair and the probe for detecting brucella based on the recombinase polymerase amplification method are designed and obtained by taking a specific conservative region of a brucella ovis pb 26 genesequence as a template. When the kit is used for detecting the pb 26 gene of the brucella by using the recombinase polymerase amplification method, the detection sensitivity reaches up to 6 CFU; meanwhile, the primer has high specificity in detection of the brucella.

The invention discloses universal primers and a probe for on-site rapid detection of Brucella and a kit. The forward primer sequence is shown as SEQIDNo.1, the reverse primer sequence is shown as SEQIDNo.2, and the probe sequence is shown as SEQIDNo.3. The universal primers, the probe and the kit provided by the invention for detection of Brucella have high sensitivity and strong specificity, minimumly can detect 6.0*10<0>cfu Brucella, and have no cross reaction with Escherichia coli O157:H7, yersinia enterocolitica O:9, Salmonella, Staphylococcus aureus and other bacteria. The universal primers, the probe and the kit provided by the invention not only can be used for detection of Brucella strains, but also can be used for detection of clinical samples, which mainly include blood, milk samples, aerosol samples, and tissue samples, etc. The kit provided by the invention is convenient to use, has no need for special equipment, can carry out sensitive, specific and rapid detection of Brucella on the crude split product of a to-be-detected sample in 25min just at 38DEG C, and is suitable for field or grassroots brucellosis quarantine work.

The invention discloses a construction method and an expression method of a coexpression vector of L7/L12, Omp31, Rs alpha and sodC Brucella immune proteins. The disclosed coexpression vector includes a double expression vector pETDuet-1 and a double expression vector pRSFDuet-1, which are both expressed in the same host bacteria, wherein the two double expression vectors are respectively connected with encoding genes of two Brucella immune proteins. On this basis, the invention also discloses the construction method and the expression method of the coexpression vector. L7/L12, Omp31, Rs alpha and sodC Brucella immune proteins all with good immunity functions can be expressed simultaneously by the coexpression vector disclosed by the invention and are effectively used for preparing subunit vaccine of the Brucella gene engineering and preventing and controlling various brucellosis.

The invention relates to a cattle and sheep brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit and a preparation method thereof, belongs to the field of detection of pathogens of zoonotic infections and aims at solving the problems of low sensitivity, poor specificity and the like in detection of Brucella by adopting other antigens. The kit is assembled by taking a recombinant Brucella BCSP31 protein with expression induced by SUMO as an expression vector in Escherichia coli to serve as an antigen-coated ELISA (enzyme-linked immunosorbent assay) plate, adding serum to be detected and rabbit anti-bovine HRP-IgG or rabbit anti-goat HRP-IgG and adding a washing buffer solution, a blocking solution, a TMB (3,3',5,5'-Tetramethylbenzidine) primer solution and a stop solution for matching. The prokaryotic expression vector, namely the SUMO, is utilized to express the Brucella BCSP31 protein in an efficient and soluble manner, the kit is used for establishing a Brucella ELISA detection method, the detection has better repeatability and specificity, and cattle and sheep Brucella can be fast and efficiently detected by utilizing the method.

The invention belongs to the field of biotechnical detection, and particularly relates to a bovine, goat, porcine and canine brucella typing fluorescent PCR (polymerase chain reaction) detection reagent kit and preparation and application thereof. The bovine, goat, porcine and canine brucella typing fluorescent PCR reagent kit contains bovine brucella detection primers and probes, goat brucella detection primers and probes, porcine brucella detection primers and probes and canine brucella detection primers and probes. The bovine, goat, porcine and canine brucella typing fluorescent PCR detection reagent kit, the preparation and the application have the advantages that the bovine, goat, porcine and canine brucella typing fluorescent PCR detection reagent kit is high in detection sensitivity, the lowest detection limit is 1*10<3> copy/ml, and the accuracy and the positive rate can reach 100%.

Live attenuated vaccines against brucellosis are described. New mutant strains of Brucella melitensis have been developed, which are attenuated via deletion of the hfq and/or purEK sites. The purEK deletion site does not include insertion of a kanamycin resistance determinant marker or any other introduced antibiotic resistance marker. The hfq deletion site preferably does not include insertion of a kanamycin resistance determinant marker or any other introduced antibiotic resistance marker.

