CN105541975A - Mixed polypeptide for producing tuberculosis associated cell factors by inducing peripheral blood mononuclear cell - Google Patents

Mixed polypeptide for producing tuberculosis associated cell factors by inducing peripheral blood mononuclear cell Download PDF

Info

Publication number
CN105541975A
CN105541975A CN201511025482.7A CN201511025482A CN105541975A CN 105541975 A CN105541975 A CN 105541975A CN 201511025482 A CN201511025482 A CN 201511025482A CN 105541975 A CN105541975 A CN 105541975A
Authority
CN
China
Prior art keywords
seqidno
tuberculosis
mixed polypeptide
polypeptide
mycobacterium tuberculosis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201511025482.7A
Other languages
Chinese (zh)
Inventor
黄曦
胡鹏男
李妍
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Diao Medical Technology Co Ltd
Original Assignee
Guangzhou Diao Medical Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Diao Medical Technology Co Ltd filed Critical Guangzhou Diao Medical Technology Co Ltd
Priority to CN201511025482.7A priority Critical patent/CN105541975A/en
Publication of CN105541975A publication Critical patent/CN105541975A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Wood Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a mixed polypeptide for producing tuberculosis associated cell factors by inducing a peripheral blood mononuclear cell. The mixed polypeptide has polypeptide sequences represented by SEQ ID NO:1-18. Compared with common stimulating proteins CFP-10, ESAT-6 and Rv1985c or a mixture of the three proteins, the mixed polypeptide has relatively efficient induction performance and the relatively good specificity. The mixed polypeptide only reacts with immunologic memory T cells produced after human body is infected by mycobacterium tuberculosis, the produced cell factors are tuberculosis associated factors including IFN-gamma, IL-2, TNF-alpha and the like with mycobacterium tuberculosis antigen specificity, and meanwhile, the stimulus response is not interfered by BCG. The specific antigen polypeptide provided by the invention is significant for the research of tuberculosis pathopoiesia and immunoprophylaxis mechanisms and therefore has positive significance to the control of tuberculosis.

