WO2016201825A1 - Mycobacterium tuberculosis antigens and applications thereof - Google Patents

Mycobacterium tuberculosis antigens and applications thereof Download PDF

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WO2016201825A1
WO2016201825A1 PCT/CN2015/091068 CN2015091068W WO2016201825A1 WO 2016201825 A1 WO2016201825 A1 WO 2016201825A1 CN 2015091068 W CN2015091068 W CN 2015091068W WO 2016201825 A1 WO2016201825 A1 WO 2016201825A1
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tuberculosis
nucleic acid
seq
immunogenic
rv0792c
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PCT/CN2015/091068
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French (fr)
Chinese (zh)
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刘军
张鹭
郭沛
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复旦大学
成都永安制药有限公司
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Priority to CN201580079653.6A priority Critical patent/CN107531764A/en
Publication of WO2016201825A1 publication Critical patent/WO2016201825A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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

Definitions

  • the invention belongs to the field of novel tuberculosis vaccines.
  • it relates to corresponding immunogenic components, vaccines or therapeutic ingredients for the prevention or treatment of pathogenic mycobacteria such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium leprae, Infection caused by Mycobacterium ulcerans.
  • the immunogenic component, vaccine or therapeutic component consists of the M. tuberculosis antigen Rv0976c, Rv1255c, Rv3160c and/or Rv0792c, or a corresponding encoding nucleic acid thereof.
  • Tuberculosis has been the most common infectious disease caused by a single cause worldwide. It is estimated that one third of the world’s population is infected with M. tuberculosis, and in 2013 alone 1.5 million people died of tuberculosis. On a global scale, effective control of tuberculosis still faces many difficulties and challenges, including the lack of rapid and accurate diagnostic techniques, the lack of effective anti-tuberculosis vaccines, and treatments for months. Co-infection of tuberculosis and HIV, as well as the spread of multiple drug resistance (MDR-TB) and extensive drug-resistant TB (XDR-TB), further complicate the prevention and control of tuberculosis.
  • MDR-TB multiple drug resistance
  • XDR-TB extensive drug-resistant TB
  • BCG Bacille Calmette-Guérin
  • M. tuberculosis vaccine is the only attenuated M. tuberculosis vaccine and is the only approved anti-tuberculosis vaccine to date.
  • BCG has two major drawbacks: one is that the protective effect on adult tuberculosis is very limited; the other is that it may cause disseminated BCG disease in people with low immunity.
  • Clinical studies have shown that BCG has more than 80% protective effect on severe tuberculosis in children including miliary tuberculosis and tuberculous meningitis. However, the protective effect on adult tuberculosis is limited, and the protective effect of clinical research is uneven (0-80%). ).
  • BCG BCG is not an ideal vaccine and has limited protection time for the human body.
  • the immunoprotective effect of BCG on newborn priming can last up to 10-20 years, so there is almost no protective effect on adult tuberculosis.
  • tuberculosis vaccines are used as an adjuvant for chemotherapy.
  • This “priming-boosting” strategy includes: after initial immunization with BCG or recombinant BCG, boosting vaccination with infants and children who have not been exposed to tuberculosis, supplemented with subunit vaccines (protein/peptide or DNA); or The unit vaccine is boosted alone; or the subunit vaccine is used as an adjuvant for chemotherapy.
  • the primary development strategy for subunit vaccines has been to find protein antigens that induce strong IFN- ⁇ release to construct subunit vaccines (protein or DNA).
  • the method used is to screen for protein antigens that induce IFN- ⁇ release by biochemical fractionation of a mixture of tuberculosis proteins, especially bacterial supernatants.
  • Esx family proteins EsxA, B, G, H, G, N
  • antigen 85 complex Ag85A, B, C
  • PE/PPE family proteins such as: PPE18, PPE14
  • H1 Ag85B-ESAT-6
  • H4 Ag85B-TB10.4
  • M72 PPE18-Rv0125
  • DNA subunit vaccines such as MVA85A (adenovirus expressing antigen 85A, Ag85A) and AERAS-402 (vaccinia virus expression) have been developed using replication-deficient viruses such as adenovirus or vaccinia virus as vectors.
  • MVA85A enhances the protective efficiency of BCG (Verreck FA, et al. (2009) MVA.85A boosting of BCG and an attenuated, phoP deficient M. tuberculosis vaccine both show protective efficacy against tuberculosis in rhesus macaques.
  • PLoS ONE 4(4): e5264 AERS-402 which has entered clinical phase IIa trials, also potentiates T cell responses after BCG priming (Magalhaes I, et al. (2008) rBCG induces strong antigen-specific T cell responses in rhesus Macaques in a prime-boost setting with an adenovirus 35 tuberculosis vaccine vector.
  • MVA85A has now completed the clinical Phase IIb trial, which is the first subunit vaccine to complete the clinical validation of the protective effect, but the results are disappointing.
  • MVA85A does not significantly improve BCG protection, anti-tuberculosis, or anti-tuberculosis infection (Tameris MD, et al. (2013) Safety and efficacy of MVA85A, a new tuberculosis vaccine , in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet 381 (9871): 1021-1028).
  • the results of MVA85A suggest that it is necessary to continue to search for new and more effective subunit vaccine candidate antigens (Geluk A, van Meijgaarden KE, Joosten SA, Commandeur S, & Ottenhoff TH (2014) Innovative Strategies to Identify M.
  • the present invention relates to an immunogenic composition, vaccine or therapeutic composition consisting of M. tuberculosis antigens Rv0976c, Rv1255c, Rv3160c and/or Rv0792c, or a coding nucleic acid corresponding thereto.
  • the invention also encompasses short/long overlapping or non-overlapping peptides produced by synthetic or recombinant methods.
  • the present invention relates to the use of the Rv0976c, Rv1255c, Rv3160c and/or Rv0792c proteins, or the coding genes corresponding to these proteins, for the diagnosis, treatment and/or prevention of M. tuberculosis infection.
  • Rv0976c, Rv1255c, Rv3610c and Rv0792c are highly immunogenic in animal models and have a protective effect against Mycobacterium tuberculosis infection. Therefore, the antigens Rv0976c, Rv1255c, Rv3610c and Rv0792c are very promising candidate antigens for the construction of anti-tuberculosis vaccines.
  • the present invention discloses an immunogenic composition, a vaccine or therapeutic composition comprising one or more of the following polypeptides:
  • the invention also discloses an immunogenic composition, a vaccine or therapeutic composition consisting of any one or more of the following nucleic acid molecules:
  • the above nucleic acid is a DNA fragment.
  • immunogenic compositions, vaccines or therapeutic compositions of the invention may have the following applications:
  • the polypeptide vaccine of the present invention is applied to humans or other mammals or animals to enhance the body against pathogenic mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium. Bovis), Mycobacterium africanum, Mycobacterium leprae or ulcer Resistance to tuberculosis infection caused by Mycobacterium ulcerans.
  • the immunogenicity of a polypeptide or immunogenic fragment may be enhanced by fusion with an adjuvant, or may be enhanced by the addition of other mycobacterial polypeptides, or other organisms such as bacteria, viruses, mammalian polypeptides.
  • the added polypeptide is also included in the composition composition and is bound to the polypeptide or immunogenic fragment in a cross-linked or non-cross-linked form.
  • the nucleic acid of the present invention is randomly inserted into a vector, such as an adenovirus or vaccinia virus vector, directly used in human or other mammals or animals as a DNA vaccine, and expresses antigen in vivo, resulting in pathogenic branching of the body.
  • Bacteria such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium leprae, or Mycobacterium ulcerans Resistance to tuberculosis infection.
  • the polypeptides and nucleic acids of the invention may constitute a therapeutic composition for use in humans or other mammals or animals to prevent and/or treat M. tuberculosis infection.
  • the present invention provides a vaccine for human or other mammalian or animal immunization against pathogenic mycobacteria such as Mycobacterium tuberculosis, Mycobacterium Bovis infection, tuberculosis caused by Mycobacterium africanum, Mycobacterium leprae or Mycobacterium ulcerans.
  • pathogenic mycobacteria such as Mycobacterium tuberculosis, Mycobacterium Bovis infection, tuberculosis caused by Mycobacterium africanum, Mycobacterium leprae or Mycobacterium ulcerans.
  • a microorganism such as a free plasmid or into a microbial genome
  • compositions, polypeptide, and nucleic acid of the present invention are used in an in vitro and in vivo assay for detecting an antibody response or a cellular immune response against Mycobacterium tuberculosis, and are suitable for diagnosis of infection or monitoring of disease progression.
  • a polypeptide may be used as an in vivo diagnostic reagent during a skin test.
  • Polypeptides can also be used for in vitro testing in ELISA or T-spot assays of blood samples from tuberculosis patients.
  • the nucleic acid or polypeptide may be used in non-human animals to extract antibodies against M. tuberculosis, which antibodies can be used to detect target antigens by in vivo or in vitro experiments.
  • the invention provides an immunogenic component comprising one or more polypeptides or immunological fragments thereof selected from the group consisting of an immunogenic component (ie, the invention provides one selected from the group consisting of Application of one or more polypeptides or immunological fragments thereof as an immunogenic component):
  • the immunological fragment is, for example, a T cell epitope.
  • the invention provides an immunogenic component comprising a nucleic acid molecule encoding the above polypeptide or an immunological fragment thereof.
  • the immunogenic component of the present invention comprises one or more nucleic acid molecules selected from the group consisting of the following as an immunogenic component (ie, the present invention provides one or more nucleic acid molecules selected from the group consisting of: Application of the original ingredients):
  • nucleic acid molecule consisting of the nucleotide sequence shown in SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8 or a complement thereof;
  • nucleic acid molecule consisting of a nucleotide sequence encoding the same amino acid sequence as the nucleic acid molecule of (a) or a complement thereof;
  • nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule as defined in (a) or (b) and which has a length of at least 10 nucleotides.
  • the present invention provides an immunogenic composition comprising the aforementioned one or more polypeptides or immunological fragments thereof, and/or one or more of the aforementioned nucleic acid molecules as an immunogenic component . That is, the present invention also provides the use of the immunogenic component described in the preparation of an immunogenic composition.
  • the immunogenic composition of the invention may further comprise an adjuvant.
  • the immunogenicity of a polypeptide or immunogenic fragment may be enhanced by fusion with an adjuvant, or by the addition of other mycobacterial polypeptides, or other organisms such as bacteria, viruses, mammalian polypeptides. strengthen.
  • the added polypeptide may also be included in the compositions of the invention to bind to the polypeptide or immunogenic fragment in a cross-linked or non-cross-linked form.
  • the present invention provides an expression vector or a non-pathogenic microorganism in which at least one copy of a DNA fragment comprising the aforementioned nucleic acid molecule of the present invention is integrated (for example, placed in a free plasmid or integrated into a microbial genome) And the DNA fragment can be expressed in the form of a polypeptide in a microorganism.
  • the expression vector or non-pathogenic microorganism is vaccinia, adenovirus, BCG or transformed cells.
  • the expression vector or non-pathogenic microorganism is used to express antibodies against mycobacteria in humans or other animals.
  • the present invention provides the immunological component, the immunogenic composition, or the expression vector or the non-pathogenic microorganism in the preparation of a medicament for anti-tuberculous mycobacterial infection.
  • the anti-tuberculous mycobacterial infection refers to enhancing the body caused by pathogenic mycobacteria, such as: Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium leprae or Mycobacterium ulcerans Resistance to tuberculosis infection.
  • the present invention also provides a medicament for infection against Mycobacterium tuberculosis, comprising the immune component of the present invention, the immunogenic composition, or the expression vector or non-pathogenic microorganism;
  • the medicament may be a vaccine.
  • the drug against M. tuberculosis infection can be applied to humans or other animals, preferably mammals, to prevent and/or treat M. tuberculosis infection.
  • the invention provides a method of immunizing against tuberculosis in a human or other animal, preferably a mammal, comprising administering to the individual (by means of intradermal, subcutaneous transdermal, muscle, or mucosal delivery)
  • the immune component, the immunogenic composition, or the expression vector or non-pathogenic microorganism of the invention are administered to the individual (by means of intradermal, subcutaneous transdermal, muscle, or mucosal delivery.
  • the invention also provides the use of the immunological component or the immunogenic composition described in the preparation of a formulation for detecting and/or diagnosing a Mycobacterium tuberculosis infection.
  • Detection of M. tuberculosis infection includes detection of an antibody response or cellular immune response against M. tuberculosis. It can be used for in vitro and/or in vivo detection for the detection of antibodies or cell-mediated immune responses against M.tb in body fluids for disease diagnosis, infection diagnosis, or monitoring disease progression.
  • Nucleic acids and polypeptides can be used to produce anti-M.tb antibodies in non-human animals.
  • the antibody can be used to detect a target antigen in vivo and/or in vitro.
  • the immunogenic components (polypeptides and nucleic acids) of the invention can be used in diagnostic methods to screen for antibodies reactive with the antigen associated with different populations (eg, active, latent infection, healthy humans). serum.
  • the experiments of the present invention show that the levels of antibodies specifically recognizing Rv0976c, Rv1255c, and Rv3160c antigens are significantly higher than those of active TB and latent infected people, and the levels of antibodies specifically binding to Rv0792c in the serum of latent infected populations are significant. Higher than active tuberculosis and healthy controls.
  • the immunogenic components of the invention can also be used in a variety of in vitro cytokine release assays to determine the level of secretion of cytokines induced by the polypeptides of the invention, including IFN-[gamma], TNF and IL-2. At least one of them.
  • the invention provides a diagnostic kit for a tuberculosis sample comprising an immunological component of the invention, or an immunogenic composition of the invention.
  • the Rv0976c, Rv1255c, Rv3160c and Rv0792c of the present invention can be used for differential diagnosis between active tuberculosis, latent tuberculosis infection and healthy individuals.
  • the levels of antibodies specifically recognizing Rv0976c, Rv1255c, and Rv3160c in the serum of active tuberculosis and latent infection were significantly higher than those in healthy people.
  • the level of antibody specifically binding to Rv0792c in the serum of latent infected population was significantly higher than that of active tuberculosis and healthy control group. .
  • the invention also provides the use of a protein (including polypeptide), nucleic acid or composition of the invention in the treatment or prevention of tuberculosis.
  • the invention also provides the use of the following proteins or nucleic acids in the preparation of a tuberculosis vaccine or in the preparation of a medicament for the diagnosis and/or detection of tuberculosis:
  • amino acid sequences of the above proteins Rv0976c, Rv1255c, Rv3160c, and Rv0792c are shown in SEQ ID Nos. 1 to 4, respectively.
  • nucleic acid sequences encoding the proteins Rv0976c, Rv1255c, Rv3160c, Rv0792c are shown in SEQ ID Nos. 5-8, respectively.
  • the invention provides a method of treating or preventing tuberculosis comprising administering a protein, nucleic acid or vaccine of the invention to a human or other animal.
  • the administration is by intradermal, subcutaneous transdermal, muscle or mucosal delivery.
  • the protein or polypeptide fragment thereof provided by the invention and the nucleic acid encoding the same have high immunogenicity against Mycobacterium tuberculosis, can provide effective protection to the organism, can be used as a vaccine candidate or prepared for diagnosis and/or detection of tuberculosis. Reagents.