The invention discloses a Brucella multi-epitope fusion protein antigen. Amino acid sequences of dominant epitopes of Brucella outer membrane proteins BP26, OMP31, OMP16 and OMP2b are in series connection to construct a fusion protein gene to express the proteins, and a Brucella multi-epitope fusion protein antigen vaccine is prepared. Mice challenge tests show that the Brucella multi-epitope fusion protein antigen vaccine plays a role in immune protection in Brucella infection.

The invention provides a plasmid pZF17-30 for constructing a Brucella mutant strain, a construction method of the plasmid and an application of the plasmid. The construction process mainly includes the steps: amplifying an rAPOBEC1-nCas9-UGI fragment from a commercial plasmid pCMV-BE3; integrating the fragment with a carrier containing a general host replicon pBBR1-MCS-5 and non-specific sgRNA byrecombination reaction; inserting a ccdB gene and a Chl resistance gene into the sgRNA; digesting the plasmid pZF17-30 by the aid of restricted enzyme BsaI; connecting specific sgRNA with linearized pZF17-30. The sequence of the constructed carrier is as shown in SEQ ID NO. 3, the carrier can be used for constructing the Brucella mutant strain, and a 478-site basic group in a CDS (coding sequence)area of a virB10 gene is mutated into T from C.

A recombinant, attenuated strain of Brucella suis or Brucella melitensis with a deficiency in carboxyl-terminal protease activity or tail-specific protease activity can be used as a vaccine for the prevention or treatment of Brucellosis. Prior exposure to the Brucella species is identified by detecting a genetic sequence for carboxyl-terminal (i.e. tail-specific) protease activity in a biological sample.

The invention relates to a brucellosis complement fixation-enzyme-linked immunosorbent assay (CF-ELISA) diagnostic kit, and the kit combines a reaction system in a complement fixation test (CFT) and an enzyme marker amplification system in an enzyme-linked immunosorbent assay (ELISA). The brucellosis complement fixation-enzyme-linked immunosorbent assay (CF-ELISA) diagnostic kit has the characteristics of high sensitivity, easy and fast use, high throughput and high degree of standardization compared with a traditional complement fixation test diagnostic reagent, has the characteristics of high specificity and capability of detecting a variety of brucellosis specific antibodies compared with a traditional ELISA diagnostic kit, and is an ideal new brucellosis diagnostic tool. The kit comprises the following main components: a lipopolysaccharide LPS antigen-coated plate, strongly-positive control serum, weakly positive control serum, negative control serum, a guinea pig complement, enzyme labeled guinea pig complement C1q-B monoclonal antibody 60G4, a substrate coloring-developing solution, a stop solution and washing liquid.

The invention provides a method for detecting a live attenuated Brucella vaccine strain and a wild strain and an application of the method, and belongs to the field of biotechnologies. The method fordetecting the live attenuated Brucella vaccine strain and the wild strain provided by the invention designs a primer according to a missing gene of the live attenuated Brucella vaccine strain, then adopts the primer to carry out PCR amplification on DNA of a sample to be detected, and judges the sample based on detection results. By the method, the primer is designed according to the missing geneof the live attenuated Brucella vaccine strain, namely the designed primer for amplification is a part or all of the missing gene, so that the primer effectively amplifies a gene of the wild strain without amplifying a gene of the live attenuated vaccine strain, thereby accurately and effectively distinguishing the live attenuated Brucella vaccine strain from the wild strain. The method has broaduniversality, high specificity and high accuracy.

The invention provides a livestock S2 vaccine immunity and Brucella melitensis/abortus infection IELISA (enzyme linked immunosorbent assay) detection reagent.The active ingredients of the IELISA detection reagent are a Brucella S2 vaccine strain gene GL_0002189 coded antigen protein and a Brucella BP26 protein, or recombinant proteins with N terminals or C terminals fused with His labels.The livestock S2 vaccine immunity and Brucella melitensis/abortus infection IELISA detection reagent is capable of identifying main epidemic strains-Brucella melitensis/abortus and a mainstream vaccine-S2 vaccine generally used in the market, thus, vaccine immunity in livestock serums and natural Brucella melitensis/abortus infection antibodies can be identified within short time, and the problem of brucellosis detection and herd purification interfered by vaccine antibodies in immune herds is solved.The livestock S2 vaccine immunity and Brucella melitensis/abortus infection IELISA detection reagent can bring social and economic benefits and make great contributions to purifying brucellosis in the herds.

