12 results about "Bovine tuberculosis" patented technology

The invention discloses a method for establishing indirect ELISA (enzyme-linked immunosorbent assay) for detecting mycobacterium bovis Lprl, which comprises the following steps: preparing mycobacterium bovis Lprl protein, taking the purified protein as an antigen, and determining conditions such as optimal antigen coating concentration, serum dilution, serum incubation time, secondary antibody dilution concentration, incubation time and the like. The established indirect ELISA detection method does not have cross reaction with other bovine pathogens, the specificity is good, the detection accuracy is high, and the total coincidence rate of test results of intradermal allergy with bovine tubercle bacillus is 95.8%; the intra-batch variation coefficient of the detected samples is 2.43%-3.61%, the inter-batch variation coefficient is 2.83%-3.96%, and the intra-batch variation coefficient and the inter-batch variation coefficient are both smaller than 10%. A new diagnosis target is developed, the established indirect ELISA method has the characteristics of high sensitivity, strong specificity, good repeatability and the like, the operation is easy, and a new method is provided for the detection work of the mycobacterium bovis.

The invention discloses a mycobacterium tuberculosis deletion region protein Rv1577c and a fusion antigen LB28 containing a mycobacterium tuberculosis RD region and a latent infection related antigen, wherein the fusion antigen comprises Rv1577c and Rv1738. The invention provides a candidate antigen Rv1577c of a tuberculosis subunit vaccine through antigen screening and experimental verification. The Rv1577c is deleted in BCG, so that the immunogenicity is better, and the Rv1577c has certain consistency with ESAT6 and CFP10 in bovine tuberculosis immunodiagnosis. And the Rv1577 is combined with a latent infection associated antigen Rv1738 to construct the tuberculosis fusion protein LB28. Under the assistance of an adjuvant DDA + PolyI: C (DP), the fusion protein LB28 shows good immunogenicity in a mouse model, and has an immune protection effect of resisting mycobacterium bovis infection. The Rv1577 and the fusion protein LB28 constructed based on the Rv1577 can be applied to animal and human tuberculosis prevention and treatment.

This invention discloses the application of the TRIM58 gene in regulating host resistance to Mycobacterium bovis infection. A bovine macrophage cell line with TRIM58 gene knockout was constructed using CRISPR / Cas9 technology. Results showed that TRIM58 knockout significantly inhibited intracellular survival of Mycobacterium bovis and reduced infection-induced cell death; reintroduction or overexpression of TRIM58 restored or enhanced bacterial viability. Therefore, this invention is the first to demonstrate that TRIM58 is a negative regulator of host resistance to Mycobacterium bovis infection, and that inhibitors of the TRIM58 gene can be used to prepare drugs against Mycobacterium bovis infection or to treat bovine tuberculosis. This invention fills a research gap in the field of TRIM58 in bovine tuberculosis and provides an effective tool for screening and studying the mechanisms of action of anti-bovine tuberculosis drugs.

The invention belongs to the technical field of biological detection, and particularly relates to an antigen protein composition for detecting bovine tuberculosis gamma-interferon and a detection kit. The antigen protein composition for detecting the bovine tuberculosis gamma-interferon is prepared from MTB8.4, ESAT6, CFP10 and Rv3615c. The antigen protein composition is used for detecting the bovine tuberculosis gamma-interferon. Through scientific matching, the antigen protein composition MTB8.4, ESAT6, CFP10 and Rv3615c is obtained, the sensitivity and efficiency are improved in combination with a light-activated chemiluminescence technology, an anti-interference system is optimized for animal samples, comprehensive breakthrough of sensitivity (96%), specificity (98%) and detection efficiency (200 samples per hour) is achieved, and an efficient solution is provided for large-scale screening of bovine tuberculosis.

The invention discloses a genetically modified bovine mycobacterium attenuated strain as well as a construction method and application thereof. Reconstruction is carried out by using a homologous recombination principle, and gene knockout of a genome RD1 region and membrane protein gene insertion are realized, so that an attenuated live vaccine candidate strain is constructed. The compound can be used as an immunotherapeutic agent or a tuberculosis attenuated live vaccine for bladder cancer treatment. Based on a mycobacterium bovis virulent strain AF2122 / 97, an RD1 region of the mycobacterium bovis virulent strain AF2122 / 97 is knocked out through gene modification so as to reduce the toxicity of the mycobacterium bovis virulent strain AF2122 / 97, but cell wall antigens (such as MPB70 / 83) are retained, and meanwhile, deletion region related antigen fragments are refilled and inserted so as to enhance the immunogenicity of the mycobacterium bovis virulent strain AF2122 / 97.