Description

A kind of mixed polypeptide producing tuberculosis relevant cell factor for inducing peripheral blood mononuclear cell
Technical field
The present invention relates to Protein Detection field, be specifically related to a kind of mixed polypeptide producing tuberculosis relevant cell factor for inducing peripheral blood mononuclear cell.
Background technology
Tuberculosis is a kind of common chronic infectious disease caused by mycobacterium tuberculosis, can invade and many internal organs, infects the most common with pulmonary Mycobacterium.The current whole world has the people of nearly 1/3 to infect tubercule bacillus, and statistic data shows had 1,500,000 people to die from tuberculosis in 2013,9,000,000 new cases, and tuberculosis has become one of principal disease of whole world adult death because of transmissible disease.China is one of 22 tuberculosis high burden countries in the whole world, and active tuberculosis patient number occupies second place of the world.Infection lungy mainly occurs in that patient is undiscovered and before treating, 1 tuberculosis patient can infect nearly 10-15 people by close contact in 1 year.Circulation way lungy is through respiratory infectious, in the lung of pulmonary tuberculosis patient, have a large amount of tubercule bacillus, if he facing to healthy population cough or sneeze just very possible by pathogen transmission to Healthy People.And before not finding sufferer, patient does not take any preventive means, with the process of the close contact such as kinsfolk, colleague, classmate, contactee is just easily by tubercle bacillus affection.Present stage, primary treatments master lungy was pharmacological agent, but such treatment can not stop propagation lungy completely.Bcg vaccination (BCG) internationally recognizedly at present the most effectively prevents immunization method lungy; newborn infant's palpus bcg vaccination is specified in the planned immunization program of China; but its protective efficacy does not also reach 100%; therefore, primary measure lungy is prevented to be the neopathy people finding as early as possible to be hidden in crowd.
Mycobacterium tuberculosis is a kind of typical intracellular infection bacterium, and human body is to the cellular immunization of its immunologic mechanism mainly based on T cell.After mycobacterium tuberculosis invades respiratory tract, non-activated dendritic cell (Dendriticcells in former alveolar, DC) anti-microbial activity is weak, engulfed M. tuberculosis growth can not be prevented, but mycobacterium tuberculosis can be engulfed and take elsewhere to, offer antigen, make T lymphocyte sensitization around, the T lymphocyte of sensitization can produce cytokine profiles (INF-γ, IL-2, IL-4 and TNF-α etc.), and these cytokine actings in conjunction, kill the mycobacterium tuberculosis in focus.According to this immunologic mechanism, by carrying out analysis contrast to the cytokine infected in the patient of mycobacterium tuberculosis and healthy human blood, the concentration finding to infect INF-γ, IL-2, TNF-α in its blood of patient of mycobacterium tuberculosis obviously raises.
For the patient infecting Mycobacterium tuberculosis, when the immune specific lymphocyte crossed contacts tubercule bacillus again, still corresponding cytokine can be secreted.For studying mechanism of causing a disease and the immunologic mechanism of mycobacterium tuberculosis, researchist uses external evoked peripheral blood mononuclear cell (PBMC) to produce the mode of tuberculosis relevant cell factor.By the peripheral blood extraction of the patient that infects mycobacterium tuberculosis, extract PBMC wherein, and adopt tuberculosis related antigen to stimulate it, the T lymphocyte in PMBC to antigen generation immune response, can produce corresponding cytokine.Carry out testing to have suitable danger owing to directly using mycobacterium tuberculosis, be unfavorable for carrying out large quantity research, therefore researchist adopts the antigen of mycobacterium tuberculosis albumen of extraction to research.
Purified protein derivative of tuberculin (PPD) is the active substance obtained from mycobacterium tuberculosis culturing filtrate, T cell can be stimulated to produce the cytokines such as IFN-γ, IL-2, but for through the sample of bacille Calmette-Guerin vaccine (BCG) immune body, the T cell that PPD can stimulate it to extract equally produces tuberculosis relevant cell factor, therefore the specificity of PPD is lower, also needs to find the better stimulator of specificity.
Summary of the invention
The object of the present invention is to provide a kind of mixed polypeptide producing tuberculosis relevant cell factor for inducing peripheral blood mononuclear cell.
The technical solution used in the present invention is:
A kind of mixed polypeptide, comprises following peptide sequence:
1)MAEMKTDAATLAQEAGNFERISGDL（SEQIDNO：1）；
2)GNFERISGDLKTQIDQVESTAGSLQ（SEQIDNO：2）；
3)QVESTAGSLQGQWRGAAGTAAQAAV（SEQIDNO：3）；
4)AAGTAAQAAVVRFQEAANKQKQELD（SEQIDNO：4）；
5)ANKQKQELDEISTNIRQAGVQYSR（SEQIDNO：5）；
6)IRQAGVQYSRADEEQQQALSSQMGF（SEQIDNO：6）；
7)MTEQQWNFAGIEAAASAIQGNVTSI（SEQIDNO：7）；
8)SAIQGNVTSIHSLLDEGKQSLTKLA（SEQIDNO：8）；
9)EGKQSLTKLAAAWGGSGSEAYQGVQ（SEQIDNO：9）；
10)SGSEAYQGVQQKWDATATELNNALQ（SEQIDNO：10）；
11)TATELNNALQNLARTISEAGQAMAS（SEQIDNO：11）；
12)ISEAGQAMASTEGNVTGMFA（SEQIDNO：12）；
13)LAALAAVVELGSFDAAAERLHVTPS（SEQIDNO：13）；
14)AAAERLHVTPSAVSQRIKSLEQQVG（SEQIDNO：14）；
15)RIKSLEQQVGQVLVVREKPCRATTA（SEQIDNO：15）；
16)REKPCRATTAGIPLLRLAAQTALLE（SEQIDNO：16）；
17)RLAAQTALLESEALAEMGGNASLKR（SEQIDNO：17）；
18)MGGNASLKRTRITIAVNADSMATWF（SEQIDNO：18）。
Preferably, above-mentioned mixed polypeptide is by following amino acid sequence encode:
1)ATGGCAGAGATGAAGACCGATGCCGCTACCCTCGCGCAGGAGGCAGGTAATTTCGAGCGGATCTCCGGCGACCTG（SEQIDNO：19）；