The invention discloses a brucella melitensis recombination strain M5-delta bp26-delta znuA as well as a preparation method and application thereof. The preparation method comprises the steps: with a brucella melitensis M5 genome as a template, carrying out PCR (Polymerase Chain Reaction) to obtain upstream and downstream homologous arms of a znuA gene, and connecting the upstream and downstream homologous arms to obtain a fragment delta znuA; cloning the fragment delta znuA to a carrier pRE112 to obtain a recombinant plasmid pRE-delta znuA; inducing the plasmid into a brucella melitensis M5-delta bp26 cell, and respectively screening through chloramphenicol and saccharose selective mediums to obtain double homologous recombinant recons, namely the recombination strain M5-delta bp26-delta znuA. Compared with an M5 parent strain, the strain is consistent in growth characteristic, stable in heredity, not easy to cause throwback, low in toxicity, favorable in immune effect retention and capable of being applied to the distinguishing and detection of natural infection or artificial immunity because of bp26 gene deletion markers. Therefore, the strain can be used as a marker vaccine with a better effect to be applied clinically.

Virulence-associated antigens involved in adhesin have been identified in several organisms: Haemophilus influenzae biogroup aegyptius; Escherichia coli K1; EHEC E.coli; Actinobacillus actinomycetencomitans; Haemophilus somnus; Haemophilus ducreyi; EPEC E.coli; EA EC E.coli; uropathogenic E.coli; Shigella flexneri; Brucella melitensis; Brucella suis; Ralstonia solanacearum; Sinorhizobium meliloti; Bradorhizobium japonicum; and Burkholderia fungorum. Although the degree of sequence identity between the adhesins is low, they share a common arrangement of domains from N-terminus to C-terminus, namely: a leader peptide; a globular head; a coiled-coil region; and a transmembrane anchor region.

The invention discloses a small RNA (ribonucleic acid) related to virulence of Brucella and application of the small RNA in preparation of the weak virulence Brucella. The application Northern blot proves an sRNA Clu7 exists in a virulent Brucella strain M28, a sRNA defective strain M28 delta Clu7 is constructed by adopting a homologous recombination method, and the affection of the sRNA on the virulence of the M28 is evaluated by taking a mouse macrophage RAW264.7 and a Balb/c mouse as models. A result shows that the replication capability and the survival capability of the virulent strain M28 in the mouse macrophage RAW264.7 and the mouse can be remarkably reduced after deficiency of the sRNA. When in a fifth week of inoculation, the M28 delta Clu7 is completely eliminated from a body ofthe mouse, and the virulence of the M28 delta Clu7 is remarkably lowered in comparison with the M28. A mouse virulence attacking protection experiment shows that the protection effect that the mouseresists the infection of the virulent strain M28 by the immune M28 delta Clu7 can reach the protection force of a vaccine strain M5-90. The results indicate that the sRNA Clu7 has a significant decisive effect on the virulence of the Brucella. The small RNA has a significant promoting effect on a mechanism of disclosing the virulence of the Brucella. According to the small RNA, a novel technical means is provided for research and development of a novel candidate vaccine strain of the Brucella.

The invention provides an EF-Tu protein monoclonal antibody MAb of Brucella Melitensis and a preparation method and application thereof. An EF-Tu gene is cloned to a pET30a vector to construct an EF-Tu protein for expressing an His-carrying label in an prokaryotic expression vector, the purified EF-Tu protein is used for immunizing a Balb/c mouse, the mouse spleen cell and myeloma SP2/0 cell are fused to prepare a hybridoma cell, and the anti-Brucella Melitensis EF-Tu MAb is obtained by specific screening, in-vitro culturing and purifying. An ELISA plate is coated by adopting EF-Tu purified soluble protein for prokaryotic expression of a GST-carrying label during screening, so that the condition that MAb recognizing His label protein is screened can be avoided, the soluble GST-EF-Tu protein has activity close to the natural Bacterium burgeri elongation factor Tu protein, and the specificity of the hybridoma cell is ensured. The Western blot result shows that the MAb can react with the GST or His label-carrying Bacterium burgeri protein antigen rather than the GST label or Escherichia coli thalli protein, and the antibody MAb has strong specificity.

