35 results about "Mycobacterium bovis" patented technology

The present invention relates to a recombinant Mycobacterium bovis BCG strain comprising a plasmid having a short guide RNA (sgRNA) target sequence for knocking out the Mb3739 gene. Also provided are vaccine compositions for eliciting an immune response against Mycobacterium tuberculosis comprising a recombinant Mycobacterium bovis BCG strain engineered to activate the NOD-1 pathway. The recombinant Mycobacterium bovis BCG strain or vaccine composition can be used in a method against an immune response initiated by Mycobacterium tuberculosis. The invention also relates to a method for obtaining the recombinant bovine mycobacterium BCG strain.

The invention provides sgRNA for detecting tuberculosis based on an RPA-CRISPR Cas12b technology, a kit, a detection method and application, and belongs to the technical field of molecular detection. The invention provides sgRNA (single guide ribonucleic acid) for detecting mycobacteria. The sgRNA comprises sgRNA-6110 of which the nucleotide sequence is shown as SEQ ID NO: 1 and / or sgRNA-1081 of which the nucleotide sequence is shown as SEQ ID NO: 2. The sgRNA provided by the invention can specifically recognize and combine high-conservative-degree target sequences IS6110 and IS1081 of mycobacterium tuberculosis and / or mycobacterium bovis, and can be used for instant test of human and livestock tuberculosis pathogenic bacteria mycobacterium tuberculosis and / or mycobacterium bovis.

Certain embodiments of the invention pertain to a method of treating an infection in a subject caused by an infectious agent other than Escherichia coli, the method comprising administering to the subject arsinothricin or a salt thereof. The infectious agent other than E. coli can be a bacterium, protozoan, helminth, archaebacterium, or a fungus. In preferred embodiments, the infectious agent is Mycobacterium tuberculosis, Mycobacterium bovis, or Enterobacter cloacae. The invention also pertains to a method of treating an infection in a subject caused by an infectious agent, comprising administering to the subject arsinothricin or a salt thereof in combination with an inhibitor of phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase. In certain such embodiments, the infectious agent expresses phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase. Further embodiments provide compositions comprising arsinothricin or a salt thereof and an inhibitor of phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase.

The application discloses application of an MgdE gene in regulating virulence of Mycobacterium bovis, a nucleotide sequence of the MgdE gene is shown as SEQ ID NO. 1, an amino acid sequence of a coded protein is shown as SEQ ID NO. 2, the protein coded by the gene is a nuclear regulatory protein, depends on two nuclear localization signal peptides (NLS) to play a nuclear localization function, and then inhibits a host inflammation level, enhances survival and colonization of the bacteria in a host cell, the two NLS are respectively located at positions 108-111 and 300-305 of the amino acid sequence shown as SEQ ID NO. 2. The MgdE gene or the two NLS is modified to obtain a weak strain of Mycobacterium bovis, it is found for the first time that the MgdE gene and the two NLS can be used as important targets for inhibiting Mycobacterium bovis infection. The application has important application value in preparation of Mycobacterium bovis bacillus Calmette-Guerin (BCG), and also makes an important contribution to exploration of a pathogenic mechanism and an immune escape strategy of Mycobacterium bovis.

Certain embodiments of the invention pertain to a method of treating an infection in a subject caused by an infectious agent other than Escherichia coli, the method comprising administering to the subject arsinothricin or a salt thereof. The infectious agent other than E. coli can be a bacterium, protozoan, helminth, archaebacterium, or a fungus. In preferred embodiments, the infectious agent is Mycobacterium tuberculosis, Mycobacterium bovis, or Enterobacter cloacae. The invention also pertains to a method of treating an infection in a subject caused by an infectious agent, comprising administering to the subject arsinothricin or a salt thereof in combination with an inhibitor of phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase. In certain such embodiments, the infectious agent expresses phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase. Further embodiments provide compositions comprising arsinothricin or a salt thereof and an inhibitor of phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase.