2)GGTAATTTCGAGCGGATCTCCGGCGACCTGAAAACCCAGATCGACCAGGTGGAGTCGACGGCAGGTTCGTTGCAG（SEQIDNO：20）；
3)CAGGTGGAGTCGACGGCAGGTTCGTTGCAGGGCCAGTGGCGCGGCGCGGCGGGGACGGCCGCCCAGGCCGCGGTG（SEQIDNO：21）；
4)GCGGGGACGGCCGCCCAGGCCGCGGTGGTGCGCTTCCAAGAAGCAGCCAATAAGCAGAAGCAGGAACTCGACGAG（SEQIDNO：22）；
5)GCCAATAAGCAGAAGCAGGAACTCGACGAGATCTCGACGAATATTCGTCAGGCCGGCGTCCAATACTCGAGGGCC（SEQIDNO：23）；
6)CGTCAGGCCGGCGTCCAATACTCGAGGGCCGACGAGGAGCAGCAGCAGGCGCTGTCCTCGCAAATGGGCTTC（SEQIDNO：24）；
7)AGCATGACAGAGCAGCAGTGGAATTTCGCGGGTATCGAGGCCGCGGCAAGCGCAATCCAGGGAAATGTCACGTCC（SEQIDNO：25）；
8)GCAAGCGCAATCCAGGGAAATGTCACGTCCATTCATTCCCTCCTTGACGAGGGGAAGCAGTCCCTGACCAAGCTC（SEQIDNO：26）；
9)GACGAGGGGAAGCAGTCCCTGACCAAGCTCGCAGCGGCCTGGGGCGGTAGCGGTTCGGAGGCGTACCAGGGTGTC（SEQIDNO：27）；
10)GGTAGCGGTTCGGAGGCGTACCAGGGTGTCCAGCAAAAATGGGACGCCACGGCTACCGAGCTGAACAACGCGCTG（SEQIDNO：28）；
11)GCCACGGCTACCGAGCTGAACAACGCGCTGCAGAACCTGGCGCGGACGATCAGCGAAGCCGGTCAGGCAATGGCT（SEQIDNO：29）；
12)ACGATCAGCGAAGCCGGTCAGGCAATGGCTTCGACCGAAGGCAACGTCACTGGGATGTTCGCATAG（SEQIDNO：30）；
13)CTGGCCGCATTGGCTGCCGTGGTCGAACTGGGCAGCTTCGATGCGGCCGCGGAGCGCCTACATGTCACCCCGTCG（SEQIDNO：31）；
14)GCCGCGGAGCGCCTACATGTCACCCCGTCGGCTGTCAGTCAGCGCATCAAGTCGTTGGAGCAGCAGGTCGGCCAG（SEQIDNO：32）；
15)ATCAAGTCGTTGGAGCAGCAGGTCGGCCAGGTGCTGGTGGTCAGGGAAAAGCCATGTCGGGCGACGACCGCAGGT（SEQIDNO：33）；
16)GAAAAGCCATGTCGGGCGACGACCGCAGGTATCCCGCTGTTGCGGTTGGCCGCGCAAACAGCGTTGCTCGAGTCC（SEQIDNO：34）；
17)TTGGCCGCGCAAACAGCGTTGCTCGAGTCCGAGGCGCTCGCTGAAATGGGTGGCAACGCGTCGCTGAAACGCACG（SEQIDNO：35）；
18)ATGGGTGGCAACGCGTCGCTGAAACGCACGCGGATCACCATTGCGGTAAACGCCGATTCCATGGCGACATGGTTT（SEQIDNO：36）。
Preferably, described mixed polypeptide produces the application of tuberculosis relevant cell factor inductor as inducing peripheral blood mononuclear cell.
Preferably, described mixed polypeptide is as the application of diagnosis antigen.
Preferably, described mixed polypeptide is as the application of tuberculosis prophylaxis treatment stimulator.
The invention has the beneficial effects as follows:
The invention provides and a kind ofly stimulatory effect T cell can produce the mixed polypeptide of IFN-γ.The present invention is by bioinformatics method conjugated antigen Antigen Epitope Prediction, filter out the polypeptide that a lot of bar length is homogeneous, and use the T cell of this mixed polypeptide to the crowd infecting mycobacterium tuberculosis to induce, detect the tuberculosis relevant cell factor of secretion, the method is compared with the mixture of conventional stimulatory protein(SP) CFP-10, ESAT-6 and Rv1985c albumen or three kinds of albumen, and its induction performance is more efficient, specificity is better.
Mixed polypeptide susceptibility of the present invention is strong, specificity is high, only with mycobacterium tuberculosis infection human body after the immunological memory T cell that produces react, the cytokine produced is the tuberculosis correlation factors such as specific IFN-γ, IL-2, TNF-α of negre antigen, and this irritant reaction is not by the interference of BCG simultaneously.
Specific antigen protein effect of stimulation provided by the invention is good, and stimulator antigen adopts the form of mixed polypeptide, and shorter aminoacid sequence more easily by the epitope identification of lymphocytic cell surface, thus reaches the effect of irritation cell secretion relevant cell factor.
Specific antigens provided by the invention tuberculosis is caused a disease and the research of immunoprophylaxis mechanism significant, thus to control lungy, there is positive meaning.
Accompanying drawing explanation
Fig. 1 is PBMC IL-2 secretion level statistics figure after mixed polypeptide stimulates of different sources;
Fig. 2 is PBMC IFN-γ secretion level ELISPOT detected result figure after mixed polypeptide stimulates of different sources;
Fig. 3 is PBMC TNF-α secretion level statistics figure after mixed polypeptide stimulates of different sources;
Fig. 4 is that different stimulated antigen is to PBMC effect of stimulation comparison diagram.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited thereto.
The Characterization of antigenic epitopes of embodiment 1, specificity mixed polypeptide and Peptide systhesis
With reference to GenBank database, bioinformatics technique is used to carry out analyses and prediction to epitope, optimize mixed polypeptide (SEQIDNO:1 ~ 18), wherein every bar polypeptide is containing 20-25 amino acid, containing 8-10 amino acid tumor-necrosis factor glycoproteins between adjacent two polypeptide.Blending ratio is not laid down hard and fast rule, and preferably, 18 polypeptide are wait mass mixing.
Further according to calculating aminoacid sequence: wherein encoded by nucleotide sequence SEQIDNO:19 ~ 36 respectively in mixed polypeptide SEQIDNO:1 ~ 18.Adopt polypeptide solid-state reaction method (SPPS), carry out operation according to synthesizer specification sheets and generate required polypeptide, and product is carried out frozen dried with for subsequent use.
Embodiment 2, density gradient centrifugation are separated human peripheral blood single nucleus cell (PBMC)
Use heparin tube or the vacuum test tube aseptic aspiration personnel to be tested peripheric venous blood 4ml of fresh Lithium heparinate or heparin sodium, put upside down and make antithrombotics and blood mix stand-by.Prepare 2 15ml centrifuge tubes, add isopyknic injection physiological saline and lymphocyte separation medium with blood sampling volume respectively.After fresh heparin anti-coagulating and physiological saline being mixed, uniform speed slow adds in parting liquid, and in 22 DEG C, the centrifugal 20min of 1800g, after centrifugal, visible PBMC cell is present in cloud layer.Draw PBMC cellular layer in new 15ml centrifuge tube, with RPMI-1640 nutrient solution polishing to 12ml, in 22 DEG C, the centrifugal 10min of 600g.Outwell supernatant, with RPMI-1640 nutrient solution polishing to 5ml, after piping and druming precipitation is gently resuspended, in 22 DEG C, the centrifugal 10min of 350g.