  • FIG. 1 Expression and purification of Rv0976c protein; wherein each lane represents: 1, molecular weight marker; 2. whole cells induced by IPTG; 3. flow through Ni 2+ affinity chromatography column; 4, 50 mM imidazole washing buffer 5, 100 mM imidazole washing buffer; 6, 150 mM imidazole elution buffer; 7, 300 mM imidazole elution buffer; 8, 300 mM imidazole elution buffer; 9, 500 mM imidazole elution buffer. Fragments eluted with 300 mM imidazole elution buffer (lanes 7 and 8) were collected as purified Rv0976c protein.
  • FIG. 1 Protein-induced T cell response of the invention: production of Th1 cytokines (TNF, IL-2, IL-12) as determined by ELISA. The data was homogenized to Ag85A, and Ag85A was included in parallel in each experiment.
  • Th1 cytokines TNF, IL-2, IL-12
  • FIG. 1 Protein-induced B cell response of the invention; wherein A is Rv0792c, B is Rv1255c, C is Rv0976c, and D is Rv3160c.
  • FIG. 1 Experimental results of protective efficacy of Rv0976c; BALB/c mice were immunized with DNA vaccine and infected with M. tuberculosis H37Rv. Five weeks after the infection, the mice were sacrificed and the number of M. tuberculosis in the lungs and spleen was counted. A is the result of counting the amount of Mycobacterium tuberculosis in the mouse lung, and B is the result of counting the amount of Mycobacterium tuberculosis in the mouse spleen. Among them, pVAX is a M.
  • pVAX: Ag85A is a M. tuberculosis count result of a mouse inoculated with plasmid DNA expressing Ag85A
  • pVAX: PPE18 is a result of counting M. tuberculosis in mice inoculated with plasmid DNA expressing PPE18
  • pVAX: Rv0976c is a result of counting M. tuberculosis in mice inoculated with plasmid DNA expressing Rv0976c.
  • FIG. 7 Experimental results of protective efficacy of Rv1255c, Rv3160c and Rv0792c; BALB/c mice were immunized with DNA vaccine and infected with M. tuberculosis H37Rv. After 9 weeks of infection, the mice were sacrificed and the number of M. tuberculosis in the lungs and spleen was counted. A is the spleen count result, and B is the lung count result. Among them, sham and pcDNA are M. tuberculosis count results of mice inoculated with PBS or empty plasmid vector (negative control), and pcDNA: Ag85A is the result of M.
  • pcDNA :HspX is the result of M. tuberculosis in mice inoculated with plasmid DNA expressing HspX
  • pcDNA: Rv1255c is the result of M. tuberculosis inoculation of a plasmid DNA expressing Rv1255c
  • pcDNA: Rv3160c is a plasmid inoculated with Rv3160c.
  • pcDNA: Rv0792c is a result of counting M. tuberculosis in mice inoculated with plasmid DNA expressing Rv0792c.
  • FIG. 8 Serological test results for active tuberculosis (TB), latent tuberculosis (LTBI), and healthy controls (HC) with selected proteins; A is Rv0792c, B is Rv0976c, C is Rv1255c, and D is Rv3160c .
  • the International Tuberculosis Vaccine Research field has conducted animal protection experiments on more than 200 antigens over the past 20 years and found that only a few antigens have potential as subunit anti-tuberculosis vaccines, including the well-known antigens Ag85A, PPE18, HspX, Esat- 6. CFP-10, etc. These antigens have been patented by relevant researchers and constructed corresponding subunit vaccines for clinical trials, such as MVA85A above.
  • the inventors of the present invention conducted an animal experiment on about 130 tuberculosis proteins that have not been used to construct a subunit anti-tuberculosis vaccine, and found that the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins of the M. tuberculosis H37Rv strain are highly immunogenic. It can produce a significant protective effect against Mycobacterium tuberculosis infection.
  • Rv0976c is a conserved putative protein with unknown biological functions in Mycobacterium tuberculosis, including Mycobacterium tuberculosis CDC1551, Mycobacterium bovis (including BCG), Mycobacterium tuberculosis, and tuberculosis Mycobacteria and Mycobacterium marinum have homologous genes among a plurality of mycobacterial species.
  • Rv1255c, Rv3160c and Rv0792c are both transcriptional regulatory proteins and are highly conserved within Mycobacterium. The amino acid sequences of Rv0976c, Rv1255c, Rv3160c and Rv0792c are shown in SEQ ID No. 1 to SEQ ID No.
  • the present invention cloned the Rv0976c, Rv1255c, Rv3160c and Rv0792c open reading frames into the pET28a plasmid, and expressed and purified using the E. coli BL21 strain using a standardized procedure (Fig. 1 and Fig. 2). 10 ⁇ g of the purified protein was mixed with Freund's incomplete adjuvant, and C57BL/6 mice were immunized every other week for 3 times to evaluate the immunogenicity of these antigens. After 8 weeks of the first immunization, the mice were sacrificed to isolate spleen cells.
  • the spleen cells were stimulated by Rv3160c and Rv0792c antigens at a concentration of 5 ⁇ g/ml and 10 ⁇ g/ml, respectively, and PBS was used as the antigen-free negative control.
  • the purified Ag85A antigen was used as a positive control, and the spleen cells were cultured under stimulation.
  • the cell culture supernatant was collected 3 days later, and the release levels of Th1 type cytokines (IFN- ⁇ , TNF- ⁇ , and IL-2) were measured by ELISA.
  • the present invention cloned the DNA sequence encoding these antigens (Table 2) into the mammalian expression vector pVAX1 (Rv0976c) or pcDNA3.1 containing the CMV promoter. (Rv1255c, Rv3160c and Rv0792c) (Fig. 5).
  • the pVAX1 and pcDNA3.1 vectors were purchased from Invitrogen.
  • the coding sequences of Ag85A were also cloned into these two vectors as experimental controls. Mice were immunized with these recombinant DNAs, followed by M.
  • tuberculosis H37Rv infection challenge experiments The specific experiment was as follows: BALB/c mice were immunized once a week with 100 ⁇ g of DNA, and immunized 3 times. After 8 weeks of the first immunization, mice were infected with M. tuberculosis (6 ⁇ 10 5 CFU/mouse) via the tail vein or aerosol (dose: 100-300 CFU/lung/mouse), and sacrificed after 5 or 9 weeks of challenge. The mice were counted for the number of M. tuberculosis in the lungs and spleen. The constructed Ag85A DNA was run in parallel as a control. In the same batch of experiments, the number of M.
  • tuberculosis in the lung and spleen of mice inoculated with Rv0976c DNA vaccine after 5 weeks of infection with M. tuberculosis was significantly lower than that of the negative control (pVAX) or the mice inoculated with Ag85A DNA vaccine.
  • the number of Mycobacterium species was similar to that of M. tuberculosis in mice inoculated with the PPE18 DNA vaccine (Fig. 6).
  • PPE18 is also a positive control, and fusion protein vaccine M72 (PPE18-Rv0125) with it as an important component has entered clinical trials (Andersen P& Kaufmann SH (2014) Novel vaccination strategies against tuberculosis. Cold Spring Harb Perspect Med 4 (6)).
  • HspX is a latent-associated antigen of M. tuberculosis and was also used to construct a multi-level subunit vaccine, which was used as a positive control in this experiment (Mir FA, Kaufmann SH, & Eddine AN (2009) A multicistronic DNA vaccine induces significant protection against Tuberculosis in mice and offers flexibility in the expressed antigen repertoire.
  • Rv0976c, Rv1255c, Rv3160c and Rv0792c can react with specific antibodies in human serum, and then distinguish between healthy people and different disease populations, the present invention will purify Rv0976c, Rv1255c, Rv3160c and Rv0792c.
  • the protein was subjected to an ELISA reaction with serum from different populations (active tuberculosis group, tuberculosis latent infection group, healthy control group). The results showed that Rv0976c, Rv1255c, Rv3160c and Rv0792c differed from those of different disease patterns in the serum.
  • serum derived from active tuberculosis and latent infections specifically recognized Rv0976c, Rv1255c, and Rv3160c antigens.
  • Antibody levels were significantly higher than those in healthy populations; the level of antibodies specifically binding to Rv0792c in the serum of latently infected populations was significantly higher than in active tuberculosis and healthy controls (Figure 8).
  • the present invention relates to the use of the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins as vaccines.
  • the protein, polypeptide or peptide of the present invention includes a native protein, polypeptide or peptide, or a homologue functionally identical to a native protein, polypeptide or peptide. These homologues include at least 60%, 70%, 80% or more (more preferably), and most preferably 90% or more (e.g., 95%, 96%, 97%, 98%) of the native protein, polypeptide or peptide contained in Table 1. Or 99%) a protein, polypeptide or peptide of amino acid sequence homology.
  • homologues include substitution, addition and deletion of one or more (eg, 1-50, 1-20, 1-10, 1-5) amino acid residues based on the amino acid sequence of the native protein, polypeptide or peptide in Table 1. a protein, polypeptide or peptide.
  • Such homologs include, inter alia, proteins, polypeptides or peptides containing conservative amino acid substitutions.
  • Nucleic acid sequence in the present invention means a nucleotide sequence encoding such a polypeptide. Further within the framework of the invention are overlapping or non-overlapping peptides which are either long or short and which have at least 70% amino acid sequence identity to any of the polypeptides of the invention.
  • the present invention provides the use of the above polypeptide or nucleic acid as a preparation of an immunogenic component (composition), vaccine or therapeutic composition, which can be combined with BCG as a prophylactic vaccine as an antipathogenic branching Bacteria, such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium leprae, Mycobacterium ulcerans. Enhanced vaccine or therapeutic vaccine.
  • the immunogenic component, vaccine or therapeutic composition of the present invention can be used as a prophylactic vaccine for a population not infected with pathogenic mycobacteria or a population inoculated with BCG, and can also be used as a therapeutic vaccine. A population infected with pathogenic mycobacteria.
  • the invention provides a class of susceptible expression vectors, such as vaccinia, adenovirus or BCG comprising a nucleic acid fragment of the invention, and a class of transformed cells comprising at least one such vector.
  • susceptible expression vectors such as vaccinia, adenovirus or BCG comprising a nucleic acid fragment of the invention
  • the present invention also relates to a method for diagnosing tuberculosis caused by pathogenic mycobacteria in an animal body including a human.
  • Intradermal injection of the polypeptide of the present invention produces a detectable positive skin reaction at the injection site of a tuberculosis individual, and no skin reaction is detected at the injection site of the individual without tuberculosis.
  • a monocyte-containing blood sample eg, T lymphocytes
  • the positive reaction may be T cell proliferation or extracellular. Release such as IFN- ⁇ -like cytokines.
  • a type of serum sample is mixed with a polypeptide sample of the invention, which may be in an individual who has been or is being infected. It was observed that the antibody in the serum sample binds to the polypeptide of the present invention.
  • a monoclonal or polyclonal antibody which specifically reacts with a polypeptide of the present invention in an immunoassay, or a specific binding fragment of the above antibody, is also within the scope of the present invention.
  • a sample of a bodily fluid or a potentially infected organ from an animal including a human is mixed with the above antibody, and the binding of the sample and the antibody can be detected in the infected individual.
  • Nucleic acid probes encoding the polypeptides of the invention can also be used in a variety of diagnostic assays to detect the presence of pathogens in a particular sample.
  • Such a tuberculosis diagnostic method can comprise at least a portion of a nucleic acid sequence, using a PCR or mature hybridization technique to hybridize an animal sample, including a human, to a nucleic acid fragment (or full length fragment) to detect the presence of a nucleic acid sequence in the sample.
  • nucleotide molecules in the DNA sequences provided herein may be modified.
  • the invention includes nucleotide modifications of sequences (or fragments thereof) that can be expressed directly in bacterial or mammalian cells. Modifications include substitutions, insertions or deletions of nucleotides, changes in the relative position of nucleotides or sequential changes.
  • Nucleic acid molecules of the invention include sequences encoding substitutions, additions or deletions that result in non-conservative amino acids in the sequence of Table 1.
  • Nucleic acid molecules (DNA and RNA) comprising corresponding functions that alter the non-conservative amino acid sequence in the amino acids of Table 1, which encode amino acid sequences with non-conservative amino acid substitutions (especially substitutions, additions, or deletions of chemical approximations, but also The amino acid sequence identical or similar to Table 1 is retained. These DNA or RNA may encode fragments or variants of the amino acid sequence shown in Table 1.
  • Fragments can be used as immunogens and immunogenic compositions. Such fragments or variants can be identified by the methods described below.
  • the nucleic acid molecule (or a fragment thereof) referred to in the present invention has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least at least the nucleic acid molecule sequence set forth in the application.
  • a consistency of 97%, at least 98%, or, preferably, 99% or 99.5% or greater, can be expressed in bacterial or mammalian cell lines.
  • Consistency refers to the similarity of two nucleotide sequences, the highest value that can be matched after sequence alignment. Consistency is calculated according to existing mature methods. For example, if a stretch of nucleotide sequence (referred to as sequence A) is 90% identical to the reference fragment of SEQ ID No. 5, then in addition to the reference nucleotide sequence of every 100 SEQ ID No. 5, the sequence Except for 10 point mutations (eg, substitutions with other nucleotides), the remaining sequences are identical.
  • Sequence identity is preferentially set such that the sequence has at least 70%, at least 80%, at least 90%, of SEQ ID No. 5 or its complement provided in the invention, At least 95%, at least 96%, at least 97%, at least 98%, or, preferably, 99% or 99.5% identity.
  • Sequence consistency first uses the GCG program in bioinformatics Perform calculations (Wisconsin University). Other programs can also be used for consistent calculations, such as the Clustal W program (better for default parameters; Thompson, JD et al., Nucleic Acid Res.
  • BLAST P Mycobacterium tuberculosis BLASTN (http:tigrblast.tigr.org/) from the Genomics Institute, Mycobacterium bovis, at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/Projects/Microbes/), Tuberculosis of Mycobacterium bovis BCG (Pastuer), Mycobacterium marinum, Mycobacterium leprae, Mycobacterium tuberculosis BLASTN, Pasterculist (http://genolist.pasteur.fr/TubercuList/) BLASTN study by the Pasteur BLASTN study, Leproma (http://genolist.pasteur.fr/Leproma/), University of Minnesota (http://www.cbc.umn.edu/ResearchProjects/Ptb/ And http://www.cbc.umn.edu/ResearchProjects/AGAC/M
  • tuberculosis BLASTN NCBI USA-(http://www.ncbi.nlm. Various BLAST studies by nih.gov/BLAST/), and GenomeNet (Bioinformatics Center - Institute of Chemistry) (http://blast.genome.ad.jp/) Research various BLAST.
  • nucleic acid sequences given in Table 2 are not only the only sequences encoding the polypeptides in Table 1.
  • the invention encompasses nucleic acid molecules having the same basic genetic information as the nucleic acid molecules of Table 2.
  • Nucleic acid molecules which produce one or more nucleotide changes (including RNA) as compared to the nucleic acid sequences in the application, but which produce the same polypeptide product as in Table 1, are also within the scope of the present invention.