The application discloses an adjuvant containing Mycobacterium vaccae surface polysaccharide and application thereof, and the adjuvant comprises: Mycobacterium vaccae surface polysaccharide, wherein the Mycobacterium vaccae surface polysaccharide comprises: LAM polysaccharide, and LM polysaccharide and / or PIMs polysaccharide. The adjuvant of the application can improve the immunization effect of a vaccine antigen component and has good safety. Therefore, the adjuvant of the application can further improve the prevention and / or treatment effect of a vaccine, and has a wide application prospect.

The invention belongs to the technical field of medicines, and particularly relates to a novel tuberculosis subunit vaccine as well as a preparation method and application thereof, and the preparation method comprises the following steps: S1, culturing a mycobacterium bovis attenuated strain; s2, extracting the cell wall of the attenuated strain of mycobacterium bovis; s3, identifying cell walls of the attenuated strains of the mycobacterium bovis; s4, preparing a cell wall and manganese adjuvant subunit vaccine; s5, subunit vaccine intranasal immunization of mice; and S6, performing a mouse challenge protection experiment. Two novel tuberculosis subunit vaccines named as CW-MnJ (alpha) and CW-MnJ (beta) are prepared by extracting cell wall components of a mycobacterium bovis attenuated strain and using the cell wall components as antigens and combining the cell wall components with manganese adjuvants (MnJ (alpha) and MnJ (beta)). A mouse immune protection experiment proves that the lung and spleen bacterium load can be remarkably reduced by the mouse intranasal immunization CW-MnJ (alpha) and CW-MnJ (beta), and the spleen bacterium load of the mouse immunized with the CW-MnJ (alpha) is remarkably reduced compared with a positive control group (BCG immunization group), so that the CW-MnJ (alpha) and CW-MnJ (beta) subunit vaccines prepared by the invention have very good immune protection effects.

The present invention relates to a method for determining the presence or absence of M. tuberculosis, M. bovis, and M. bovis BCG in a sample comprising a nucleic acid molecule. A method according to the present invention can detect the individual presence of M. tuberculosis, M. bovis, and M. bovis BCG, and the co-presence of two thereof.

The invention discloses an application of a small molecule drug ATWLPPR Peptide TFA in resisting mycobacterium bovis infection, and belongs to the field of biological medicine, the small molecule drug ATWLPPR Peptide TFA is an agonist of a target host factor NRP1, and then the inhibition effect of the ATWLPPR Peptide TFA on mycobacterium bovis in vitro and in vivo is researched. An in-vitro bacterial loading titration result shows that the ATWLPPR Peptide TFA treatment can present dose-dependent inhibition of the mycobacterium bovis to adhere to A549 cells. Mouse experiment results show that ATWLPPR Peptide TFA treatment can inhibit mice from being infected with mycobacterium bovis, pathological injuries of mouse lung tissues are effectively relieved, and a brand new direction is provided for prevention and control of mycobacterium bovis.

The invention relates to the technical field of pathogenic microorganism detection, and particularly discloses a primer group for detecting pathogenic microorganisms of rodent experimental animals and application of the primer group. The primer group disclosed by the invention is used for simultaneously detecting corynebacterium pneumophila, staphylococcus xylosus and staphylococcus sciuri of rodent experimental animals; the primer group comprises a primer pair aiming at Corynebacterium bovis, wherein the sequences of the primer pair are SEQ ID NO: 1 and SEQ ID NO: 2; the sequences of the primer pair aiming at the staphylococcus xylosus are SEQ ID NO: 3 and SEQ ID NO: 4; the sequences of the primer pair aiming at the staphylococcus sciuri are SEQ ID NO: 5 and SEQ ID NO: 6. The primer group and the detection method disclosed by the invention can realize rapid detection of three bacteria on a molecular level, have the advantages of simplicity and convenience in operation, high detection efficiency and accurate result, and are suitable for rapid screening and monitoring of health conditions of experimental animals.