Abandon supernatant, add 0.5ml-1ml serum free medium re-suspended cell.Adopt Trypan Blue manual count method or use suitable equipment Auto-counting.According to count results, being diluted with serum free medium is the cell suspension of desired concn.
Embodiment 3, mixed polypeptide induction human peripheral blood single nucleus cell secretion IL-2 level
Choose the heparin sodium anti-freezing peripheral vein blood sample of 20 routine Sputum smears or Sputum culturing inspection positive patient and 20 routine PPD skin test negative patients respectively, density stratification method is separated PBMC, uses serum free medium to be diluted to 2.5 × 10 after cell counting 6the concentration of individual/ml, seeds cells in 96 orifice plates and cultivates, every hole inoculation 2.5 × 10 5individual cell, each sample arranges 3 detect aperture, be blank control wells (adding 50 μ l serum free mediums) respectively, experimental port (adds the mixed polypeptide that 50 μ l concentration are 2 μ g/ml, serum free medium is used to dilute) and Positive control wells (adding 50 μ l phytohemagglutinin PHA), 37 DEG C, 5%CO 2condition hatches 18-24 hour.Hatch rear collection each hole culture supernatant, use ELISA method to detect the burst size of IL-2 in supernatant.
Adopt and wrap by the elisa plate of anti-human IL-2 monoclonal antibody in advance, cell culture supernatant is added respectively in each hole, incubated at room 2 hours, elisa plate is cleaned 3-5 time with Washbuffer, anti-IL-2 antibody is added in detect aperture, 37 DEG C of incubated at room 1 hour, then antibody is removed, elisa plate is cleaned 3-5 time with Washbuffer, add HRP mark again two resist, incubated at room 1 hour, then remove two to resist, elisa plate is cleaned 3-5 time with Washbuffer, add the TMB chromogenic substrate prepared, lucifuge colour developing 5-10 minute under room temperature, finally add stop buffer color development stopping, interpretation of result is carried out by microplate reader in 30 minutes.Fig. 1 is shown in by ELISA detected result statistical graph.
In Fig. 1, relatively Sputum smears or Sputum culturing check that the secretory volume of difference and PPD skin test negative patient PBMC IL-2 after stimulating through mixed polypeptide that the secretory volume of the PBMC of positive patient IL-2 after stimulating through mixed polypeptide deducts the IL-2 secretory volume numerical value of blank control wells deducts the difference of the IL-2 secretory volume numerical value of blank control wells, carry out statistical study to it.Experimental result show in 20 examples have 19 routine Sputum smears or Sputum culturing to check the difference level of positive patient is obviously all higher than the difference level of PPD skin test negative patient group, illustrates that Sputum smears or Sputum culturing check that the PBMC of positive patient is significantly increased in the secretory volume of its IL-2 after mixed polypeptide stimulation.
Embodiment 4, mixed polypeptide induction human peripheral blood single nucleus cell secretion of gamma-IFN level
Choose the heparin sodium anti-freezing peripheral vein blood sample of 20 routine Sputum smears or Sputum culturing inspection positive patient and 20 routine PPD skin test negative patients respectively, density stratification method is separated PBMC, uses serum free medium to be diluted to 2.5 × 10 after cell counting 6individual/ml concentration is for subsequent use., as Sptting plate, every hole adds 2.5 × 10 to select pvdf membrane check-out console (use anti-human IFN-γ monoclonal antibody bag to be spent the night in advance, 4 DEG C of conditions are preserved) 5individual PBMC, each sample arranges 3 detect aperture, blank control wells (adding 50 μ l serum free mediums), experimental port (adding the mixed polypeptide that 50 μ l concentration are 2 μ g/ml) and Positive control wells (adding 50 μ l phytohemagglutinin PHA) respectively, 37 DEG C, 5%CO 218-24 hour is hatched in incubator.Within second day, take out check-out console, abandon culture supernatant, rinse check-out console 3-4 time with PBS, then wash plate 1-2 time with PBS, rock 5 times gently back and forth at every turn, pat dry after washing plate for the last time.In each hole, add traget antibody working fluid, put 37 DEG C and hatch 1 hour.Abandon liquid, wash plate 3-5 time with PBS, rock 5 times gently back and forth at every turn, pat dry after washing plate for the last time, every hole adds enzyme conjugates working fluid, puts 37 DEG C and hatches 1 hour.Abandon liquid, plate is washed 5 times with PBS, rock 5 times gently back and forth at every turn, pat dry after washing plate for the last time, every hole adds the nitrite ion of 100 μ l, put 37 DEG C of lucifuges colour developing 5-10 minute high-visible to spot size naked eyes, abandon liquid, with deionized water or tap water check-out console pros and cons and base 3-5 time, pat dry the water in hole, color development stopping, plate is placed ventilation drying at room temperature 1-2 hour, colour developing result CTLImmunoSpotS6ELISpot analyser carries out automatic analysis, the results are shown in Figure 2, only show the result of a wherein example herein, numeral wherein in figure is positive spots statistics.
As shown in Figure 2: 20 routine Sputum smears or Sputum culturing check that the ELISOPT detected result of positive patient is all in obviously positive, and the ELISOPT detected result of 20 routine PPD skin test negative patients is all in obviously negative, experimental result shows that Sputum smears or Sputum culturing check that the secretory volume of the PBMC of positive patient its IFN-γ after stimulating through mixed polypeptide is significantly improved.