  • nucleotide functional equivalents encoding the above polypeptides which can be detected by conventional DNA-DNA or DNA-RNA hybridization techniques are also within the scope of the present invention.
  • the DNA of the present invention has sufficient sequence identity with the nucleic acid molecule provided in the application, and hybridization can be achieved under strict hybridization conditions (low salt washing solution: about 0.2% SSC, reaction temperature of 50-65 ° C). Common hybridization techniques).
  • the invention also encompasses nucleic acid molecules that are capable of hybridizing to one or more of the sequences in Table 2 or its complements. This nucleic acid molecule achieves hybridization under highly stringent conditions (Sambrook et al. Molecular Cloning: A Laboratory Manual, Most Recent Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). It is preferred to use a low salt wash (about 0.2% SSC) at a reaction temperature of about 50-65 °C.
  • the preparation of vaccines is well known. Typically, injectable liquid solutions or suspensions; solid forms which are readily dissolved or suspended in the liquid prior to injection, may be prepared by emulsifying, proteolipidization.
  • the immunogenic composition is often mixed with a pharmaceutically acceptable excipient that is compatible with the active composition. Suitable excipients include: water, physiological saline, dextrose, glycerol, ethanol or mixtures thereof.
  • the vaccine may contain other minor excipients, such as infiltrants or emulsifiers, pH buffers, and/or adjuvants that enhance the effectiveness of the vaccine, if desired.
  • the active ingredients of the adjuvant may include, but are not limited to, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine ( CGP 19835A, known as MTP-PE) and RIBI.
  • thr-MDP N-acetyl-muramyl-L-threonyl-D-isoglutamine
  • CGP 11637 known as nor-MDP
  • CGP 19835A known as MTP-PE
  • RIBI RIBI
  • RIBI is extracted three kinds of bacterial components: monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in a mixture of 2% squalene / Tween 80 TM manufactured by emulsion An adjuvant.
  • the efficiency of the adjuvant is assessed by the level of antibody directly induced by the immunogenic polypeptide containing the immunogenic sequence of M. tuberculosis, immunized with a mixture of different adjuvants.
  • the vaccine is vaccinated either subcutaneously or intramuscularly, as well as suppositories or oral formulations.
  • conventional binders or carriers include, for example, polyethylene glycol, triglycerides, and such suppositories are mixtures containing from 0.5% to 10% (1% to 2% by weight) of the active ingredient.
  • Oral formulations include excipients that are normally used, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These ingredients are in the form of solutions, suspensions, tablets, pills, capsules, sustained release agents or powders with from 10% to 95% (25% to 70% by weight) of the active ingredient.
  • Appropriate vaccine immunization doses are administered depending on the purpose of the prophylactic and/or therapeutic vaccine.
  • the vaccine can be administered in a single dose regimen, or preferably in a multiple dose regimen.
  • a multi-dose regimen is one in which the initial vaccination procedure can be used in 1-10 separate doses, followed by administration of other doses at subsequent intervals required to maintain and or boost the immune response, for example, at 1-4 months. Two doses, if needed, are given a subsequent dose a few months later.
  • the dosage regimen is at least partially determined by the individual and depends on the judgment of the practitioner.
  • the vaccine may be used in conjunction with other immunoglobulin-like immunomodulators, and a sub-part of the invention is also a multivalent vaccine formulation consisting of a mixture of the vaccine and other vaccines, particularly BCG or recombinant BCG.
  • the therapeutic component (pharmaceutical composition) of the present invention is used for the treatment or prevention of a mammal against Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium leprae or Mycobacterium ulcerans, and is also used for Treatment of degenerative diseases, abnormal physiological conditions, such as malignant tumors.
  • compositions can be administered to humans or other animals in the form of tablets, sprays, tracheal perfusions or intravenous injections.
  • FIG. 15 The open reading frame of Rv0976c by using the upstream PCR primer 5'-TAGGATCCGTGCGTATCGGAAACTGCTCG-3' (SEQ ID No. 15) and the downstream PCR primer 5'-TAGAAGCTTTCACAAC AGGGTCTCCGG GATCT-3' (SEQ ID No. 16) was amplified.
  • the PCR amplification primers for Rv1255c, Rv3160c and Rv0792c are: 5'-CATGGATCCATGGCGGGTACCGACTGGCTG-3' (upstream, Rv1255c, SEQ ID No. 17), 5'-CTGAAGCTTTCACTCGGGTCCAGGGTGAC-3' (downstream, Rv1255c, SEQ ID No. 18) ; 5'-CGTGGATCCATGCCGAGGCAGGCCGGCCGCTG-3' (upstream, Rv3160c, SEQ ID No. 19), 5'-GCAAAGCTTCTAGAGCCCGCGGTCGGGGGGTGCG-3' (downstream, Rv3160c, SEQ ID No.
  • PCR reaction using M. tuberculosis H37Rv (ATCC93009) genomic DNA as a template (50 ⁇ l system) containing template DNA (10 ng), upstream and downstream primers 0.5 ⁇ M each, 0.2 mM dNTPs, 1 ⁇ reaction buffer, 1.25 units of PrimeSTAR HS DNA polymerase (Clontech). Cycling conditions: denaturation at 95 ° C for 5 min; denaturation at 30 cycles (98 ° C, 10 sec), annealing (65 ° C, 20 sec), extension (72 ° C, 2 min); last 72 ° C extension for 5 min, 4 ° C cooling.
  • the PCR amplification product was subjected to agarose gel electrophoresis and purified using a gel purification kit (Qiagen).
  • the product was purified by digestion with BamHI and HindIII at 37 ° C for 3 h, and the digested target fragment was purified using a gel purification kit (Qiagen).
  • the pET28a plasmid (Novagen) was recovered by restriction enzyme digestion under the same conditions.
  • the ligation reaction system total 10 ⁇ l included: 2 ⁇ l of PCR fragment, 2 ⁇ l of pET28a fragment, 1 ⁇ l of 10 ⁇ T4 ligase buffer, and 1 ⁇ l of DNA T4 ligase (NEB).
  • the reaction was stopped by ligation at room temperature for 3 h and incubation at 65 ° C for 20 min.
  • the plasmid-gene fragment ligation products pET28a-Rv0976c, pET28a-Rv1255c, pET28a-Rv3160c and pET28a-Rv0792c were transformed into E. coli DH5 ⁇ , respectively.
  • the ligation reaction mixture was mixed with E. coli DH5 ⁇ competent cells, plated on LB plates containing kanamycin (50 ⁇ g/ml), cultured at 37 ° C overnight, and randomly selected for inoculation into LB liquid medium.
  • Recombinant plasmids pET28a-Rv0976c, pET28a-Rv1255c, pET28a-Rv3160c and pET28a-Rv0792c were extracted from E. coli DH5 ⁇ cells using Qiagen Miniprep kit, and the insertion sequence was verified by DNA sequencing.
  • the recombinant plasmids pET28a-Rv0976c, pET28a-Rv1255c, pET28a-Rv3160c and pET28a-Rv0792c were separately transformed into E. coli BL21 and plated on LB plates containing kanamycin (50 ⁇ g/ml), 37 Incubate overnight at °C, randomly select the monoclonal inoculation into LB liquid medium, and expand to 1L. After incubation at 26 ° C for 3 h, 1 mM IPTG was added. The culture was induced overnight at 26 °C. The cells were collected by centrifugation at 12,000 rpm for 10 min at 4 ° C and resuspended in BugBuster (Novagen) protein extract.
  • mice 50 ⁇ l of the purified protein of Example 1 (10 ⁇ g) and 50 ⁇ l of Freund's incomplete adjuvant (Sigma) were thoroughly mixed, and subcutaneously injected with C57BL/6 mice (Shanghai Slack, SPF grade, 6-8 weeks female) (each group) 4)). Immunization was performed once every other week for 3 times to evaluate the immunogenicity of these antigens. After 8 weeks of the first immunization, the mice were sacrificed and the spleens were isolated.
  • lymphocytes After obtaining spleen lymphocytes, 1 ⁇ 10 6 lymphocytes were added to 24-well cell culture plates and stimulated with Rv0976c, Rv1255c, Rv3160c and Rv0792c antigens respectively, and the stimulation concentrations were 5 ⁇ g/ml and 10 ⁇ g/ml, respectively; The antigen served as an antigen-free negative control. Incubate for 60 hours at 37 ° C, 5% CO 2 , 100% saturated humidity.
  • the cell culture supernatant was collected by centrifugation, and the levels of Th1 type cytokines IFN- ⁇ , TNF- ⁇ , and IL-2 were measured, and the OptEIATM pre-coated ELISA kit (BD Biosciences) was used, and the procedure recommended by the kit was followed.
  • Purified Ag85A was used as a reference, and the expression results of different antigenic cytokines were homogenized according to the results of Ag85A. The results showed that the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins were highly immunogenic and capable of inducing Th1 type cytokine secretion levels close to or stronger than Ag85A (Fig. 3).
  • mice sera were collected and antigen-specific antibody levels were measured by ELISA.
  • the serum was serially diluted (1:400 to 1:51200) and added to a 96-well plate pre-coated with Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins.
  • ELISA assays for antibody subtypes were performed using HRP-labeled anti-mouse IgG1 (Youinwei, sc2969) or IgG2c (Youinwei, ab97255) secondary antibody.
  • the results showed that the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins induced high levels of antigen-specific antibodies in the immunized mice, showing strong B cell reactivity (Fig. 4).
  • Candidate gene clones enter mammalian expression vector
  • the scheme for cloning the gene encoding Rv0976c, Rv1255c, Rv3160c and Rv0792c into the mammalian expression vector pVAX1 or pcDNA3.1 is shown in FIG.
  • the primers for the Rv0792c clone into pVAX1 were: 5'-TAGAATTCGCCACCATGGGCATGCGTATCGGAAACTG-3' (upstream, SEQ ID No. 29) and 5'-TAACTGCAGCTAGTGATGGTGATGGTGATGCAACAGGGTCTCCG-3' (downstream, SEQ ID No. 30).
  • the system and reaction conditions of the PCR were the same as in Example 1.
  • the PCR product was digested with EcoRI and PstI, and ligated into the same
  • the digested pVAX1 plasmid (Invitrogen, Cat. No. V260-20) produced pVAX1-Rv0976c.
  • Plasmids pVAX-Ag85A and pVAX-PPE18 were constructed in the same manner.
  • Rv1255c, Rv3160c and Rv0792c were cloned into pcDNA3.1, and the corresponding PCR primers were: 5'-ATATACTTAAGGCCGCCACCATGGCGGGTACCGACTGGCTGTC-3' (upstream, Rv1255c, SEQ ID No. 31), 5'-AATATTCTAGATCAATGGTGATGGTGATGATGCTCGGGTCCAGGGTGACCGGC-3' (downstream, Rv1255c, SEQ ID No. 32); 5'-ATATACTTAAGGCCGCCACCATGCCGAGGCAGGCCGGCCG-3' (upstream, Rv3160c, SEQ ID No.
  • the PCR product was digested with AflII and XbaI, and ligated into the same digested pcDNA3.1 plasmid (Invitrogen, Cat. No. V790-20) to produce pcDNA-Rv1255c, pcDNA-Rv3160c and pcDNA-Rv0792c.
  • the plasmids pcDNA-Ag85A and pcDNA-HspX were constructed in the same manner.
  • mice Human Fukang, SPF grade, 6-8 weeks female, 6 rats per group
  • mice were immunized every other week with 100 ⁇ g/mouse of pVAX-Rv0796c, pVAX-Ag85A or pVAX-PPE18.
  • M. tuberculosis H37Rv strain purchased from the China Microbial Culture Collection, ATCC93009, Shanghai Pulmonary Hospital, cultured and cultured
  • mice After 5 weeks of infection, the mice were sacrificed, and the organ homogenate was serially diluted, and then coated with 7H11 agar plates (adding OADC, namely Middlebrook OADC, mixed nutrient additive, 10% ratio), and counting the number of lung and spleen bacteria. .
  • the results showed that the protective effect of Rv0976c against M. tuberculosis was stronger than that of Ag85A, which was close to that of PPE18: the number of M. tuberculosis in the lung and spleen of mice infected with Rv0976c DNA vaccine was significantly lower than that after 5 weeks of infection with M. tuberculosis.
  • the number of M. tuberculosis in the control (pVAX) or mice inoculated with the Ag85A DNA vaccine was similar to the number of M. tuberculosis in the mice inoculated with the PPE18 DNA vaccine (Fig. 6).
  • mice were immunized every week with 100 ⁇ g/mouse of pcDNA-Rv1255c, pcDNA-Rv3160c, pcDNA-Rv0792c, pcDNA-Ag85A or pcDNA-HspX (Hua Fukang, SPF grade, 6-8 weeks female, each group) 6), a total of 3 immunizations.
  • M. tuberculosis H37Rv strain 100 CFU/lung was infected as an aerosol.
  • mice After 9 weeks of infection, the mice were sacrificed, and the organ homogenate was serially diluted, and then coated on a 7H11 agar plate (adding OADC, ibid.), and the number of lung and spleen organs was counted.
  • Example 4 Serological detection of active tuberculosis, latent tuberculosis, and healthy controls with selected antigens
  • the present invention purified the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins of Example 1 from different populations.
  • the serum was subjected to an ELISA reaction.
  • the experiment included three groups of population samples: active tuberculosis group (20 patients), tuberculosis latent infection group (25 samples), and healthy control group (24 individuals).
  • the active tuberculosis group included 11 sputum-positive patients and 9 patients with clinical signs of X-ray examination.
  • Tuberculosis latent sensation refers to those who have long-term, continuous close contact with TB patients (2-8 years, mean 4.3 years) but do not show the clinical symptoms of active tuberculosis.
  • a healthy individual is a population that does not have a history of close contact with TB patients, no history of tuberculosis or clinical TB symptoms. All individuals were from the same geographical area and had a history of BCG vaccination.
  • Each purified antigen (Rv0976c, Rv1255c, Rv3160c, and Rv0792c) was added to a 96-well plate at 0.25 ⁇ g/well; the serum from the experimental group of active tuberculosis, latent tuberculosis, and healthy controls was 1 : 100 dilution, added to the antigen-coated wells, antigen-specific antibody levels were detected by standard ELISA methods, and the results are shown in FIG.
  • Rv0976c, Rv1255c, Rv3160c and Rv0792c differed from those of different disease patterns in the serum.
  • serum derived from active tuberculosis and latent infections specifically recognized Rv0976c, Rv1255c, and Rv3160c antigens.
  • Antibody levels were significantly higher than in healthy populations (B, C, D in Figure 8).
  • the level of antibodies that specifically bind Rv0792c in the serum of latently infected populations was significantly higher than that of active tuberculosis and healthy controls (Figure 8 A).

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Abstract

Provided are an immunogenicity composition comprising mycobacterium tuberculosis antigens Rv0976c, Rv1255c, Rv3160c and/or Rv0792c and coding genes thereof, vaccines or a treatment composition.