The invention discloses a multiplex PCR (Polymerase Chain Reaction) detection kit and a multiplex PCR detection method for distinguishing bovine type, human type and avian type mycobacteria. The multiplex PCR detection kit comprises a primer pair 16sRNA-F and 16sRNA-R, a primer pair 16sRNA-F, a primer pair 16sRNA-R and a primer pair 16sRNA-R, wherein the primer pair 16sRNA-F and 16sRNA-R are used for specifically amplifying mycobacterium genes 16sRNA; the primer pair is used for specifically amplifying the gene of the mycobacterium tuberculosis guup16810, and the primer pair is used for specifically amplifying the gene of the mycobacterium tuberculosis guup16810-F and the gene of the mycobacterium tuberculosis guup16810-R; the primer pair Rv1506c-F and the primer pair Rv1506c-R are used for specifically amplifying the mycobacterium bovis Rv1506c gene; the kit is characterized in that the kit is a primer pair of the guup32724-F and the guup32724-R which are used for specifically amplifying the avian mycobacterium gene guup32724, and the kit is a primer pair of the guup32724-F and the guup32724-R. The multiplex primer provided by the invention has the advantages of difficulty in forming a dimer and a hairpin structure, uniform Tm value, moderate primer length and GC content and the like, and is beneficial to obtaining a high-quality amplification product, so that a detection result is accurate and reliable.

The application provides an sgRNA, a kit, a detection method and application for detecting tuberculosis based on RPA-CRISPR Cas12b technology, and belongs to the technical field of molecular detection. The application provides an sgRNA for detecting mycobacterium, which comprises sgRNA-6110 with a nucleotide sequence as shown in SEQ ID NO: 1 and / or sgRNA-1081 with a nucleotide sequence as shown in SEQ ID NO: 2. The sgRNA can specifically recognize and combine with the high-conservation target sequence IS6110 and IS1081 of Mycobacterium tuberculosis and / or Mycobacterium bovis, and can be used for instant detection of Mycobacterium tuberculosis and / or Mycobacterium bovis which are pathogenic bacteria of human and livestock tuberculosis.

The invention relates to pediococcus pentosaceus R11 and application thereof. The preservation number of the pediococcus pentosaceus R11 is CGMCC No.32715, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is November 20, 2024, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The pediococcus pentosaceus R11 and the culture thereof provided by the invention are helpful for preventing and controlling mycobacterium tuberculosis bovine variant infection.

The invention relates to the technical field of microbiological detection, in particular to a Brucella bovis and Mycobacterium bovis dual-fluorescent microsphere test strip and a preparation method and application thereof, and the Brucella bovis and Mycobacterium bovis dual-fluorescent microsphere test strip comprises a bottom plate, a sample pad, a nitrocellulose membrane coated with recombinant protein and absorbent paper, the nitrocellulose membrane coated with the recombinant protein is provided with a brucella abortus lipopolysaccharide detection line T1, a mycobacterium bovis rMPB70 protein detection line T2 and a recombinant streptococcus G (rSPG) protein control line C, and the brucella abortus lipopolysaccharide detection line T1, the mycobacterium bovis rMPB70 protein detection line T2 and the rSPG protein control line C are arranged in parallel. The detection time is short, the stability is good, the operation is simple, the sensitivity is high, complex instruments and professional personnel are not needed, and the result judgment is visual and reliable.

The invention discloses application of an STX6 gene as a target spot in prevention and treatment of tuberculosis, the survival of mycobacterium bovis, mycobacterium tuberculosis, BCG and mycobacterium smegmatis in host cells can be remarkably inhibited by knocking out or knocking down the expression of the STX6 gene, the host cell death induced by the mycobacterium bovis and the mycobacterium tuberculosis is inhibited, and the normal proliferation of the host cells is not influenced. Meanwhile, the STX6 is highly conserved in mammals, so that the STX6 can be widely applied to prevention and treatment of tuberculosis caused by humans and animals, can also be used as a target for gene editing animal design, is applied to genetic therapy and disease-resistant breeding, and has rich application and economic values.

The application provides a recombinant protein antigen and application thereof in preparation of a kit for detecting bovine gamma interferon, and belongs to the field of biomedical technology. The LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein are obtained through modification and design of CFP10, ESAT6 and Rv3615c; and the recombinant protein antigen is obtained by mixing the three fusion proteins. The recombinant protein antigen obtained by the application is compared with avian and bovine PPD, and it is found that the recombinant protein antigen obtained by the application has higher bovine mycobacterium specificity and sensitivity; and the recombinant protein culture tube prepared by using the culture composition has the advantages of ready-to-use blood sampling anticoagulation, simultaneous incubation and long-distance transportation of plasma after centrifugation.