Embodiment 5, mixed polypeptide induction human peripheral blood single nucleus cell TNF secretion-alpha levels
Choose the heparin sodium anti-freezing peripheral vein blood sample of 20 routine Sputum smears or Sputum culturing inspection positive patient and 20 routine PPD skin test negative patients, density stratification method is separated PBMC, uses serum free medium to be diluted to 2.5 × 10 after cell counting 6the concentration of individual/ml, seeds cells in 96 orifice plates and cultivates, every hole inoculation 2.5 × 10 5individual cell.Each sample arranges 3 detect aperture, is blank control wells (adding 50 μ l serum free mediums), experimental port (adding the mixed polypeptide that 50 μ l concentration are 2 μ g/ml) and Positive control wells (adding 50 μ l phytohemagglutinin PHA) respectively, 37 DEG C, 5%CO 2hatch 18-24 hour in incubator, hatched rear collection each hole culture supernatant, use ELISA method to detect the burst size of supernatant TNF-α.Adopt and wrap by the elisa plate of anti-human TNF-α monoclonal antibody in advance, add cell culture supernatant, incubated at room 2 hours, elisa plate is cleaned 3-5 time with Washbuffer, anti-TNF-α antibody is added in detect aperture, incubated at room 1 hour, then antibody is removed, elisa plate is cleaned 3-5 time with Washbuffer, add HRP mark again two resist, incubated at room 1 hour, then remove two to resist, elisa plate is cleaned 3-5 time with Washbuffer, add the TMB chromogenic substrate prepared, lucifuge colour developing 5-10 minute under room temperature, finally add stop buffer color development stopping, interpretation of result is carried out by microplate reader in 30 minutes.Fig. 3 is shown in by ELISA detected result statistical graph.
In Fig. 3, relatively Sputum smears or Sputum culturing check that the secretory volume of difference and PPD skin test negative patient PBMC IL-2 after stimulating through mixed polypeptide that the secretory volume of the PBMC of positive patient TNF-α after stimulating through mixed polypeptide deducts the IL-2 secretory volume numerical value of blank control wells deducts the difference of the TNF-α secretory volume numerical value of blank control wells, carry out statistical study to it.Experimental result shows 19 routine Sputum smears or Sputum culturing checks that the difference level of positive patient is obviously all higher than the difference level of PPD skin test negative patient group, illustrates that Sputum smears or Sputum culturing check that the secretory volume of the PBMC of positive patient its TNF-α after stimulating through mixed polypeptide is significantly increased.
Embodiment 6, specificity mixed polypeptide and CFP-10, ESAT-6 and Rv1985c albumen and composition thereof contrast human peripheral blood single nucleus cell effect of stimulation
The stimulatory protein(SP) for inducing peripheral blood mononuclear cell conventional at present has CFP-10, ESAT-6 and Rv1985c etc., and the mixed polypeptide prepared with the present invention and CFP-10, ESAT-6 and Rv1985c albumen and composition thereof carry out inducibility contrast experiment.
Concrete operations are as follows: the heparin sodium anti-freezing peripheral vein blood sample choosing Sputum smears or Sputum culturing inspection positive patient, density stratification method is separated PBMC, uses serum free medium to be diluted to 2.5 × 10 after cell counting 6the concentration of individual/ml is for subsequent use., as Sptting plate, every hole adds 2.5 × 10 to select pvdf membrane check-out console (use anti-human IFN-γ monoclonal antibody bag to be spent the night in advance, 4 DEG C of conditions are preserved) 5individual PBMC.Each sample arranges 6 detect aperture, blank control wells (adding 50 μ l serum free mediums) respectively, it is the mixed polypeptide of 2ug/ml that experimental port 1(adds 50 μ l concentration), experimental port 2(adds the ESAT-6 albumen that 50 μ l concentration are 2ug/ml), experimental port 3(adds the CFP-10 albumen that 50 μ l concentration are 2ug/ml), experimental port 4(adds the Rv1985c albumen that 50 μ l concentration are 2ug/ml), experimental port 5(adds the mixture of 50 μ l3 kind albumen, the concentration of often kind of albumen is 2ug/ml) and Positive control wells (adding phytohemagglutinin PHA), 37 DEG C, 5%CO 218-24 hour is hatched in incubator.Within second day, take out check-out console, abandon culture supernatant, rinse check-out console 3-5 time with PBS, rock 5 times gently back and forth at every turn, pat dry after washing plate for the last time.In each hole, add traget antibody working fluid, put 37 DEG C and hatch 1 hour.Abandon liquid, wash plate 3-5 time with PBS, rock 5 times gently back and forth at every turn, pat dry after washing plate for the last time, every hole adds enzyme conjugates working fluid, puts 37 DEG C and hatches 1 hour.Abandon liquid, wash plate 5 times with PBS, rock 5 times gently back and forth at every turn, pat dry after washing plate for the last time, every hole adds 100 μ l nitrite ions, and it is high-visible to spot size naked eyes to put 37 DEG C of lucifuges colour developing 5-10 minute, abandons liquid, with deionized water or tap water check-out console pros and cons and base 3-5 time, water in button dry hole, color development stopping, places ventilation drying at room temperature 1-2 hour by plate, colour developing result CTLImmunoSpotS6ELISpot analyser carries out automatic analysis, the results are shown in Figure 4.In Fig. 4, the T cell of the experimental result display PPD positive is maximum at the spot of mixed polypeptide group (experimental group 1), and namely the effect of stimulation of this group is best.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