Description

结核分枝杆菌抗原及其应用Mycobacterium tuberculosis antigen and its application 技术领域Technical field
本发明属于新型的结核疫苗领域。特别涉及相应的免疫原性成分、疫苗或治疗成分，用来预防或治疗由致病性分枝杆菌，如：结核分枝杆菌、牛型分枝杆菌、非洲分枝杆菌、麻风分枝杆菌、溃疡分枝杆菌，引起的感染。免疫原性成分、疫苗或治疗成分由结核分枝杆菌抗原Rv0976c、Rv1255c、Rv3160c和/或Rv0792c，或其对应的编码核酸组成。The invention belongs to the field of novel tuberculosis vaccines. In particular, it relates to corresponding immunogenic components, vaccines or therapeutic ingredients for the prevention or treatment of pathogenic mycobacteria such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium leprae, Infection caused by Mycobacterium ulcerans. The immunogenic component, vaccine or therapeutic component consists of the M. tuberculosis antigen Rv0976c, Rv1255c, Rv3160c and/or Rv0792c, or a corresponding encoding nucleic acid thereof.
背景技术Background technique
结核病一直是世界范围内单病因导致的死亡率最高的一种传染病。据估计世界三分之一人口受到结核分枝杆菌感染，仅2013年就有150万人死于结核。在全球范围内，结核病的有效控制依然面临许多困难和挑战，包括缺乏快速准确的诊断技术、缺乏有效的抗结核疫苗、以及疗程长达数月等。而结核菌和HIV的协同感染以及越来越多的多重抗药性(MDR-TB)和广泛抗药性TB(XDR-TB)的传播也使得结核病的防控工作进一步复杂化。基于目前情况，迫切需要快速、准确的诊断方法和更有效的疫苗。因此，急需安全有效的抗结核新疫苗，预计这类疫苗每年可以减少800-1000万以上新发结核病例。Tuberculosis has been the most common infectious disease caused by a single cause worldwide. It is estimated that one third of the world’s population is infected with M. tuberculosis, and in 2013 alone 1.5 million people died of tuberculosis. On a global scale, effective control of tuberculosis still faces many difficulties and challenges, including the lack of rapid and accurate diagnostic techniques, the lack of effective anti-tuberculosis vaccines, and treatments for months. Co-infection of tuberculosis and HIV, as well as the spread of multiple drug resistance (MDR-TB) and extensive drug-resistant TB (XDR-TB), further complicate the prevention and control of tuberculosis. Based on the current situation, there is an urgent need for rapid and accurate diagnostic methods and more effective vaccines. Therefore, there is an urgent need for safe and effective new anti-tuberculosis vaccines, which are expected to reduce new tuberculosis cases by more than 8 million to 10 million per year.
Bacille Calmette-Guérin(BCG)，即卡介苗，作为一种减毒的牛结核分枝杆菌，是迄今为止唯一被批准使用的抗结核疫苗。但是，卡介苗存在两个主要的缺陷：一是对成人肺结核的保护效果非常有限；二是在免疫能力低下人群中可能引起播散性BCG疾病。临床研究结果显示，BCG对儿童严重结核包括粟粒性肺结核和结核性脑膜炎有80％以上的保护效果，然而，对成人肺结核的保护效果有限，临床研究的保护效果参差不齐(0-80％)。推测原因如下：临床实验中使用的BCG菌株的差异、临床实验方法的不同、临床实验人群对环境分枝杆菌的不同接触史、不同人群营养状态和遗传背景差别、临床结核分枝杆菌菌株的异质性。现在已经清楚，BCG不是一种理想的疫苗，对人体的保护时间有限。BCG对新生儿初免所产生的的免疫保护效果最多能维持10-20年，因此对成人肺结核几乎没有保护效果。Bacille Calmette-Guérin (BCG), BCG, is the only attenuated M. tuberculosis vaccine and is the only approved anti-tuberculosis vaccine to date. However, BCG has two major drawbacks: one is that the protective effect on adult tuberculosis is very limited; the other is that it may cause disseminated BCG disease in people with low immunity. Clinical studies have shown that BCG has more than 80% protective effect on severe tuberculosis in children including miliary tuberculosis and tuberculous meningitis. However, the protective effect on adult tuberculosis is limited, and the protective effect of clinical research is uneven (0-80%). ). The reasons are presumed as follows: differences in BCG strains used in clinical trials, differences in clinical trial methods, different exposure history of clinical experimental populations to environmental mycobacteria, differences in nutritional status and genetic background among different populations, and differences in clinical M. tuberculosis strains Qualitative. It is now clear that BCG is not an ideal vaccine and has limited protection time for the human body. The immunoprotective effect of BCG on newborn priming can last up to 10-20 years, so there is almost no protective effect on adult tuberculosis.
当前，普遍认为新一代最有效的结核疫苗是采用初免-加强策略来强化由BCG引起的免疫应答。这种“初免-加强”策略包括：利用BCG或重组BCG初始免疫之后，对尚未接触结核菌的婴儿和儿童，辅以亚单位疫苗(蛋白质/肽或DNA)加强接种；或者对青少年以亚单位疫苗单独加强免疫；或者以亚单位疫苗作为化疗的辅助剂。Currently, it is widely believed that the most effective new generation of tuberculosis vaccines is the use of prime-boost strategies to strengthen the immune response caused by BCG. This “priming-boosting” strategy includes: after initial immunization with BCG or recombinant BCG, boosting vaccination with infants and children who have not been exposed to tuberculosis, supplemented with subunit vaccines (protein/peptide or DNA); or The unit vaccine is boosted alone; or the subunit vaccine is used as an adjuvant for chemotherapy.
亚单位疫苗开发中一个关键问题是抗原的选择。多数研究都集中在几个能够诱导强IFN-γ释放的抗原上。机体抗结核菌主要依赖细胞免疫应答，虽然机制尚未完全清楚，但含有包括CD4+和CD8+T细胞、非传统的γδT细胞、CD1-限制性αβT细胞在内的多种复合组分。迄今为止， 尚无有效的评价疫苗保护效果或保护性免疫的“生物标识物”。BCG主要通过诱导CD4+T细胞分泌IFN-γ，诱发Th1型细胞免疫反应(Black GF,et al.(2002)BCG-induced increase in interferon-gamma response to mycobacterial antigens and efficacy of BCG vaccination in Malawi and the UK:two randomised controlled studies.Lancet 359(9315):1393-1401)。IFN-γ在控制结核方面的关键作用已经在小鼠和人中得到证实。据此，主要由CD4+T细胞产生的抗原特异性IFN-γ被广泛地用做保护性免疫的评价指标，虽然单凭IFN-γ不足以提供全部的抗结核保护(Hanekom WA,et al.(2008)Immunological outcomes of new tuberculosis vaccine trials:WHO panel recommendations.PLoS Med 5(7):e145)。一直以来，亚单位疫苗的主要研发策略是寻找能够诱导强IFN-γ释放的蛋白抗原来构建亚单位疫苗(蛋白或DNA)。采用的方法是通过将结核菌蛋白混合物尤其是菌液上清液进行生化分提，筛选诱导IFN-γ释放的蛋白抗原。运用这种方法已经找到了包括Esx家族蛋白(EsxA、B、G、H、G、N)，抗原85复合体(Ag85A、B、C)，PE/PPE家族蛋白(如：PPE18、PPE14)在内的几个抗原。根据这些研究结果构建的三个蛋白融合体H1(Ag85B-ESAT-6)、H4[Ag85B-TB10.4(EsxH)]和M72(PPE18-Rv0125)是目前以蛋白为主的亚单位疫苗中研究最为深入的。这几个融合蛋白单独使用都显示出接近，但不高于BCG的保护效果。H1、H4和M72都已进入临床IIa期实验。A key issue in the development of subunit vaccines is the choice of antigen. Most studies have focused on several antigens that are capable of inducing strong IFN-γ release. The body's anti-tuberculosis mainly depends on the cellular immune response, although the mechanism is not fully understood, but contains a variety of composite components including CD4 + and CD8 + T cells, non-traditional γδT cells, CD1-restricted αβ T cells. So far, There are no effective "biomarkers" for evaluating vaccine protection or protective immunity. BCG induces Th1 type cellular immune responses mainly by inducing secretion of IFN-γ by CD4+ T cells (Black GF, et al. (2002) BCG-induced increase in interferon-gamma response to mycobacterial antigens and efficacy of BCG vaccination in Malawi and the UK: two randomised controlled studies. Lancet 359 (9315): 1393-1401). The key role of IFN-γ in controlling tuberculosis has been confirmed in mice and humans. Accordingly, antigen-specific IFN-γ produced mainly by CD4+ T cells is widely used as an indicator for the evaluation of protective immunity, although IFN-γ alone is insufficient to provide all anti-tuberculosis protection (Hanekom WA, et al. (2008) Immunological outcomes of new tuberculosis vaccine trials: WHO panel recommendations. PLoS Med 5(7): e145). The primary development strategy for subunit vaccines has been to find protein antigens that induce strong IFN-γ release to construct subunit vaccines (protein or DNA). The method used is to screen for protein antigens that induce IFN-γ release by biochemical fractionation of a mixture of tuberculosis proteins, especially bacterial supernatants. Using this method, we have found Esx family proteins (EsxA, B, G, H, G, N), antigen 85 complex (Ag85A, B, C), PE/PPE family proteins (such as: PPE18, PPE14) Several antigens within. Based on these findings, three protein fusions H1 (Ag85B-ESAT-6), H4 [Ag85B-TB10.4 (EsxH)], and M72 (PPE18-Rv0125) are currently studied in protein-based subunit vaccines. The most in-depth. These fusion proteins were shown to be close to each other but not higher than the protective effect of BCG. H1, H4 and M72 have all entered clinical phase IIa experiments.
为了增强抗原被CD8+T细胞识别，以复制缺陷病毒，如腺病毒或牛痘病毒为载体，开发了DNA亚单位疫苗，如MVA85A(腺病毒表达抗原85A，Ag85A)；AERAS-402(牛痘病毒表达抗原85A、85B和TB10.4(EsxH))。在猴子模型中，MVA85A能够加强BCG的保护效率(Verreck FA,et al.(2009)MVA.85A boosting of BCG and an attenuated,phoP deficient M.tuberculosis vaccine both show protective efficacy against tuberculosis in rhesus macaques.PLoS ONE 4(4):e5264)，已经进入临床IIa期实验的AERAS-402也能够加强BCG初免后的T细胞应答(Magalhaes I,et al.(2008)rBCG induces strong antigen-specific T cell responses in rhesus macaques in a prime-boost setting with an adenovirus 35 tuberculosis vaccine vector.PLoS ONE 3(11):e3790；Andersen P&Kaufmann SH(2014)Novel vaccination strategies against tuberculosis.Cold Spring Harb Perspect Med 4(6))。MVA85A目前已经完成了临床IIb期实验，这是第一株完成保护效果临床验证的亚单位疫苗，但结果却令人失望。对已经接种卡介苗的南非婴儿，MVA85A并不能显著提高BCG的保护效果、抗结核病发病能力或抗结核分枝杆菌感染能力(Tameris MD,et al.(2013)Safety and efficacy of MVA85A,a new tuberculosis vaccine,in infants previously vaccinated with BCG:a randomised,placebo-controlled phase 2b trial.Lancet 381(9871):1021-1028)。MVA85A的实验结果提示我们，有必要继续寻找新型更有效的亚单位疫苗候选抗原(Geluk A,van Meijgaarden KE,Joosten SA,Commandeur S,&Ottenhoff TH(2014)Innovative Strategies to Identify M.tuberculosis Antigens and Epitopes Using Genome-Wide Analyses.Front Immunol 5:256；Singh S,Saraav I,&Sharma S(2014) Immunogenic potential of latency associated antigens against Mycobacterium tuberculosis.Vaccine 32(6):712-716；Aagaard C,et al.(2011)A multistage tuberculosis vaccine that confers efficient protection before and after exposure.Nat Med 17(2):189-194)。In order to enhance the recognition of antigens by CD8+ T cells, DNA subunit vaccines such as MVA85A (adenovirus expressing antigen 85A, Ag85A) and AERAS-402 (vaccinia virus expression) have been developed using replication-deficient viruses such as adenovirus or vaccinia virus as vectors. Antigens 85A, 85B and TB 10.4 (EsxH)). In the monkey model, MVA85A enhances the protective efficiency of BCG (Verreck FA, et al. (2009) MVA.85A boosting of BCG and an attenuated, phoP deficient M. tuberculosis vaccine both show protective efficacy against tuberculosis in rhesus macaques. PLoS ONE 4(4): e5264), AERS-402, which has entered clinical phase IIa trials, also potentiates T cell responses after BCG priming (Magalhaes I, et al. (2008) rBCG induces strong antigen-specific T cell responses in rhesus Macaques in a prime-boost setting with an adenovirus 35 tuberculosis vaccine vector.PLoS ONE 3(11):e3790; Andersen P&Kaufmann SH(2014)Novel vaccination strategies against tuberculosis.Cold Spring Harb Perspect Med 4(6)). MVA85A has now completed the clinical Phase IIb trial, which is the first subunit vaccine to complete the clinical validation of the protective effect, but the results are disappointing. For South African infants who have been vaccinated with BCG, MVA85A does not significantly improve BCG protection, anti-tuberculosis, or anti-tuberculosis infection (Tameris MD, et al. (2013) Safety and efficacy of MVA85A, a new tuberculosis vaccine , in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet 381 (9871): 1021-1028). The results of MVA85A suggest that it is necessary to continue to search for new and more effective subunit vaccine candidate antigens (Geluk A, van Meijgaarden KE, Joosten SA, Commandeur S, & Ottenhoff TH (2014) Innovative Strategies to Identify M. tuberculosis Antigens and Epitopes Using Genome-Wide Analyses.Front Immunol 5:256; Singh S, Saraav I, & Sharma S (2014) Immunogenic potential of latency associated antigens against Mycobacterium tuberculosis. Vaccine 32(6): 712-716; Aagaard C, et al. (2011) A multistage tuberculosis vaccine that confers efficient protection before and after exposure. Nat Med 17(2): 189 -194).
因此，为诊断或治疗结核病，仍然需要提供来自于结核杆菌的抗原的改进的成分和方法。Therefore, for the diagnosis or treatment of tuberculosis, there is still a need for improved compositions and methods for providing antigens from Mycobacterium tuberculosis.
发明内容Summary of the invention
本发明涉及一种由结核分枝杆菌抗原Rv0976c、Rv1255c、Rv3160c和/或Rv0792c、或与其对应的编码核酸所组成的免疫原性组合物、疫苗或治疗组合物。同时，本发明也包括用合成或重组的方法所产生的短/长的重叠或不重叠的肽。另外，本发明涉及使用Rv0976c、Rv1255c、Rv3160c和/或Rv0792c蛋白、或者是这些蛋白对应的编码基因，在结核分枝杆菌感染的诊断、治疗和/或预防上的应用。The present invention relates to an immunogenic composition, vaccine or therapeutic composition consisting of M. tuberculosis antigens Rv0976c, Rv1255c, Rv3160c and/or Rv0792c, or a coding nucleic acid corresponding thereto. At the same time, the invention also encompasses short/long overlapping or non-overlapping peptides produced by synthetic or recombinant methods. Further, the present invention relates to the use of the Rv0976c, Rv1255c, Rv3160c and/or Rv0792c proteins, or the coding genes corresponding to these proteins, for the diagnosis, treatment and/or prevention of M. tuberculosis infection.