The invention relates to the technical field of biological detection, in particular to a primer and probe composition for detecting mycobacterium tuberculosis and mycobacterium bovis and application of the primer and probe composition. The invention provides a primer and probe composition for detecting mycobacterium tuberculosis and mycobacterium bovis, and the primer and probe composition comprises a composition 1 for detecting the mycobacterium tuberculosis and a composition 2 for detecting the mycobacterium bovis, wherein the composition 1 for detecting mycobacterium tuberculosis comprises a primer and a probe with nucleotide sequences shown as SEQ ID NO.1-3; wherein the composition 2 for detecting the mycobacterium bovis comprises a primer and a probe which have nucleotide sequences as shown in SEQ ID NO.4-6. The primer and probe composition provided by the invention is high in detection sensitivity and strong in specificity.

The present invention relates to a method for determining the presence of Mycobacterium bovis in a subject. The method comprises: (i) determining the level of one or more biomarkers in a sample from the subject; and (ii) comparing the level of said one or more biomarkers with the level of said one or more metabolites in a control sample to determine whether Mycobacterium bovis is present in the subject. The biomarkers are selected from: dihydro-leukotriene B4, 3-hydroxyisovaleric acid, sphingomyelin [SM(d18:2(4E,14Z) / 24:0)], sphingomyelin [SM(d18:1 / 24:1(15Z))], 18:3 cholesteryl ester, an unknown compound 389.21933 m / z (under negative ionisation), an unknown compound 886.71063 m / z (under negative ionisation), citric acid, MG(0:0 / 18:3(6Z,9Z,12Z) / 0:0), pyrocatechol, N-(docosanoyl)-heptadecasphing-4-enine-1-phosphocholine and an unknown compound 359.22107 m / z (under negative ionisation).

The invention describes a recombinant Mycobacterium strain such as M. smegmatis that is recombinantly converted to express the M. tb complex ESX protein expression system, e.g. M. tb complex ESX-1 protein expression system. The recombinant strain exports virulence effector proteins (such as EspA, EspB, EspC, EsxA or EsxB) similar to M. tb, when administered to a subject, and is therefore useful as a potential tuberculosis vaccine candidate. The recombinant strain is non-pathogenic and the vaccinated subject does not give a false positive result when tested with any standard tuberculosis diagnostic test e.g. CCT, as the recombinant strain does not sensitize the vaccinated host to M. tb antigens or protein derivate of M. bovis.

Certain embodiments of the invention pertain to a method of treating an infection in a subject caused by an infectious agent other than Escherichia coli, the method comprising administering to the subject arsinothricin or a salt thereof. The infectious agent other than E. coli can be a bacterium, protozoan, helminth, archaebacterium, or a fungus. In preferred embodiments, the infectious agent is Mycobacterium tuberculosis, Mycobacterium bovis, or Enterobacter cloacae. The invention also pertains to a method of treating an infection in a subject caused by an infectious agent, comprising administering to the subject arsinothricin or a salt thereof in combination with an inhibitor of phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase. In certain such embodiments, the infectious agent expresses phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase. Further embodiments provide compositions comprising arsinothricin or a salt thereof and an inhibitor of phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase.

Certain embodiments of the invention pertain to a method of treating an infection in a subject caused by an infectious agent other than Escherichia coli, the method comprising administering to the subject arsinothricin or a salt thereof. The infectious agent other than E. coli can be a bacterium, protozoan, helminth, archaebacterium, or a fungus. In preferred embodiments, the infectious agent is Mycobacterium tuberculosis, Mycobacterium bovis, or Enterobacter cloacae. The invention also pertains to a method of treating an infection in a subject caused by an infectious agent, comprising administering to the subject arsinothricin or a salt thereof in combination with an inhibitor of phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase. In certain such embodiments, the infectious agent expresses phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase. Further embodiments provide compositions comprising arsinothricin or a salt thereof and an inhibitor of phosphinothricin N-acetyltransferase or arsinothricin N-acetyltransferase.