<110> Guangzhou Di Ao medical science and technology company limited
<120> mono-kind produces the mixed polypeptide of tuberculosis relevant cell factor for inducing peripheral blood mononuclear cell
<130>
<160>36
<170>PatentInversion3.5
<210>1
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>1
MetAlaGluMetLysThrAspAlaAlaThrLeuAlaGlnGluAlaGly
151015
AsnPheGluArgIleSerGlyAspLeu
2025
<210>2
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>2
GlyAsnPheGluArgIleSerGlyAspLeuLysThrGlnIleAspGln
151015
ValGluSerThrAlaGlySerLeuGln
2025
<210>3
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>3
GlnValGluSerThrAlaGlySerLeuGlnGlyGlnTrpArgGlyAla
151015
AlaGlyThrAlaAlaGlnAlaAlaVal
2025
<210>4
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>4
AlaAlaGlyThrAlaAlaGlnAlaAlaValValArgPheGlnGluAla
151015
AlaAsnLysGlnLysGlnGluLeuAsp
2025
<210>5
<211>24
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>5
AlaAsnLysGlnLysGlnGluLeuAspGluIleSerThrAsnIleArg
151015
GlnAlaGlyValGlnTyrSerArg
20
<210>6
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>6
IleArgGlnAlaGlyValGlnTyrSerArgAlaAspGluGluGlnGln
151015
GlnAlaLeuSerSerGlnMetGlyPhe
2025
<210>7
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>7
MetThrGluGlnGlnTrpAsnPheAlaGlyIleGluAlaAlaAlaSer
151015
AlaIleGlnGlyAsnValThrSerIle
2025
<210>8
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>8
SerAlaIleGlnGlyAsnValThrSerIleHisSerLeuLeuAspGlu
151015
GlyLysGlnSerLeuThrLysLeuAla
2025
<210>9
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>9
GluGlyLysGlnSerLeuThrLysLeuAlaAlaAlaTrpGlyGlySer
151015
GlySerGluAlaTyrGlnGlyValGln
2025
<210>10
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>10
SerGlySerGluAlaTyrGlnGlyValGlnGlnLysTrpAspAlaThr
151015
AlaThrGluLeuAsnAsnAlaLeuGln
2025
<210>11
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>11
ThrAlaThrGluLeuAsnAsnAlaLeuGlnAsnLeuAlaArgThrIle
151015
SerGluAlaGlyGlnAlaMetAlaSer
2025
<210>12
<211>20
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>12
IleSerGluAlaGlyGlnAlaMetAlaSerThrGluGlyAsnValThr
151015
GlyMetPheAla
20
<210>13
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>13
LeuAlaAlaLeuAlaAlaValValGluLeuGlySerPheAspAlaAla
151015
AlaGluArgLeuHisValThrProSer
2025
<210>14
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>14
AlaAlaAlaGluArgLeuHisValThrProSerAlaValSerGlnArg
151015
IleLysSerLeuGluGlnGlnValGly
2025
<210>15
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>15
ArgIleLysSerLeuGluGlnGlnValGlyGlnValLeuValValArg
151015
GluLysProCysArgAlaThrThrAla
2025
<210>16
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>16
ArgGluLysProCysArgAlaThrThrAlaGlyIleProLeuLeuArg
151015
LeuAlaAlaGlnThrAlaLeuLeuGlu
2025
<210>17
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>17
ArgLeuAlaAlaGlnThrAlaLeuLeuGluSerGluAlaLeuAlaGlu
151015
MetGlyGlyAsnAlaSerLeuLysArg
2025
<210>18
<211>25
<212>PRT
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>18
MetGlyGlyAsnAlaSerLeuLysArgThrArgIleThrIleAlaVal
151015
AsnAlaAspSerMetAlaThrTrpPhe
2025
<210>19
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>19
atggcagagatgaagaccgatgccgctaccctcgcgcaggaggcaggtaatttcgagcgg60
atctccggcgacctg75
<210>20
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>20
ggtaatttcgagcggatctccggcgacctgaaaacccagatcgaccaggtggagtcgacg60
gcaggttcgttgcag75
<210>21
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>21
caggtggagtcgacggcaggttcgttgcagggccagtggcgcggcgcggcggggacggcc60
gcccaggccgcggtg75
<210>22
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>22
gcggggacggccgcccaggccgcggtggtgcgcttccaagaagcagccaataagcagaag60
caggaactcgacgag75
<210>23
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>23
gccaataagcagaagcaggaactcgacgagatctcgacgaatattcgtcaggccggcgtc60
caatactcgagggcc75
<210>24
<211>72
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>24
cgtcaggccggcgtccaatactcgagggccgacgaggagcagcagcaggcgctgtcctcg60
caaatgggcttc72
<210>25
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>25
agcatgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccag60
ggaaatgtcacgtcc75
<210>26
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>26
gcaagcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcag60
tccctgaccaagctc75
<210>27
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>27
gacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggag60
gcgtaccagggtgtc75
<210>28
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>28
ggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgag60
ctgaacaacgcgctg75
<210>29
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>29
gccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagcc60
ggtcaggcaatggct75
<210>30
<211>66
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>30
acgatcagcgaagccggtcaggcaatggcttcgaccgaaggcaacgtcactgggatgttc60
gcatag66
<210>31
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>31
ctggccgcattggctgccgtggtcgaactgggcagcttcgatgcggccgcggagcgccta60
catgtcaccccgtcg75
<210>32
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>32
gccgcggagcgcctacatgtcaccccgtcggctgtcagtcagcgcatcaagtcgttggag60
cagcaggtcggccag75
<210>33
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>33
atcaagtcgttggagcagcaggtcggccaggtgctggtggtcagggaaaagccatgtcgg60
gcgacgaccgcaggt75
<210>34
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>34
gaaaagccatgtcgggcgacgaccgcaggtatcccgctgttgcggttggccgcgcaaaca60
gcgttgctcgagtcc75
<210>35
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>35
ttggccgcgcaaacagcgttgctcgagtccgaggcgctcgctgaaatgggtggcaacgcg60
tcgctgaaacgcacg75
<210>36
<211>75
<212>DNA
<213> mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>36
atgggtggcaacgcgtcgctgaaacgcacgcggatcaccattgcggtaaacgccgattcc60
atggcgacatggttt75