本案发明人发现Rv0976c、Rv1255c、Rv3610c和Rv0792c在动物模型中具有高度的免疫原性，并具有抗结核分枝杆菌感染的保护效果。因此，抗原Rv0976c、Rv1255c、Rv3610c和Rv0792c是构建抗结核疫苗非常有前景的候选抗原。The inventors of the present invention found that Rv0976c, Rv1255c, Rv3610c and Rv0792c are highly immunogenic in animal models and have a protective effect against Mycobacterium tuberculosis infection. Therefore, the antigens Rv0976c, Rv1255c, Rv3610c and Rv0792c are very promising candidate antigens for the construction of anti-tuberculosis vaccines.
本发明公开一种免疫原性组合物，一种疫苗或治疗性组合物，它们包含了下述的一种或多种多肽：The present invention discloses an immunogenic composition, a vaccine or therapeutic composition comprising one or more of the following polypeptides:
(a)：表1所示的氨基酸序列；(SEQ ID Nos.1～4，分别对应于Rv0976c、Rv1255c、Rv3160c和Rv0792c)(a): the amino acid sequence shown in Table 1; (SEQ ID Nos. 1-4, corresponding to Rv0976c, Rv1255c, Rv3160c, and Rv0792c, respectively)
(b)：(a)序列中具有免疫原性的片段，比如T细胞表位；和/或(b): (a) an immunogenic fragment of the sequence, such as a T cell epitope; and/or
(c)：与(a)或(b)中任一序列具有70％以上一致性的氨基酸序列。(c): an amino acid sequence having 70% or more identity with any of the sequences of (a) or (b).
本发明也公开一种免疫原性组合物，一种疫苗或治疗性组合物，它们由下述的任一种或多种核酸分子组成：The invention also discloses an immunogenic composition, a vaccine or therapeutic composition consisting of any one or more of the following nucleic acid molecules:
(a)：表2所示的核酸序列；(SEQ ID Nos.5～8，分别为Rv0976c、Rv1255c、Rv3160c和Rv0792c的编码核酸)(a): the nucleic acid sequence shown in Table 2; (SEQ ID Nos. 5 to 8, respectively, encoding nucleic acids of Rv0976c, Rv1255c, Rv3160c, and Rv0792c)
(b)：与(a)中核苷酸编码相同氨基酸序列的核苷酸序列，或者其互补序列；或者(b): a nucleotide sequence encoding the same amino acid sequence as the nucleotide in (a), or a complementary sequence thereof;
(c)：能够在严格杂交条件下与(a)或(b)中的核苷酸序列发生杂交的10个以上的核苷酸序列。(c): 10 or more nucleotide sequences capable of hybridizing to the nucleotide sequence in (a) or (b) under strict hybridization conditions.
优选地，上述核酸为DNA片段。Preferably, the above nucleic acid is a DNA fragment.
本发明的免疫原性组合物、疫苗或治疗性组合物可具有如下应用：The immunogenic compositions, vaccines or therapeutic compositions of the invention may have the following applications:
应用1，本发明中的多肽疫苗应用于人类或其他哺乳动物或动物中，以增强机体对由致病性分枝杆菌，如：结核分枝杆菌(Mycobacterium tuberculosis)、牛型分枝杆菌(Mycobacterium bovis)、非洲分枝杆菌(Mycobacterium africanum)、麻风分枝杆菌(Mycobacterium leprae)或溃疡分 枝杆菌(Mycobacterium ulcerans)引起的结核感染的抗性。包括由纯化的多肽或其免疫原性片段组成的免疫原性组分的使用方法。多肽或免疫原性片段的免疫原性可能通过与佐剂的融合被加强，也可以通过添加其他分枝杆菌多肽，或其他生物，如细菌、病毒、哺乳动物多肽来加强。添加的多肽也被包含在组成组合物当中，以交联或非交联形式结合到多肽或免疫原性片段上。 Application 1, the polypeptide vaccine of the present invention is applied to humans or other mammals or animals to enhance the body against pathogenic mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium. Bovis), Mycobacterium africanum, Mycobacterium leprae or ulcer Resistance to tuberculosis infection caused by Mycobacterium ulcerans. A method of using an immunogenic component consisting of a purified polypeptide or an immunogenic fragment thereof. The immunogenicity of a polypeptide or immunogenic fragment may be enhanced by fusion with an adjuvant, or may be enhanced by the addition of other mycobacterial polypeptides, or other organisms such as bacteria, viruses, mammalian polypeptides. The added polypeptide is also included in the composition composition and is bound to the polypeptide or immunogenic fragment in a cross-linked or non-cross-linked form.
应用2，本发明中的核酸随机***到载体，如腺病毒或牛痘病毒载体中，以DNA疫苗形式直接用于人类或其他哺乳动物或动物，在体内表达抗原，导致机体对致病性分枝杆菌，如：结核分枝杆菌(Mycobacterium tuberculosis),、牛型分枝杆菌(Mycobacterium bovis)、非洲分枝杆菌(Mycobacterium africanum)、麻风分枝杆菌(Mycobacterium leprae)或溃疡分枝杆菌(Mycobacterium ulcerans)引起的结核感染的抗性。因此，本发明中的多肽和核酸可以构成治疗性组合物，应用于人类或其他哺乳动物或动物中，来预防和/或治疗结核分枝杆菌感染。 Application 2, the nucleic acid of the present invention is randomly inserted into a vector, such as an adenovirus or vaccinia virus vector, directly used in human or other mammals or animals as a DNA vaccine, and expresses antigen in vivo, resulting in pathogenic branching of the body. Bacteria, such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium leprae, or Mycobacterium ulcerans Resistance to tuberculosis infection. Thus, the polypeptides and nucleic acids of the invention may constitute a therapeutic composition for use in humans or other mammals or animals to prevent and/or treat M. tuberculosis infection.
应用3，本发明提供了一种用于人类或其他哺乳动物或动物免疫的疫苗，以抵抗由致病性分枝杆菌，如：结核分枝杆菌(Mycobacterium tuberculosis)、牛型分枝杆菌(Mycobacterium bovis)、非洲分枝杆菌(Mycobacterium africanum)、麻风分枝杆菌(Mycobacterium leprae)或溃疡分枝杆菌(Mycobacterium ulcerans)引起的结核感染。作为非致病性微生物的有效组成组合物，至少含有编码上述多肽的DNA片段的一个拷贝，整合进微生物中(如游离质粒中或进入微生物基因组)，令微生物表达这段多肽。 Application 3, the present invention provides a vaccine for human or other mammalian or animal immunization against pathogenic mycobacteria such as Mycobacterium tuberculosis, Mycobacterium Bovis infection, tuberculosis caused by Mycobacterium africanum, Mycobacterium leprae or Mycobacterium ulcerans. As an effective constituent composition of a non-pathogenic microorganism, at least one copy of the DNA fragment encoding the above polypeptide is contained, and integrated into a microorganism (such as a free plasmid or into a microbial genome) to allow the microorganism to express the polypeptide.
应用4，本发明中的组合物、多肽、核酸在体内外实验中用于检测针对结核分枝杆菌的抗体反应或细胞免疫反应，适用于感染的诊断或监测疾病进程。例如，多肽可能在皮试时作为体内诊断试剂。多肽也能在结核病人血样本的ELISA或T-spot检测时用于体外测试。或者，核酸或多肽可能在非人动物中用来提取抗结核分枝杆菌的抗体，此抗体可以用来通过体内或体外实验来检测靶位抗原。 Application 4, the composition, polypeptide, and nucleic acid of the present invention are used in an in vitro and in vivo assay for detecting an antibody response or a cellular immune response against Mycobacterium tuberculosis, and are suitable for diagnosis of infection or monitoring of disease progression. For example, a polypeptide may be used as an in vivo diagnostic reagent during a skin test. Polypeptides can also be used for in vitro testing in ELISA or T-spot assays of blood samples from tuberculosis patients. Alternatively, the nucleic acid or polypeptide may be used in non-human animals to extract antibodies against M. tuberculosis, which antibodies can be used to detect target antigens by in vivo or in vitro experiments.
从而，一方面，本发明提供了一种免疫原性成分，其包括选自以下的一种或多种多肽或其免疫性片段作为免疫原性成分(即，本发明提供了选自以下的一种或多种多肽或其免疫性片段作为免疫原性成分的应用)：Thus, in one aspect, the invention provides an immunogenic component comprising one or more polypeptides or immunological fragments thereof selected from the group consisting of an immunogenic component (ie, the invention provides one selected from the group consisting of Application of one or more polypeptides or immunological fragments thereof as an immunogenic component):
(a)由SEQ ID No.1、SEQ ID No.2、SEQ ID No.3或SEQ ID No.4所示的氨基酸序列组成的多肽或其免疫性片段；(a) a polypeptide consisting of the amino acid sequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4 or an immunological fragment thereof;
(b)与(a)限定的氨基酸序列具有至少70％一致性且与(a)具有相同功能的由(a)衍生的多肽或其免疫性片段；(b) a polypeptide derived from (a) or an immunological fragment thereof having at least 70% identity with the defined amino acid sequence of (a) and having the same function as (a);
所述免疫性片段例如T细胞表位。The immunological fragment is, for example, a T cell epitope.
另一方面，本发明还提供了一种免疫原性成分，其包括编码上述多肽或其免疫性片段的核酸分子。具体而言，本发明的免疫原性成分，其包括选自以下的一种或多种核酸分子作为免疫原性成分(即，本发明提供了选自以下的一种或多种核酸分子作为免疫原性成分的应用)： In another aspect, the invention provides an immunogenic component comprising a nucleic acid molecule encoding the above polypeptide or an immunological fragment thereof. Specifically, the immunogenic component of the present invention comprises one or more nucleic acid molecules selected from the group consisting of the following as an immunogenic component (ie, the present invention provides one or more nucleic acid molecules selected from the group consisting of: Application of the original ingredients):
(a)由SEQ ID No.5、SEQ ID No.6、SEQ ID No.7或SEQ ID No.8所示的核苷酸序列或其互补序列组成的核酸分子；(a) a nucleic acid molecule consisting of the nucleotide sequence shown in SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8 or a complement thereof;
(b)由与(a)中核酸分子编码相同氨基酸序列的核苷酸序列或其互补序列组成的核酸分子；或者(b) a nucleic acid molecule consisting of a nucleotide sequence encoding the same amino acid sequence as the nucleic acid molecule of (a) or a complement thereof; or
(c)在严格条件下与(a)或(b)限定的核酸分子杂交且具有至少10个核苷酸长度的核酸分子。(c) a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule as defined in (a) or (b) and which has a length of at least 10 nucleotides.
另一方面，本发明还提供了一种免疫原性组合物，其包含前述的一种或多种多肽或其免疫性片段、和/或前述的一种或多种核酸分子作为免疫原性成分。即，本发明还提供了所述的免疫原性成分在制备具有免疫原性组合物中的应用。本发明的免疫原性组合物还可进一步包含佐剂。例如，如前所述，多肽或免疫原性片段的免疫原性可能通过与佐剂的融合被加强，也可以通过添加其他分枝杆菌多肽，或其他生物，如细菌、病毒、哺乳动物多肽来加强。添加的多肽也可被包含在本发明的组合物当中，以交联或非交联形式结合到多肽或免疫原性片段上。In another aspect, the present invention provides an immunogenic composition comprising the aforementioned one or more polypeptides or immunological fragments thereof, and/or one or more of the aforementioned nucleic acid molecules as an immunogenic component . That is, the present invention also provides the use of the immunogenic component described in the preparation of an immunogenic composition. The immunogenic composition of the invention may further comprise an adjuvant. For example, as previously described, the immunogenicity of a polypeptide or immunogenic fragment may be enhanced by fusion with an adjuvant, or by the addition of other mycobacterial polypeptides, or other organisms such as bacteria, viruses, mammalian polypeptides. strengthen. The added polypeptide may also be included in the compositions of the invention to bind to the polypeptide or immunogenic fragment in a cross-linked or non-cross-linked form.
另一方面，本发明还提供了一种表达载体或非致病性微生物，其中整合有至少一个拷贝的包含前述本发明的核酸分子的DNA片段(例如，放在游离质粒中或整合进入微生物基因组)，且DNA片段能够以多肽形式在微生物中表达。具体地，所述表达载体或非致病性微生物是牛痘、腺病毒、BCG或转化细胞。所述表达载体或非致病性微生物用于在人或其它动物体内表达针对分枝杆菌的抗体。In another aspect, the present invention provides an expression vector or a non-pathogenic microorganism in which at least one copy of a DNA fragment comprising the aforementioned nucleic acid molecule of the present invention is integrated (for example, placed in a free plasmid or integrated into a microbial genome) And the DNA fragment can be expressed in the form of a polypeptide in a microorganism. In particular, the expression vector or non-pathogenic microorganism is vaccinia, adenovirus, BCG or transformed cells. The expression vector or non-pathogenic microorganism is used to express antibodies against mycobacteria in humans or other animals.
另一方面，本发明还提供了所述的免疫组分、所述的免疫原性组合物、或所述的表达载体或非致病性微生物在制备用于抗结核分枝杆菌感染的药物中的应用。所述的抗结核分枝杆菌感染是指可增强机体对致病性分枝杆菌，如：结核分枝杆菌、牛型分枝杆菌、非洲分枝杆菌、麻风分枝杆菌或溃疡分枝杆菌引起的结核感染的抗性。In another aspect, the present invention provides the immunological component, the immunogenic composition, or the expression vector or the non-pathogenic microorganism in the preparation of a medicament for anti-tuberculous mycobacterial infection. Applications. The anti-tuberculous mycobacterial infection refers to enhancing the body caused by pathogenic mycobacteria, such as: Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium leprae or Mycobacterium ulcerans Resistance to tuberculosis infection.
本发明还提供了一种抗结核分枝杆菌感染的药物，该药物包含本发明所述的免疫组分、所述的免疫原性组合物、或所述的表达载体或非致病性微生物；优选地，所述药物可以为疫苗。抗结核分枝杆菌感染的药物可应用于人类或其他动物优选哺乳动物中，来预防和/或治疗结核分枝杆菌感染。The present invention also provides a medicament for infection against Mycobacterium tuberculosis, comprising the immune component of the present invention, the immunogenic composition, or the expression vector or non-pathogenic microorganism; Preferably, the medicament may be a vaccine. The drug against M. tuberculosis infection can be applied to humans or other animals, preferably mammals, to prevent and/or treat M. tuberculosis infection.
另一方面，本发明还提供了一种人类或其他动物优选哺乳类动物的抗结核的免疫方法，该方法包括施予个体(通过皮内、皮下透皮、肌肉、或粘膜递送的方式)本发明所述的免疫组分、所述的免疫原性组合物、或所述的表达载体或非致病性微生物。In another aspect, the invention provides a method of immunizing against tuberculosis in a human or other animal, preferably a mammal, comprising administering to the individual (by means of intradermal, subcutaneous transdermal, muscle, or mucosal delivery) The immune component, the immunogenic composition, or the expression vector or non-pathogenic microorganism of the invention.