The application provides a polypeptide antigen composition of bovine Mycobacterium tuberculosis specific T cells and application thereof in preparation of a bovine gamma interferon kit, and belongs to the field of biomedical technology. The polypeptide antigen composition is obtained by mixing 15 short peptides with 20-50 amino acids designed based on the amino acid sequences of ESAT6, CFP10 and Rv3615c in a specific ratio, and has good specificity and can be applied to the differential detection of early bovine tuberculosis of BCG vaccinated bovine and NTM Mycobacterium avium subspecies paratuberculosis positive bovine. The polypeptide antigen composition obtained by the application is compared with avian and bovine PPD, and it is found that the polypeptide antigen composition obtained by the application has higher bovine Mycobacterium tuberculosis specificity and sensitivity; and the polypeptide culture tube prepared by the application has the advantages of ready-to-use blood sampling, simultaneous incubation and long-distance transportation of plasma after centrifugation.

This invention discloses NmrAL2_Bovine The application of genes as targets in the prevention and control of bovine tuberculosis, including knockout or knockdown. NmrAL2_ Bovine The gene can significantly inhibit the survival of Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium smegmae in host cells without affecting the normal proliferation of host cells. Therefore, NmrAL2_Bovine Gene inhibitors can be used to prepare drugs for the prevention and treatment of bovine tuberculosis. NmrAL2_Bovine Genes can also be used as targets in the design of transgenic cattle for breeding against bovine tuberculosis or for genetic therapy research.

The application discloses a Mycobacterium bovis non-coding small RNA, and belongs to the field of biology. Mycobacterium bovis , M.bovis The nucleotide sequence of the non-coding small RNA is shown as SEQ ID NO:1. Research finds that the survival ability of a BCG strain with overexpressed ncBCG201 is obviously enhanced under carbon starvation culture conditions, the survival ability is obviously decreased under membrane pressure culture conditions, the overexpressed strain shows a slower growth speed in a logarithmic growth phase, and the biofilm forming capacity is obviously reduced. The above results show that the non-coding small RNA can reduce the pathogenicity and resistance of the Mycobacterium bovis, and has a good application prospect in preparation of a bovine tuberculosis vaccine.

The present invention relates to the field of biotechnology and provides a recombinant protein, a preparation method thereof, and its application in bovine tuberculosis detection. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the recombinant protein is shown in SEQ ID NO.2. The recombinant protein is obtained by serially combining ESAT-6, CFP-10, and BFH using a linker in the manner of ESAT-6-CFP-10-BFH. Through the above technical solution, the problem of low sensitivity of the EC method for detecting bovine tuberculosis in the prior art is solved.

The invention discloses a bovine tuberculosis gamma-interferon ELISA (enzyme-linked immunosorbent assay) detection kit and application thereof. The kit contains a stimulating protein for detecting bovine tuberculosis and a monoclonal antibody for resisting bovine gamma-interferon; wherein the stimulating protein for bovine tuberculosis detection is an ESAT-6-CFP-10 recombinant protein, and is obtained by connecting an optimized mycobacterium tuberculosis ESAT-6 antigen gene and an optimized mycobacterium tuberculosis CFP-10 antigen gene in series, inserting a prokaryotic expression vector and then performing prokaryotic expression; the monoclonal antibody for resisting the bovine gamma-interferon is secreted by a hybridoma cell with the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.45834. Compared with tubercle bacillus, the sensitivity of the ESAT-6-CFP-10 recombinant protein for stimulating T lymphocytes to secrete gamma-interferon is higher than that of bovine tubercle bacillus derivatives, interference of tuberculosis bacteria in the external environment can be eliminated, and the diagnosis accuracy is improved.

The invention provides a specific primer and probe for detecting bovine tuberculosis and a kit composed of the specific primer and probe, and belongs to the technical field of biological detection. Specific primer pairs and probes are designed for cattle CXCL10, IFN-gamma and GAPDH genes, and quantitative detection of the transcription level of CXCL10 and IFN-gamma in cattle in-vitro whole blood is realized by using a qPCR technology, so that cattle tuberculosis is diagnosed. The method greatly shortens the detection time (incubation only needs 4 hours), simplifies the operation process, eliminates the system error in the sample adding process by using the GAPDH gene as the internal reference, and improves the accuracy of the detection result.

The invention discloses a screening method and application of bovine tuberculosis diagnostic markers, and belongs to the technical field of bovine tuberculosis diagnosis. According to the invention, system screening is carried out by utilizing machine learning, and clinical sample verification is carried out, so that three marker genes with specific diagnostic value on bTB infection, namely genes ADGRA3, PRELP and HBB, are successfully identified as bovine tuberculosis diagnostic markers in numerous genes which are not publicly reported yet. The marker genes obtained through screening are combined, a multi-parameter combined diagnosis model (AUC = 0.959 and AUC = 0.847) is constructed, the comprehensive distinguishing capacity of the model is improved compared with a traditional single-gene detection method, the comprehensive distinguishing capacity of the model is also at a higher level compared with an existing multi-gene detection method, and a new solution is provided for precise diagnosis of bTB.