Claims (4)

1. a mixed polypeptide, is characterized in that, comprises following peptide sequence:
1）MAEMKTDAATLAQEAGNFERISGDL（SEQIDNO：1）；
2）GNFERISGDLKTQIDQVESTAGSLQ（SEQIDNO：2）；
3）QVESTAGSLQGQWRGAAGTAAQAAV（SEQIDNO：3）；
4）AAGTAAQAAVVRFQEAANKQKQELD（SEQIDNO：4）；
5）ANKQKQELDEISTNIRQAGVQYSR（SEQIDNO：5）；
6）IRQAGVQYSRADEEQQQALSSQMGF（SEQIDNO：6）；
7）MTEQQWNFAGIEAAASAIQGNVTSI（SEQIDNO：7）；
8）SAIQGNVTSIHSLLDEGKQSLTKLA（SEQIDNO：8）；
9）EGKQSLTKLAAAWGGSGSEAYQGVQ（SEQIDNO：9）；
10）SGSEAYQGVQQKWDATATELNNALQ（SEQIDNO：10）；
11）TATELNNALQNLARTISEAGQAMAS（SEQIDNO：11）；
12）ISEAGQAMASTEGNVTGMFA（SEQIDNO：12）；
13）LAALAAVVELGSFDAAAERLHVTPS（SEQIDNO：13）；
14）AAAERLHVTPSAVSQRIKSLEQQVG（SEQIDNO：14）；
15）RIKSLEQQVGQVLVVREKPCRATTA（SEQIDNO：15）；
16）REKPCRATTAGIPLLRLAAQTALLE（SEQIDNO：16）；
17）RLAAQTALLESEALAEMGGNASLKR（SEQIDNO：17）；
18）MGGNASLKRTRITIAVNADSMATWF（SEQIDNO：18）。
2. mixed polypeptide according to claim 1 produces the application of tuberculosis relevant cell factor inductor as inducing peripheral blood mononuclear cell.
3. mixed polypeptide according to claim 1 is as the application of diagnosis antigen.
4. mixed polypeptide according to claim 1 is as the application of tuberculosis prophylaxis treatment stimulator.
CN201511025482.7A 2015-12-29 2015-12-29 Mixed polypeptide for producing tuberculosis associated cell factors by inducing peripheral blood mononuclear cell Pending CN105541975A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201511025482.7A CN105541975A (en) 2015-12-29 2015-12-29 Mixed polypeptide for producing tuberculosis associated cell factors by inducing peripheral blood mononuclear cell