另一方面，本发明还提供了所述的免疫组分或所述的免疫原性组合物在制备用于检测和/或诊断结核分枝杆菌感染的制剂中的应用。检测结核分枝杆菌感染包括检测机体针对结核分枝杆菌的抗体反应或细胞免疫反应。可用于在体外和/或体内检测，用于检测体液中针对M.tb的抗体或细胞介导的免疫反应，以进行疾病诊断、感染诊断，或监测疾病进展。核酸和多肽可用于在非人动物体内产生抗M.tb抗体。所述抗体可用于体内和/或体外检测靶抗原。在本发明的一个 方面，本发明的所述免疫原性成分(多肽和核酸)可被用于诊断方法，以筛选不同人群(例如，活动性、潜伏感染、健康人)相关的与所述抗原有抗体反应的抗血清。本发明的实验表明，源于活动性结核和潜伏感染人群的血清，特异性识别Rv0976c、Rv1255c、Rv3160c抗原的抗体水平显著高于健康人群血清；潜伏感染人群血清中特异性结合Rv0792c的抗体水平显著高于活动性结核和健康对照组。本发明的免疫原性成分还可被用于各种体外细胞因子释放检测，测定由本发明的多肽诱导产生的细胞因子的分泌水平，所述的细胞因子包括IFN-γ、TNF和IL-2中的至少一种。In another aspect, the invention also provides the use of the immunological component or the immunogenic composition described in the preparation of a formulation for detecting and/or diagnosing a Mycobacterium tuberculosis infection. Detection of M. tuberculosis infection includes detection of an antibody response or cellular immune response against M. tuberculosis. It can be used for in vitro and/or in vivo detection for the detection of antibodies or cell-mediated immune responses against M.tb in body fluids for disease diagnosis, infection diagnosis, or monitoring disease progression. Nucleic acids and polypeptides can be used to produce anti-M.tb antibodies in non-human animals. The antibody can be used to detect a target antigen in vivo and/or in vitro. One in the present invention In aspects, the immunogenic components (polypeptides and nucleic acids) of the invention can be used in diagnostic methods to screen for antibodies reactive with the antigen associated with different populations (eg, active, latent infection, healthy humans). serum. The experiments of the present invention show that the levels of antibodies specifically recognizing Rv0976c, Rv1255c, and Rv3160c antigens are significantly higher than those of active TB and latent infected people, and the levels of antibodies specifically binding to Rv0792c in the serum of latent infected populations are significant. Higher than active tuberculosis and healthy controls. The immunogenic components of the invention can also be used in a variety of in vitro cytokine release assays to determine the level of secretion of cytokines induced by the polypeptides of the invention, including IFN-[gamma], TNF and IL-2. At least one of them.
另一方面，本发明还提供了一种用于结核样本的诊断试剂盒，其中包括本发明所述的免疫组分、或本发明所述的免疫原性组合物。本发明的Rv0976c、Rv1255c、Rv3160c和Rv0792c可用于活动性结核、结核潜伏感染和健康个体间的鉴别诊断。活动性结核和潜伏感染人群的血清中特异性识别Rv0976c、Rv1255c、Rv3160c的抗体水平显著高于健康人群血清；潜伏感染人群血清中特异性结合Rv0792c的抗体水平显著高于活动性结核和健康对照组。In another aspect, the invention provides a diagnostic kit for a tuberculosis sample comprising an immunological component of the invention, or an immunogenic composition of the invention. The Rv0976c, Rv1255c, Rv3160c and Rv0792c of the present invention can be used for differential diagnosis between active tuberculosis, latent tuberculosis infection and healthy individuals. The levels of antibodies specifically recognizing Rv0976c, Rv1255c, and Rv3160c in the serum of active tuberculosis and latent infection were significantly higher than those in healthy people. The level of antibody specifically binding to Rv0792c in the serum of latent infected population was significantly higher than that of active tuberculosis and healthy control group. .
本发明还提供本发明的蛋白(包括多肽)、核酸或组合物在治疗或预防结核病中的应用。The invention also provides the use of a protein (including polypeptide), nucleic acid or composition of the invention in the treatment or prevention of tuberculosis.
本发明还提供以下蛋白或核酸在制备结核疫苗或制备用于诊断和/或检测结核病的试剂中的用途：The invention also provides the use of the following proteins or nucleic acids in the preparation of a tuberculosis vaccine or in the preparation of a medicament for the diagnosis and/or detection of tuberculosis:
(a)结核分枝杆菌蛋白Rv0976c、Rv1255c、Rv3160c、Rv0792c；(a) Mycobacterium tuberculosis proteins Rv0976c, Rv1255c, Rv3160c, Rv0792c;
(b)编码上述蛋白的核酸。(b) a nucleic acid encoding the above protein.
上述蛋白Rv0976c、Rv1255c、Rv3160c、Rv0792c的氨基酸序列分别如SEQ ID Nos.1～4所示。The amino acid sequences of the above proteins Rv0976c, Rv1255c, Rv3160c, and Rv0792c are shown in SEQ ID Nos. 1 to 4, respectively.
优选地，编码蛋白Rv0976c、Rv1255c、Rv3160c、Rv0792c的核酸序列分别如SEQ ID Nos.5～8所示。Preferably, the nucleic acid sequences encoding the proteins Rv0976c, Rv1255c, Rv3160c, Rv0792c are shown in SEQ ID Nos. 5-8, respectively.
在另一个方面，本发明提供一种治疗或预防结核病的方法，包括向人或其它动物施用本发明的蛋白、核酸或疫苗。In another aspect, the invention provides a method of treating or preventing tuberculosis comprising administering a protein, nucleic acid or vaccine of the invention to a human or other animal.
优选地，所述施用为皮内、皮下透皮、肌肉或粘膜递送方式。Preferably, the administration is by intradermal, subcutaneous transdermal, muscle or mucosal delivery.
本发明提供的蛋白或其多肽片段及其编码核酸具有针对结核杆菌的高的免疫原性，能够为生物体提供有效的保护性，可以作为疫苗候选物或者制备用于诊断和/或检测结核病的试剂。The protein or polypeptide fragment thereof provided by the invention and the nucleic acid encoding the same have high immunogenicity against Mycobacterium tuberculosis, can provide effective protection to the organism, can be used as a vaccine candidate or prepared for diagnosis and/or detection of tuberculosis. Reagents.
附图说明DRAWINGS
图1、本发明的蛋白的克隆、表达和纯化方案；Figure 1. Cloning, expression and purification scheme of the protein of the present invention;
图2、Rv0976c蛋白的表达和纯化；其中，各泳道表示：1、分子量标记；2、由IPTG诱导的全细胞；3、流过Ni2+亲和层析柱；4、50mM咪唑清洗缓冲液；5、100mM咪唑清洗缓冲液；6、150mM咪唑洗提缓冲液；7、300mM咪唑洗提缓冲液；8、300mM咪唑洗提缓冲液；9、500mM咪唑洗提缓冲液。收集用300mM咪唑洗提缓冲液洗提的片段(泳道7和8)作为纯化 的Rv0976c蛋白。Figure 2. Expression and purification of Rv0976c protein; wherein each lane represents: 1, molecular weight marker; 2. whole cells induced by IPTG; 3. flow through Ni 2+ affinity chromatography column; 4, 50 mM imidazole washing buffer 5, 100 mM imidazole washing buffer; 6, 150 mM imidazole elution buffer; 7, 300 mM imidazole elution buffer; 8, 300 mM imidazole elution buffer; 9, 500 mM imidazole elution buffer. Fragments eluted with 300 mM imidazole elution buffer (lanes 7 and 8) were collected as purified Rv0976c protein.
图3、本发明蛋白诱导的T细胞应答：通过ELISA测定的Th1细胞因子(TNF,IL-2,IL-12)的生产。数据被均一化为Ag85A，Ag85A平行地包括在每一个实验中。Figure 3. Protein-induced T cell response of the invention: production of Th1 cytokines (TNF, IL-2, IL-12) as determined by ELISA. The data was homogenized to Ag85A, and Ag85A was included in parallel in each experiment.
图4、本发明蛋白诱导的B细胞应答；其中，A为Rv0792c，B为Rv1255c，C为Rv0976c，D为Rv3160c。Figure 4. Protein-induced B cell response of the invention; wherein A is Rv0792c, B is Rv1255c, C is Rv0976c, and D is Rv3160c.
图5、将本发明核酸克隆进入哺乳动物表达载体pVAX1或pcDNA3.1；Rv0976c核酸克隆至pVAX1，Rv1255c、Rv3160c和Rv0792c克隆至pcDNA3.1；Ag85A克隆至pVAX1和pcDNA3.1，PPE18克隆至pVAX1，HspX克隆至pcDNA3.1。Figure 5. Cloning of the nucleic acid of the present invention into mammalian expression vector pVAX1 or pcDNA3.1; Rv0976c nucleic acid cloned into pVAX1, Rv1255c, Rv3160c and Rv0792c cloned into pcDNA3.1; Ag85A cloned into pVAX1 and pcDNA3.1, PPE18 cloned into pVAX1, HspX was cloned into pcDNA3.1.
图6、Rv0976c的保护效力实验结果；以DNA疫苗免疫BALB/c小鼠，并用M.tuberculosis H37Rv感染。感染5周后,处死小鼠并计数肺、脾脏器中结核分枝杆菌数量。A为小鼠肺器中结核分枝杆菌栽菌量计数结果，B为小鼠脾脏器中结核分枝杆菌栽菌量计数结果。其中，pVAX为接种用于克隆的空质粒载体的小鼠的结核分枝杆菌计数结果(阴性对照)，pVAX:Ag85A为接种表达Ag85A的质粒DNA的小鼠的结核分枝杆菌计数结果，pVAX:PPE18为接种表达PPE18的质粒DNA的小鼠的结核分枝杆菌计数结果,pVAX:Rv0976c为接种表达Rv0976c的质粒DNA的小鼠的结核分枝杆菌计数结果。Figure 6. Experimental results of protective efficacy of Rv0976c; BALB/c mice were immunized with DNA vaccine and infected with M. tuberculosis H37Rv. Five weeks after the infection, the mice were sacrificed and the number of M. tuberculosis in the lungs and spleen was counted. A is the result of counting the amount of Mycobacterium tuberculosis in the mouse lung, and B is the result of counting the amount of Mycobacterium tuberculosis in the mouse spleen. Among them, pVAX is a M. tuberculosis count result of a mouse inoculated with an empty plasmid vector for cloning (negative control), and pVAX: Ag85A is a M. tuberculosis count result of a mouse inoculated with plasmid DNA expressing Ag85A, pVAX: PPE18 is a result of counting M. tuberculosis in mice inoculated with plasmid DNA expressing PPE18, and pVAX: Rv0976c is a result of counting M. tuberculosis in mice inoculated with plasmid DNA expressing Rv0976c.
图7、Rv1255c、Rv3160c和Rv0792c的保护效力实验结果；以DNA疫苗免疫BALB/c小鼠，并用M.tuberculosis H37Rv感染。感染9周后，处死小鼠并计数肺、脾脏器中结核分枝杆菌数量。A为脾计数结果，B为肺计数结果。其中，sham和pcDNA分别为接种PBS或者空质粒载体的小鼠的结核分枝杆菌计数结果(阴性对照)，pcDNA:Ag85A为接种表达Ag85A的质粒DNA的小鼠的结核分枝杆菌计数结果，pcDNA:HspX为接种表达HspX的质粒DNA的小鼠的结核分枝杆菌计数结果，pcDNA:Rv1255c为接种表达Rv1255c的质粒DNA的小鼠的结核分枝杆菌计数结果，pcDNA:Rv3160c为接种表达Rv3160c的质粒DNA的小鼠的结核分枝杆菌计数结果，pcDNA:Rv0792c为接种表达Rv0792c的质粒DNA的小鼠的结核分枝杆菌计数结果。Figure 7. Experimental results of protective efficacy of Rv1255c, Rv3160c and Rv0792c; BALB/c mice were immunized with DNA vaccine and infected with M. tuberculosis H37Rv. After 9 weeks of infection, the mice were sacrificed and the number of M. tuberculosis in the lungs and spleen was counted. A is the spleen count result, and B is the lung count result. Among them, sham and pcDNA are M. tuberculosis count results of mice inoculated with PBS or empty plasmid vector (negative control), and pcDNA: Ag85A is the result of M. tuberculosis in mice inoculated with plasmid DNA expressing Ag85A, pcDNA :HspX is the result of M. tuberculosis in mice inoculated with plasmid DNA expressing HspX, pcDNA: Rv1255c is the result of M. tuberculosis inoculation of a plasmid DNA expressing Rv1255c, and pcDNA: Rv3160c is a plasmid inoculated with Rv3160c. As a result of M. tuberculosis count of mice of DNA, pcDNA: Rv0792c is a result of counting M. tuberculosis in mice inoculated with plasmid DNA expressing Rv0792c.
图8、用选定蛋白对活动性结核(TB)、潜伏结核感染者(LTBI)、健康对照者(HC)的血清学检测结果；A为Rv0792c，B为Rv0976c，C为Rv1255c，D为Rv3160c。Figure 8. Serological test results for active tuberculosis (TB), latent tuberculosis (LTBI), and healthy controls (HC) with selected proteins; A is Rv0792c, B is Rv0976c, C is Rv1255c, and D is Rv3160c .
具体实施方式detailed description
当前结核疫苗研究面临的一个主要挑战就是缺乏适用于保护性免疫评价的指标或保护效果评价的生物标志。1998年，Cole等完成了结核分枝杆菌H37Rv的全基因组测序，预测有4000个开放阅读框，揭示了其核苷酸序列和推测的蛋白序列(Wang X,et al.(2015)Protection against Mycobacterium tuberculosis Infection Offered by a New Multistage Subunit Vaccine Correlates with Increased Number of IFN-gamma+IL-2+CD4+and IFN-gamma+CD8+T Cells.PLoS ONE  10(3):e0122560)。然而，这一序列信息无法预测蛋白的抗原性，而且，考虑到当前缺乏保护性相关的免疫指标，仅凭一个蛋白具有抗原性这一信息也不足以预测出/判断出这是一个好的疫苗候选。本领域人员都很清楚，判断一个蛋白是否是一个好的疫苗候选的唯一办法，就像下文提出的，是用这个蛋白免疫接种动物后，能否增强动物机体对结核分枝杆菌感染的抗性。国际结核疫苗研究领域在过去20年中对200多个抗原进行了动物的保护实验研究，发现仅仅有少数抗原具有作为亚单位抗结核疫苗的潜力，包括熟知的抗原Ag85A、PPE18、HspX、Esat-6、CFP-10等，这些抗原已经被相关研究人员申请专利保护并构建相应的亚单位疫苗进行临床实验，如上述的MVA85A。A major challenge facing current tuberculosis vaccine research is the lack of biomarkers for indicators or protective effects evaluations that are useful for protective immune evaluation. In 1998, Cole et al completed the genome-wide sequencing of M. tuberculosis H37Rv, predicting 4,000 open reading frames, revealing its nucleotide sequence and predicted protein sequence (Wang X, et al. (2015) Protection against Mycobacterium Tuberculosis Infection Offered by a New Multistage Subunit Vaccine Correlates with Increased Number of IFN-gamma+IL-2+CD4+and IFN-gamma+CD8+T Cells.PLoS ONE 10(3): e0122560). However, this sequence of information cannot predict the antigenicity of the protein, and given the current lack of protectively relevant immune indicators, the information that only one protein is antigenic is not sufficient to predict/determine that this is a good vaccine. Candidate. It is well known in the art that the only way to determine if a protein is a good vaccine candidate is as follows, is it possible to enhance the animal's resistance to M. tuberculosis infection after immunizing the animal with this protein? . The International Tuberculosis Vaccine Research field has conducted animal protection experiments on more than 200 antigens over the past 20 years and found that only a few antigens have potential as subunit anti-tuberculosis vaccines, including the well-known antigens Ag85A, PPE18, HspX, Esat- 6. CFP-10, etc. These antigens have been patented by relevant researchers and constructed corresponding subunit vaccines for clinical trials, such as MVA85A above.