The invention relates to construction and application of an A19 Brucella GroES deleted mutant strain and belongs to the field of Brucella vaccine research. By designing PCR primers of an upstream region, a downstream region and a kana resistance gene of GroES, amplifying homologous nucleotide fragments of two lengths of the upstream and the downstream of a target gene and the kana resistance gene,directionally connecting the homologous nucleotide fragments with the kana resistance gene by adopting a PCR amplification method, defective homologous nucleotide fragments of the target gene are obtained, and the obtained defective homologous nucleotide fragments of the target gene are connected into a cloning vector to obtain a recombinant vector containing the defective homologous nucleotide fragments of the target gene, the constructed recombinant vector containing the defective homologous nucleotide fragments of the target gene is transformed into Brucella A19, positive clones are screened to obtain the Brucella A19 attenuated vaccine mutant strain of the deleted target gene. The vaccine mutant strain is expected to be developed into a marked attenuated vaccine, and plays a positivepromoting role in controlling the epidemic and transmission of brucellosis and the healthy development of national economy.

The invention relates to a method for preparing 3 alpha-hydroxyl-7-oxo-5 beta-cholanic acid by utilizing a bio-enzyme catalysis technology, and a 7 alpha-hydroxysteroid dehydrogenase for preparing 3 alpha-hydroxyl-7-oxo-5 beta-cholanic acid. The method comprises the steps of catalyzing chenodeoxycholic acid by using the 7 alpha-hydroxysteroid dehydrogenase in the presence of NAD, a lactic dehydrogenase, sodium pyruvate and a buffer solution by taking chenodeoxycholic acid as a substrate to prepare 3 alpha-hydroxyl-7-oxo-5 beta-cholanic acid, wherein the 7 alpha-hydroxysteroid dehydrogenase is from Brucella melitensis. The method is simple in operation, mild and easily controlled in reaction condition and short in reaction time; the substrate conversion rate reaches up to more than 99.8%; and the content of an obtained product is more than 96.8%.

The invention provides a reagent kit capable of simultaneously detecting virulent viruses and bacteria and a detecting method. The reagent kit is mainly in accordance with 4 pathogenic microorganismsof ebola viruses, Sinkiang hemorrhagic fever viruses, Brucella and anthrax bacillus. The reagent kit comprises a specific primer probe combination for detecting the ebola viruses, the Sinkiang hemorrhagic fever viruses, the Brucella and the anthrax bacillus, a micro-fluidic solid-phase PCR chip on which a specificity probe is fixed, and an RNase-resistant high-stability positive quality control product consisting of virus-like particles containing viral nucleic acid and thalli containing plasmid carrying the specificity nucleic acid. According to the reagent kit, simultaneous detection of theebola viruses, the Sinkiang hemorrhagic fever viruses, the Brucella and the anthrax bacillus can be realized, and the reagent kit has the advantages of being high in detection flux, high in sensitivity, high in specificity, good in repeatability, short in detection time, low in detection cost, low in operation technique requirements, not liable to pollute and the like, and has great application prospects in the field of quick simultaneous detection of various pathogenic microorganisms.

The invention provides a hybridoma cell strain 17C8 CGMCC No. 4971 obtained from immune mice with Brucella whole cell bacteria as an antigen, and a monoclonal antibody generated from the hybridoma cell strain 17C8. The preparation method of the monoclonal antibody comprises steps that: (1) the Brucella whole cell bacteria antigen is adopted as an immunogen to immunize an animal; (2) spleen cells of the immune animal are separated, and are fused with myeloma cells, such that hybridoma cells are obtained; (3) the hybridoma cells are screened and cultured; and (4) monoclonal antibodies are separated and purified from cell culture fluid or ascitic fluid of an animal vaccinated with the hybridoma cells. As a result of experiments, the monoclonal antibody generated from the hybridoma cell strain 17C8 CGMCC No. 4971 can subject to reactions with all Brucella strains (strains and vaccine strains), and has high specificity. Therefore, the monoclonal antibody can be used in Brucella detections. According to the characteristics of the monoclonal antibody strain provided by the invention, methods for detecting Brucella antigen and blood serum can be established. Therefore, the application prospect of the monoclonal antibody is wide.

A series of genes from Brucella spp are shown to encode products which are implicated in virulence. The identification of these genes therefore allows attenuated microorganisms to be produced. Furthermore, the genes or their encoded products can be used in the manufacture of vaccines for therapeutic application.