The invention discloses preparation and application of a novel mycobacterium tuberculosis subunit vaccine LK35. The mycobacterium tuberculosis subunit vaccine is composed of fusion protein LK35 and a DP adjuvant. The fusion protein LK35 is prepared by sequentially connecting EsxL, EsxK and Rv2656c with one another. The invention also provides a preparation method of the fusion protein. The subunit vaccine can be used as a preventive vaccine for diseases related to mycobacterium tuberculosis / environmental mycobacterium, and can also be used as a therapeutic vaccine after mycobacterium tuberculosis / environmental mycobacterium infection. The vaccine immunized mice can provide good antigen-specific immune response, and the vaccine combined with fusion proteins LT20, LT57 and LT28 can provide effective long-term protective efficacy against mycobacterium bovis. Therefore, the subunit vaccine involved in the invention is expected to become a novel tuberculosis vaccine, and has good application and development prospects.

The invention discloses a multiple PCR (Polymerase Chain Reaction) primer group and a detection kit for simultaneously detecting four pathogens, and also discloses a method for carrying out multiple PCR detection by utilizing the primer group and application of the primer group. Sensitivity test results show that the sensitivity of the mycoplasma bovis, mannheimia haemolytica, pasteurella multocida and mycobacterium bovis detected by PCR has a wide detection range, and the sensitivity can be detected in samples in a certain range. Specific detection results show that only mycoplasma bovis, mannheimia haemolytica, pasteurella multocida and mycobacterium bovis can be well amplified, and other bacteria cannot be amplified, so that the primer group has good specificity.

The application discloses a preparation method of a mycobacterium bovis polysaccharide antigen, the polysaccharide antigen is a glycosyl part of a phenolic glycolipid PGL in a cell wall of mycobacterium bovis, the polysaccharide antigen has a clear and single chemical structure, can be prepared in a large scale and efficiently through a chemical synthesis method, and effectively solves problems of a complex natural extraction process, non-uniform structure, limited yield and the like. The polysaccharide antigen can be used as a core immunogen and is used for preparation of a mycobacterium PGL polysaccharide conjugate vaccine, provides a stable and controllable key raw material for tuberculosis vaccine research and development, and has a good industrial application prospect.

The invention discloses application of an MmpE gene in regulating and controlling virulence of mycobacterium bovis, the nucleotide sequence of the MmpE gene is shown as SEQ ID NO.1, the amino acid sequence of encoded protein is shown as SEQ ID NO.2, the protein encoded by the MmpE gene is nuclear regulatory protein and is positioned in a host cell nucleus, and the MmpE gene can be used for regulating and controlling the virulence of mycobacterium bovis. The MmpE protein can inhibit the inflammation level of a host and enhance the survival and colonization functions of the mycobacterium bovis in host cells after entering the nucleus, the MmpE protein plays a nuclear localization function by depending on two nuclear localization signal sequences (NLS), the two NLSs are respectively located at the 20th-22th site and the 460th-462th site of the amino acid sequence shown as SEQ ID NO.2, and then the MmpE gene or the two NLSs thereof is modified, so that the activity of the MmpE protein in the host cells is improved, and the activity of the MmpE protein in the host cells is improved. And obtaining the mycobacterium bovis attenuated strain. The invention finds an important target capable of inhibiting mycobacterium bovis infection, and has important application value in preparation of novel mycobacterium bovis vaccines and drugs.