本案发明人对约130个尚未用于构建亚单位抗结核疫苗的结核菌蛋白进行了动物实验，结果发现：结核分枝杆菌H37Rv菌株的Rv0976c、Rv1255c、Rv3160c和Rv0792c蛋白具有高度的免疫原性，能产生显著的抗结核分枝杆菌感染的保护效果。The inventors of the present invention conducted an animal experiment on about 130 tuberculosis proteins that have not been used to construct a subunit anti-tuberculosis vaccine, and found that the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins of the M. tuberculosis H37Rv strain are highly immunogenic. It can produce a significant protective effect against Mycobacterium tuberculosis infection.
本案申请日前的现有技术中，Rv0976c是结核分枝杆菌中生物功能未知的保守的假定蛋白，在包括结核分枝杆菌CDC1551、牛型分枝杆菌(包括BCG)、溃疡分枝杆菌、副结核分枝杆菌和海分枝杆菌在内的多个分枝杆菌种中具有同源基因。Rv1255c、Rv3160c和Rv0792c都是转录调控蛋白并且在分枝杆菌属内高度保守。Rv0976c、Rv1255c、Rv3160c和Rv0792c的氨基酸序列分别参见表1中的SEQ ID No.1到SEQ ID No.4，相对应的编码核酸序列参见表2的SEQ ID No.5到SEQ ID No.8。Rv0976c、Rv1255c、Rv3160c和Rv0792c的序列信息来源于结核分枝杆菌H37Rv菌株，网址：http://genodb.pasteur.fr/cgi-bin/WebObjects/GenoList。表3列出了本发明中用到的几种对比蛋白的序列信息。In the prior art of the present application, Rv0976c is a conserved putative protein with unknown biological functions in Mycobacterium tuberculosis, including Mycobacterium tuberculosis CDC1551, Mycobacterium bovis (including BCG), Mycobacterium tuberculosis, and tuberculosis Mycobacteria and Mycobacterium marinum have homologous genes among a plurality of mycobacterial species. Rv1255c, Rv3160c and Rv0792c are both transcriptional regulatory proteins and are highly conserved within Mycobacterium. The amino acid sequences of Rv0976c, Rv1255c, Rv3160c and Rv0792c are shown in SEQ ID No. 1 to SEQ ID No. 4 in Table 1, respectively, and the corresponding encoding nucleic acid sequences are shown in SEQ ID No. 5 to SEQ ID No. 8 of Table 2. The sequence information of Rv0976c, Rv1255c, Rv3160c and Rv0792c was derived from the M. tuberculosis H37Rv strain at http://genodb.pasteur.fr/cgi-bin/WebObjects/GenoList. Table 3 lists the sequence information of several comparative proteins used in the present invention.
表1.选定抗原的氨基酸序列Table 1. Amino acid sequences of selected antigens
Figure PCTCN2015091068-appb-000001
Figure PCTCN2015091068-appb-000001
Figure PCTCN2015091068-appb-000002
Figure PCTCN2015091068-appb-000002
表2.选定抗原的DNA序列Table 2. DNA sequences of selected antigens
Figure PCTCN2015091068-appb-000003
Figure PCTCN2015091068-appb-000003
Figure PCTCN2015091068-appb-000004
Figure PCTCN2015091068-appb-000004
表3：几种对比蛋白的序列信息Table 3: Sequence information for several comparative proteins
Figure PCTCN2015091068-appb-000005
Figure PCTCN2015091068-appb-000005
Figure PCTCN2015091068-appb-000006
Figure PCTCN2015091068-appb-000006
为了评价免疫原性，本发明将Rv0976c、Rv1255c、Rv3160c和Rv0792c开放读码框分别克隆进入pET28a质粒，用标准化程序利用E.coli BL21菌株表达、纯化(图1和图2)。分别用10μg纯化好的蛋白与Freund’s不完全佐剂混合，隔周免疫C57BL/6小鼠，共免疫3次以评价这些抗原的免疫原性。首次免疫8周后处死小鼠分离脾细胞。分别用对应的Rv0976c、Rv1255c、 Rv3160c和Rv0792c抗原刺激脾细胞，刺激浓度分别为5μg/ml、10μg/ml，以PBS代替抗原作为无抗原阴性对照，以纯化的Ag85A抗原为阳性对照，刺激下培养脾细胞。3天后收集细胞培养上清用ELISA检测Th1型细胞因子(IFN-γ、TNF-α和IL-2)释放水平。结果显示：Rv0976c、Rv1255c、Rv3160c和Rv0792c具有高度免疫原性，能够诱导出接近于或更高于Ag85A的Th1型细胞因子(图3)。Rv0976c、Rv1255c、Rv3160c和Rv0792c也能在免疫鼠中诱导出高水平抗原特异性抗体，产生强B细胞免疫反应(图4)。In order to evaluate immunogenicity, the present invention cloned the Rv0976c, Rv1255c, Rv3160c and Rv0792c open reading frames into the pET28a plasmid, and expressed and purified using the E. coli BL21 strain using a standardized procedure (Fig. 1 and Fig. 2). 10 μg of the purified protein was mixed with Freund's incomplete adjuvant, and C57BL/6 mice were immunized every other week for 3 times to evaluate the immunogenicity of these antigens. After 8 weeks of the first immunization, the mice were sacrificed to isolate spleen cells. Use the corresponding Rv0976c, Rv1255c, The spleen cells were stimulated by Rv3160c and Rv0792c antigens at a concentration of 5 μg/ml and 10 μg/ml, respectively, and PBS was used as the antigen-free negative control. The purified Ag85A antigen was used as a positive control, and the spleen cells were cultured under stimulation. The cell culture supernatant was collected 3 days later, and the release levels of Th1 type cytokines (IFN-γ, TNF-α, and IL-2) were measured by ELISA. The results showed that Rv0976c, Rv1255c, Rv3160c and Rv0792c were highly immunogenic and capable of inducing Th1 type cytokines close to or higher than Ag85A (Fig. 3). Rv0976c, Rv1255c, Rv3160c and Rv0792c also induced high levels of antigen-specific antibodies in the immunized mice, resulting in a strong B cell immune response (Fig. 4).
为了评价Rv0976c、Rv1255c、Rv3160c和Rv0792c的抗结核分枝杆菌保护效果，本发明将编码这些抗原的DNA序列(表2)克隆到哺乳动物表达载体pVAX1(Rv0976c)或包含CMV启动子的pcDNA3.1(Rv1255c,Rv3160c和Rv0792c)中(图5)。pVAX1和pcDNA3.1载体购自Invitrogen公司。Ag85A的编码序列也分别克隆进入这两个载体作为实验对照。用这些重组DNA对小鼠进行免疫，之后进行结核分枝杆菌H37Rv感染攻毒实验。具体实验如下：BALB/c小鼠用100μg构建的DNA隔周免疫一次，共免疫3次。首次免疫8周后用结核分枝杆菌(6×105CFU/鼠)通过尾静脉或气溶胶(剂量：100-300CFU/肺/鼠)途径感染小鼠，攻击5周或9周后，处死小鼠并计数肺、脾脏器中结核分枝杆菌数量。构建的Ag85A DNA作为对照平行进行实验。同一批实验中，接种Rv0976c DNA疫苗的小鼠在感染结核分枝杆菌5周后的肺、脾脏器中结核分枝杆菌数量明显低于阴性对照(pVAX)或者接种Ag85ADNA疫苗的小鼠中结核分枝杆菌数量，与接种PPE18DNA疫苗的小鼠中结核分枝杆菌数量相近(图6)。PPE18也是阳性对照，以它为重要组分的融合蛋白疫苗M72(PPE18-Rv0125)已经进入临床实验(Andersen P&Kaufmann SH(2014)Novel vaccination strategies against tuberculosis.Cold Spring Harb Perspect Med 4(6))。另一批次实验中，Rv1255c、Rv3160c和Rv0792c被证明具有与Ag85A或HspX相同或接近的保护能力(图7)。HspX是结核分枝杆菌潜伏相关抗原，也被用于构建多级亚单位疫苗，在本次实验中用作阳性对照(Mir FA,Kaufmann SH,&Eddine AN(2009)A multicistronic DNA vaccine induces significant protection against tuberculosis in mice and offers flexibility in the expressed antigen repertoire.Clin Vaccine Immunol 16(10):1467-1475；Wang X,et al.(2015)Protection against Mycobacterium tuberculosis Infection Offered by a New Multistage Subunit Vaccine Correlates with Increased Number of IFN-gamma+IL-2+CD4+and IFN-gamma+CD8+T Cells.PLoS ONE 10(3):e0122560)。迄今为止，Rv0976c、Rv1255c、Rv3160c和Rv0792c还没有被作为疫苗直接评价过，与目前首选的Ag85A、PPE18和HspX抗原相比，它们具有更强的免疫原性和更好或同等的保护效果，表明Rv0976c、Rv1255c、Rv3160c和Rv0792c可以作为构建亚单位疫苗的新型抗原。To evaluate the anti-tuberculosis protective effect of Rv0976c, Rv1255c, Rv3160c and Rv0792c, the present invention cloned the DNA sequence encoding these antigens (Table 2) into the mammalian expression vector pVAX1 (Rv0976c) or pcDNA3.1 containing the CMV promoter. (Rv1255c, Rv3160c and Rv0792c) (Fig. 5). The pVAX1 and pcDNA3.1 vectors were purchased from Invitrogen. The coding sequences of Ag85A were also cloned into these two vectors as experimental controls. Mice were immunized with these recombinant DNAs, followed by M. tuberculosis H37Rv infection challenge experiments. The specific experiment was as follows: BALB/c mice were immunized once a week with 100 μg of DNA, and immunized 3 times. After 8 weeks of the first immunization, mice were infected with M. tuberculosis (6×10 5 CFU/mouse) via the tail vein or aerosol (dose: 100-300 CFU/lung/mouse), and sacrificed after 5 or 9 weeks of challenge. The mice were counted for the number of M. tuberculosis in the lungs and spleen. The constructed Ag85A DNA was run in parallel as a control. In the same batch of experiments, the number of M. tuberculosis in the lung and spleen of mice inoculated with Rv0976c DNA vaccine after 5 weeks of infection with M. tuberculosis was significantly lower than that of the negative control (pVAX) or the mice inoculated with Ag85A DNA vaccine. The number of Mycobacterium species was similar to that of M. tuberculosis in mice inoculated with the PPE18 DNA vaccine (Fig. 6). PPE18 is also a positive control, and fusion protein vaccine M72 (PPE18-Rv0125) with it as an important component has entered clinical trials (Andersen P& Kaufmann SH (2014) Novel vaccination strategies against tuberculosis. Cold Spring Harb Perspect Med 4 (6)). In another batch of experiments, Rv1255c, Rv3160c, and Rv0792c were shown to have the same or similar protection capabilities as Ag85A or HspX (Figure 7). HspX is a latent-associated antigen of M. tuberculosis and was also used to construct a multi-level subunit vaccine, which was used as a positive control in this experiment (Mir FA, Kaufmann SH, & Eddine AN (2009) A multicistronic DNA vaccine induces significant protection against Tuberculosis in mice and offers flexibility in the expressed antigen repertoire. Clin Vaccine Immunol 16(10): 1467-1475; Wang X, et al. (2015) Protection against Mycobacterium tuberculosis Infection Offered by a New Multistage Subunit Vaccine Correlates with Increased Number of IFN-gamma+IL-2+CD4+and IFN-gamma+CD8+T Cells.PLoS ONE 10(3):e0122560). To date, Rv0976c, Rv1255c, Rv3160c and Rv0792c have not been directly evaluated as vaccines, and they have greater immunogenicity and better or equivalent protection than the currently preferred Ag85A, PPE18 and HspX antigens, indicating Rv0976c, Rv1255c, Rv3160c and Rv0792c can be used as novel antigens for the construction of subunit vaccines.
为了评价Rv0976c、Rv1255c、Rv3160c和Rv0792c是否能够与人血清中的特异性抗体反应，继而区分健康人群和不同疾病人群，本发明将纯化的Rv0976c、Rv1255c、Rv3160c和Rv0792c 蛋白与来自不同人群(活动性结核组、结核潜伏感染组、健康对照组)的血清进行ELISA反应。实验结果显示，Rv0976c、Rv1255c、Rv3160c和Rv0792c与不同疾病模式的人群的血清反应能力存在差异，特别的是，源于活动性结核和潜伏感染人群的血清，特异性识别Rv0976c、Rv1255c、Rv3160c抗原的抗体水平显著高于健康人群血清；潜伏感染人群血清中特异性结合Rv0792c的抗体水平显著高于活动性结核和健康对照组(图8)。这些结果表明，Rv0976c、Rv1255c、Rv3160c和Rv0792c是具有开发前景的诊断候选抗原，可用于活动性结核、结核潜伏感染和健康个体间的鉴别诊断。In order to evaluate whether Rv0976c, Rv1255c, Rv3160c and Rv0792c can react with specific antibodies in human serum, and then distinguish between healthy people and different disease populations, the present invention will purify Rv0976c, Rv1255c, Rv3160c and Rv0792c. The protein was subjected to an ELISA reaction with serum from different populations (active tuberculosis group, tuberculosis latent infection group, healthy control group). The results showed that Rv0976c, Rv1255c, Rv3160c and Rv0792c differed from those of different disease patterns in the serum. In particular, serum derived from active tuberculosis and latent infections specifically recognized Rv0976c, Rv1255c, and Rv3160c antigens. Antibody levels were significantly higher than those in healthy populations; the level of antibodies specifically binding to Rv0792c in the serum of latently infected populations was significantly higher than in active tuberculosis and healthy controls (Figure 8). These results indicate that Rv0976c, Rv1255c, Rv3160c and Rv0792c are promising diagnostic candidate antigens for the differential diagnosis of active tuberculosis, latent tuberculosis infection and healthy individuals.