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201511025482.7A CN105541975A (en) 2015-12-29 2015-12-29 Mixed polypeptide for producing tuberculosis associated cell factors by inducing peripheral blood mononuclear cell

Publications (1)

Publication Number Publication Date
CN105541975A true CN105541975A (en) 2016-05-04

Family

ID=55821574

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201511025482.7A Pending CN105541975A (en) 2015-12-29 2015-12-29 Mixed polypeptide for producing tuberculosis associated cell factors by inducing peripheral blood mononuclear cell

Country Status (1)

Country Link
CN (1) CN105541975A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107632155A (en) * 2017-09-06 2018-01-26 上海兰卫医学检验所股份有限公司 A kind of blood cell is to the immunoreactive detection method of tubercle bacillus specific
WO2018076404A1 (en) * 2016-10-24 2018-05-03 广州迪澳医疗科技有限公司 Method for in vitro detection of active tuberculosis
CN110129372A (en) * 2018-09-30 2019-08-16 北京鼎成肽源生物技术有限公司 A kind of construction method of RFFT1 cell

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101661044A (en) * 2008-08-27 2010-03-03 复旦大学附属华山医院 Diagnostic reagent of tuberculosis and kit
CN102004155A (en) * 2010-02-12 2011-04-06 复旦大学附属华山医院 Kit and method for detecting mycobacterium tuberculosis infection and application
CN105131125A (en) * 2015-09-11 2015-12-09 广州迪澳医疗科技有限公司 Fusion protein for inducing peripheral blood mononuclear cells (PBMC) to generate tuberculosis-related cytokines

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101661044A (en) * 2008-08-27 2010-03-03 复旦大学附属华山医院 Diagnostic reagent of tuberculosis and kit
CN102004155A (en) * 2010-02-12 2011-04-06 复旦大学附属华山医院 Kit and method for detecting mycobacterium tuberculosis infection and application
CN105131125A (en) * 2015-09-11 2015-12-09 广州迪澳医疗科技有限公司 Fusion protein for inducing peripheral blood mononuclear cells (PBMC) to generate tuberculosis-related cytokines

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JIAZHEN CHEN ET AL.: "Rv1985c, a promising novel antigen for diagnosis of tuberculosis infection from BCG-vaccinated controls", 《BMC INFECTIOUS DISEASES》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018076404A1 (en) * 2016-10-24 2018-05-03 广州迪澳医疗科技有限公司 Method for in vitro detection of active tuberculosis
CN107632155A (en) * 2017-09-06 2018-01-26 上海兰卫医学检验所股份有限公司 A kind of blood cell is to the immunoreactive detection method of tubercle bacillus specific
CN110129372A (en) * 2018-09-30 2019-08-16 北京鼎成肽源生物技术有限公司 A kind of construction method of RFFT1 cell
CN110129372B (en) * 2018-09-30 2021-06-18 北京鼎成肽源生物技术有限公司 Construction method of RFFT1 cells

Similar Documents

Publication Publication Date Title
CN110981944B (en) African swine fever virus T cell antigen polypeptide and ELISPOT detection method for screening antigen epitope thereof
Tyrrell et al. Some further virus isolations from common colds
CN106546737A (en) A kind of method of vitro detection active tuberculosis
CN102004155B (en) Kit and method for detecting mycobacterium tuberculosis infection and application
CN105541975A (en) Mixed polypeptide for producing tuberculosis associated cell factors by inducing peripheral blood mononuclear cell
CN105131125A (en) Fusion protein for inducing peripheral blood mononuclear cells (PBMC) to generate tuberculosis-related cytokines
CN105601747A (en) Mycobacterium tuberculosis fusion protein and application thereof in induction of peripheral blood mononuclear cells to generate cytokines
CN107216373A (en) Detect antigen polypeptide pond and its application of mycobacterium tuberculosis infection
CN107011418A (en) Detect antigen polypeptide pond and its application of mycobacterium tuberculosis infection
CN113999865B (en) Mycobacterium tuberculosis fusion protein AR2 and construction, expression and purification method and application thereof
RU2607006C1 (en) Test strain leptospira of interrogans serogroup icterohaemorrhagiae serovar copenhageni for detection of antibodies to l icterohaemorrhagiae
CN102279257B (en) Kit used for diagnosing immune associated diseases of human or animals
CN102746381B (en) Helicobacter pylori antigen HLA restrictive immunodominance epitope peptide and preparation method and application thereof
CN106248934A (en) Antigen of mycobacterium tuberculosis albumen Rv0446c and the application of t cell epitope peptide thereof
CN112717128B (en) Combined vaccine for preventing hand-foot-mouth disease and preparation method and application thereof
CN103323585A (en) ELISA kit used for detecting fish streptococcus agalactiae IgM antibody, and preparation method thereof
CN106198971A (en) The application of antigen of mycobacterium tuberculosis albumen Rv2351c
CN106405107A (en) Use of mycobacterium tuberculosis antigen protein Rv2941 and its T cell epitope peptide
CN102924576A (en) Helicobacter pylori immunodominance epitope peptide and preparation method and application thereof
CN102516356B (en) Epitope polypeptide applicable to mycobacterium tuberculosis infection detection and application thereof
CN104127444A (en) Method and device for establishment of Mycobacterium tuberculosis naturally infected non-human primate model
CN106480003A (en) Helicobacter pylori dominant antigen combination based on CD4+T cellular immunization and screening technique
CN107267430A (en) Brucella 104M vaccine strains knock out recombinant bacterium and the application of Omp25 genes
CN116615234A (en) Combined vaccine for preventing hand-foot-mouth disease and preparation method and application thereof
RU2341288C1 (en) MEDICINE FORMING CELLULAR IMMUNITY FOR MYCOBACTERIUM TUBERCULOSIS H37 Rv, METHOD OF PRODUCTION THEREOF (VERSIONS), RECOMBINANT STRAIN AND TUBERCULOSIS DIAGNOSTIC AGENT

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20160504