The invention discloses a reagent or a kit for detecting bovine respiratory syndrome pathogenic bacteria and application of the reagent or the kit, and belongs to the field of animal husbandry pathogenic bacteria detection. The technical problem to be solved by the invention is how to detect bovine respiratory syndrome pathogenic bacteria. The reagent or the kit disclosed by the invention contains primer pairs as shown in SEQ ID No.1 and 2, primer pairs as shown in SEQ ID No.4 and 5, primer pairs as shown in SEQ ID No.7 and 8, primer pairs as shown in SEQ ID No.10 and 11, probes as shown in SEQ ID No.3, 6, 9 and 12, a bovine mycoplasma positive reference substance, a bovine mycobacterium positive reference substance, a bovine pasteurella multocida positive reference substance and a bovine klebsiella pneumoniae positive reference substance and / or a bovine klebsiella pneumoniae negative reference substance. The reagent or the kit disclosed by the invention can be used for successfully detecting the mycoplasma bovis, the mycobacterium bovis, the bovine pasteurella multocida and the bovine klebsiella pneumoniae.

The invention relates to the technical field of bioengineering, in particular to a preparation method and application of an immunodiagnosis antigen for identifying mycobacterium hominis infection and mycobacterium bovis infection, and a fusion protein LB30 is successfully constructed and prepared by using a genetic engineering technology based on mycobacterium bovis deletion antigens Rv2645 and Rv3120 in an RD region. The fusion protein LB30 in the invention induces strong cellular immune response in tuberculosis patients, and the immunogenicity is superior to that of a single protein. ESAT6-CFP10-IGRA (EC-IGRA) is used for detecting tuberculosis infection of high-risk crowds and cattle herds in the pasturing area, LB30-IGRA is used for identifying mycobacterium hominis and bovine infection, it is found that the EC-IGRA positive and LB30-IGRA negative ratio of the high-risk crowds in the pasturing area is higher than that of common crowds, and it is prompted that the mycobacterium bovine infection rate in the pasturing area is higher than that of the common crowds; if EC-IGRA positive and LB30-IGRA positive cattle exist in the cattle herds, it is indicated that mycobacterium tuberculosis infection exists in the cattle herds. The method can be used for preliminarily identifying human type and bovine type mycobacterium infection, and is expected to be applied to epidemiological investigation of human-animal tuberculosis infection in the later period.

There is provided a skin test diagnostic reagent comprising: at least one 40mer polypeptide consisting of any one of SEQ ID NOs: 1, 3 or 4; at least one 40mer polypeptide consisting of any one of SEQ ID NOs: 7 or 8; and at least one 40mer polypeptide consisting of any one of SEQ ID NOs: 10, 11 or 12, characterised in that the reagent elicits a positive result when administered in a skin test to an animal infected with Mycobacterium bovis or Mycobacterium tuberculosis.

The present invention discloses a recombinant Helicobacter pylori vaccine and its preparation method and application. The recombinant Helicobacter pylori vaccine comprises a fusion antigen and an adjuvant. The fusion antigen is a fusion protein containing Helicobacter pylori antigen protein and an immunocyte factor, and the adjuvant is an intracellular metabolite of Mycobacterium bovis BCG. The beneficial effects of the present invention are as follows: The vaccine provided by the present invention uses, for the first time, a fusion protein containing Helicobacter pylori antigen protein and an immunocyte factor as the fusion antigen, supplemented with an intracellular metabolite of Mycobacterium bovis BCG as the adjuvant. This fusion antigen has natural protein activity, can produce excellent immunogenicity, and can trigger an effective anti-Helicobacter pylori immune response in immunized individuals. After further combining with the adjuvant, it can stimulate a more efficient immune response against Helicobacter pylori antigen. The fusion antigen is expressed by genetically engineered bacteria, and the bacteria used for extracting the adjuvant have high safety, stable and controllable sources, and can be mass-produced on a large scale.

The invention provides a recombinant protein antigen and application thereof in preparation of a kit for detecting bovine gamma interferon, and belongs to the technical field of biomedicine. According to the present invention, the CFP10, the ESAT6 and the Rv3615c are modified and designed to obtain the LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein, and the three fusion proteins are mixed to obtain the recombinant protein antigen; compared with poultry type and bovine type PPD, the recombinant protein antigen obtained by the invention is found to have higher specificity and sensitivity of mycobacterium bovis; the recombinant protein culture tube prepared from the culture composition has the advantages that blood sampling and anticoagulation can be achieved immediately, and meanwhile long-distance plasma transportation is achieved after incubation and centrifugation.