本发明涉及Rv0976c、Rv1255c、Rv3160c和Rv0792c蛋白作为疫苗的使用。本发明中的蛋白、多肽或肽包括天然蛋白、多肽或肽，或者与天然蛋白、多肽或肽功能相同的同系物。这些同系物包括与表1中包含的天然蛋白、多肽或肽具有至少60％，70％、80％以上(较好)、最好90％以上(如95％、96％、97％、98％或99％)的氨基酸序列同源性的蛋白、多肽或肽。这些同系物包括在表1中天然蛋白、多肽或肽的氨基酸序列基础上替换、增加和缺失一个或更多(如：1-50、1-20、1-10、1-5)个氨基酸残基的蛋白、多肽或肽。这类同系物尤其包括含有保守氨基酸替换的蛋白、多肽或肽。The present invention relates to the use of the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins as vaccines. The protein, polypeptide or peptide of the present invention includes a native protein, polypeptide or peptide, or a homologue functionally identical to a native protein, polypeptide or peptide. These homologues include at least 60%, 70%, 80% or more (more preferably), and most preferably 90% or more (e.g., 95%, 96%, 97%, 98%) of the native protein, polypeptide or peptide contained in Table 1. Or 99%) a protein, polypeptide or peptide of amino acid sequence homology. These homologues include substitution, addition and deletion of one or more (eg, 1-50, 1-20, 1-10, 1-5) amino acid residues based on the amino acid sequence of the native protein, polypeptide or peptide in Table 1. a protein, polypeptide or peptide. Such homologs include, inter alia, proteins, polypeptides or peptides containing conservative amino acid substitutions.
“本发明中的核酸序列”是指编码这种多肽的核苷酸序列。本发明框架内进一步指或长或短的重叠/非重叠肽，它们与本发明中的任何一种多肽具有至少70％的氨基酸序列一致性。"Nucleic acid sequence in the present invention" means a nucleotide sequence encoding such a polypeptide. Further within the framework of the invention are overlapping or non-overlapping peptides which are either long or short and which have at least 70% amino acid sequence identity to any of the polypeptides of the invention.
在应用1中，本发明提供了上述多肽或核酸作为制备免疫原性成分(组合物)、疫苗或治疗性组合物的应用，它们能与BCG联合作为预防性疫苗，作为抗致病性分枝杆菌，如结核分枝杆菌(Mycobacterium tuberculosis)、牛型分枝杆菌(Mycobacterium bovis)、非洲分枝杆菌(Mycobacterium africanum)、麻风分枝杆菌(Mycobacterium leprae)、溃疡分枝杆菌(Mycobacterium ulcerans.)感染的加强疫苗或治疗性疫苗。本发明中的免疫原性成分、疫苗或治疗性组合物能作为预防性疫苗用于未被致病性分枝杆菌感染的人群或接种过BCG的人群，也能作为治疗性疫苗用于被致病性分枝杆菌感染的群体。In Application 1, the present invention provides the use of the above polypeptide or nucleic acid as a preparation of an immunogenic component (composition), vaccine or therapeutic composition, which can be combined with BCG as a prophylactic vaccine as an antipathogenic branching Bacteria, such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium leprae, Mycobacterium ulcerans. Enhanced vaccine or therapeutic vaccine. The immunogenic component, vaccine or therapeutic composition of the present invention can be used as a prophylactic vaccine for a population not infected with pathogenic mycobacteria or a population inoculated with BCG, and can also be used as a therapeutic vaccine. A population infected with pathogenic mycobacteria.
在应用2中，本发明提供了一类易感染的表达载体，如包含本发明中的核酸片段的牛痘、腺病毒或BCG，以及一类含有至少一个这样载体的转化细胞。In Application 2, the invention provides a class of susceptible expression vectors, such as vaccinia, adenovirus or BCG comprising a nucleic acid fragment of the invention, and a class of transformed cells comprising at least one such vector.
本发明也涉及由致病性分枝杆菌在包括人在内的动物体引起的结核病的诊断方法。用本发明中的多肽皮内注射，在结核病患病个体的注射部位会产生可检测的阳性皮肤反应，在未患结核病个体的注射部位则不会检测到皮肤反应。The present invention also relates to a method for diagnosing tuberculosis caused by pathogenic mycobacteria in an animal body including a human. Intradermal injection of the polypeptide of the present invention produces a detectable positive skin reaction at the injection site of a tuberculosis individual, and no skin reaction is detected at the injection site of the individual without tuberculosis.
在应用3中，源于包括人在内的动物体的含有单核细胞的血样本(如：T淋巴细胞)与发明中的一个多肽样本混合，阳性反应可能是T细胞增殖，或者是胞外诸如IFN-γ类细胞因子的释放。In Application 3, a monocyte-containing blood sample (eg, T lymphocytes) derived from an animal including a human is mixed with a polypeptide sample in the invention, and the positive reaction may be T cell proliferation or extracellular. Release such as IFN-γ-like cytokines.
在应用4中，一类血清样本与发明中的一个多肽样本混合，可以在曾经或正在感染的个体 中观察到血清样本中抗体与本发明中的多肽结合。In Application 4, a type of serum sample is mixed with a polypeptide sample of the invention, which may be in an individual who has been or is being infected. It was observed that the antibody in the serum sample binds to the polypeptide of the present invention.
在免疫分析中与本发明中多肽发生特异性反应的单克隆或多克隆抗体，或者上述抗体的特异性结合片段，也属于本发明保护范围。来自于包括人在内的动物的体液或潜在感染器官样本与上述抗体混合，在被感染个体中能够检测到样本和抗体的结合。A monoclonal or polyclonal antibody which specifically reacts with a polypeptide of the present invention in an immunoassay, or a specific binding fragment of the above antibody, is also within the scope of the present invention. A sample of a bodily fluid or a potentially infected organ from an animal including a human is mixed with the above antibody, and the binding of the sample and the antibody can be detected in the infected individual.
编码本发明中多肽的核酸探针也能被用作多种诊断试验，检测特定样本中病原体的存在。这种结核诊断方法可以包含至少部分核酸序列，利用PCR或成熟的杂交技术，使来自于包括人在内的动物样本与核酸片段(或全长片段)杂交，检测样本中核酸序列的存在。Nucleic acid probes encoding the polypeptides of the invention can also be used in a variety of diagnostic assays to detect the presence of pathogens in a particular sample. Such a tuberculosis diagnostic method can comprise at least a portion of a nucleic acid sequence, using a PCR or mature hybridization technique to hybridize an animal sample, including a human, to a nucleic acid fragment (or full length fragment) to detect the presence of a nucleic acid sequence in the sample.
核酸分子的多样化Diversification of nucleic acid molecules
修饰Modification
应该具有的共识是，本申请提供的DNA序列中的核苷酸分子可能被修饰。本发明包括能够直接在细菌或哺乳动物细胞表达的序列(或其片段)的核苷酸修饰。修饰包括核苷酸的置换、***或缺失，核苷酸相对位置的改变或顺序变化。There should be a consensus that the nucleotide molecules in the DNA sequences provided herein may be modified. The invention includes nucleotide modifications of sequences (or fragments thereof) that can be expressed directly in bacterial or mammalian cells. Modifications include substitutions, insertions or deletions of nucleotides, changes in the relative position of nucleotides or sequential changes.
本发明的核酸分子包括编码表1序列中保守和沉默氨基酸的核苷酸分子的变化。保守氨基酸置换的鉴定方法如文献所示：Wu,Thomas D.“Discovering Empirically Conserved Amino Acid Substitution Groups in Databases of Protein Families”(http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi？cmd＝Retrieve&db＝PubMed&list_uids＝8877523&dopt＝Abstract)。Nucleic acid molecules of the invention include variations in nucleotide molecules encoding conservative and silencing amino acids in the sequence of Table 1. Methods for the identification of conservative amino acid substitutions are shown in the literature: Wu, Thomas D. "Discovering Empirically Conserved Amino Acid Substitution Groups in Databases of Protein Families" (http://www.ncbi.nlm.nih.gov:80/entrez/query .fcgi?cmd=Retrieve&db=PubMed&list_uids=8877523&dopt=Abstract).
本发明的核酸分子包括编码导致表1序列中非保守氨基酸的置换、添加或缺失的序列。包括使表1氨基酸中非保守氨基酸序列改变的对应功能的核酸分子(DNA和RNA)，它们编码的氨基酸序列具有非保守氨基酸置换(尤其是化学近似物的置换)、添加、或缺失，但也保留与表1相同或相似的氨基酸序列。这些DNA或RNA可能编码表1所示氨基酸序列的片段或变异体。Nucleic acid molecules of the invention include sequences encoding substitutions, additions or deletions that result in non-conservative amino acids in the sequence of Table 1. Nucleic acid molecules (DNA and RNA) comprising corresponding functions that alter the non-conservative amino acid sequence in the amino acids of Table 1, which encode amino acid sequences with non-conservative amino acid substitutions (especially substitutions, additions, or deletions of chemical approximations, but also The amino acid sequence identical or similar to Table 1 is retained. These DNA or RNA may encode fragments or variants of the amino acid sequence shown in Table 1.
片段可以作为免疫原和免疫原性组合物。这样的片段或变异体能通过下述方法鉴定。Fragments can be used as immunogens and immunogenic compositions. Such fragments or variants can be identified by the methods described below.
序列一致性Sequence consistency
本发明中所涉及的核酸分子(或其片段)，与申请书中列出的核酸分子序列具有至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％，或者，最好99％或99.5％或更高的一致性，能够在细菌或哺乳动物细胞系中表达。一致性是指两个核苷酸序列的相似性，序列对齐之后能够匹配的最高值。一致性根据现有成熟方法计算。例如，如果一段核苷酸序列(称之为序列A)与SEQ ID No.5的参照片段具有90％一致性，则除了在每100个SEQ ID No.5的参照核苷酸序列中，序列A存在10个点突变(例如和其他核苷酸的置换)外，其余序列完全一致。The nucleic acid molecule (or a fragment thereof) referred to in the present invention has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least at least the nucleic acid molecule sequence set forth in the application. A consistency of 97%, at least 98%, or, preferably, 99% or 99.5% or greater, can be expressed in bacterial or mammalian cell lines. Consistency refers to the similarity of two nucleotide sequences, the highest value that can be matched after sequence alignment. Consistency is calculated according to existing mature methods. For example, if a stretch of nucleotide sequence (referred to as sequence A) is 90% identical to the reference fragment of SEQ ID No. 5, then in addition to the reference nucleotide sequence of every 100 SEQ ID No. 5, the sequence Except for 10 point mutations (eg, substitutions with other nucleotides), the remaining sequences are identical.
序列一致性(每个结构都不含有编码核苷酸的***)被优先设置成：序列与发明中提供的SEQ ID No.5或其互补序列具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％，或者，最好99％或99.5％一致性。序列一致性首先用生物信息学中GCG程序 进行计算(Wisconsin大学)。其他的程序也可以用于一致性计算，例如Clustal W程序(较好地用于缺省参数；Thompson,JD et al.,Nucleic Acid Res.22:4673-4680)，BLAST P，BLAST X算法，基因组研究所的鸟分枝杆菌BLASTN(http:tigrblast.tigr.org/)，Wellcome Trust Sanger研究所(http://www.sanger.ac.uk/Projects/Microbes/)的牛型分枝杆菌、牛型分枝杆菌BCG(Pastuer)、海分枝杆菌、麻风分枝杆菌、结核分枝杆菌BLASTN，Pasterur研究所(Tuberculist)(http://genolist.pasteur.fr/TubercuList/)的结核分枝杆菌BLASTN研究，Pasterur研究所(Leproma)(http://genolist.pasteur.fr/Leproma/)的麻风分枝杆菌研究，Minnesota大学(http://www.cbc.umn.edu/ResearchProjects/Ptb/和http://www.cbc.umn.edu/ResearchProjects/AGAC/Mptb/Mptbhome.html)微生物基因组计划的副结核分枝杆菌BLASTN，在美国NCBI USA-(http://www.ncbi.nlm.nih.gov/BLAST/)的各种BLAST研究，以及GenomeNet(生物信息学中心-化学研究所)(http://blast.genome.ad.jp/)的各种BLAST研究。Sequence identity (an insertion of each of the constructs that does not contain a coding nucleotide) is preferentially set such that the sequence has at least 70%, at least 80%, at least 90%, of SEQ ID No. 5 or its complement provided in the invention, At least 95%, at least 96%, at least 97%, at least 98%, or, preferably, 99% or 99.5% identity. Sequence consistency first uses the GCG program in bioinformatics Perform calculations (Wisconsin University). Other programs can also be used for consistent calculations, such as the Clustal W program (better for default parameters; Thompson, JD et al., Nucleic Acid Res. 22: 4673-4680), BLAST P, BLAST X algorithm, Mycobacterium tuberculosis BLASTN (http:tigrblast.tigr.org/) from the Genomics Institute, Mycobacterium bovis, at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/Projects/Microbes/), Tuberculosis of Mycobacterium bovis BCG (Pastuer), Mycobacterium marinum, Mycobacterium leprae, Mycobacterium tuberculosis BLASTN, Pasterculist (http://genolist.pasteur.fr/TubercuList/) BLASTN study by the Pasteur BLASTN study, Leproma (http://genolist.pasteur.fr/Leproma/), University of Minnesota (http://www.cbc.umn.edu/ResearchProjects/Ptb/ And http://www.cbc.umn.edu/ResearchProjects/AGAC/Mptb/Mptbhome.html) Microbial Genome Project of M. tuberculosis BLASTN, NCBI USA-(http://www.ncbi.nlm. Various BLAST studies by nih.gov/BLAST/), and GenomeNet (Bioinformatics Center - Institute of Chemistry) (http://blast.genome.ad.jp/) Research various BLAST.
由于遗传密码子的简并性，表2中所给出的核酸序列不仅仅是编码表1中多肽的唯一序列。本发明包含与表2中核酸分子具有相同基本遗传信息的核酸分子。与申请书中的核酸序列相比出现1个或更多个核苷酸变化(包括RNA)，但产生了与表1中相同的多肽产物的核酸分子也在本发明保护范围之内。Due to the degeneracy of the genetic code, the nucleic acid sequences given in Table 2 are not only the only sequences encoding the polypeptides in Table 1. The invention encompasses nucleic acid molecules having the same basic genetic information as the nucleic acid molecules of Table 2. Nucleic acid molecules which produce one or more nucleotide changes (including RNA) as compared to the nucleic acid sequences in the application, but which produce the same polypeptide product as in Table 1, are also within the scope of the present invention.
其他能够用常规的DNA-DNA或DNA-RNA杂交技术检测出来的，编码上述多肽的核苷酸功能等价物也在本发明保护范围之内。Other nucleotide functional equivalents encoding the above polypeptides which can be detected by conventional DNA-DNA or DNA-RNA hybridization techniques are also within the scope of the present invention.
杂交Hybridization
本发明涉及的DNA与申请中提供的核酸分子具有足够的序列一致性，在严格的杂交条件(用低盐洗涤液：约0.2％SSC，50-65℃反应温度)下能够实现杂交(本领域常用的杂交技术)。本发明也包含能与表2中的序列或其互补序列中的一个或多个发生杂交的核酸分子。这种核酸分子在高度严格条件下实现杂交(Sambrook et al.Molecular Cloning:A Laboratory Manual,Most Recent Edition,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.)。最好用低盐洗涤液(约0.2％SSC)，约50-65℃反应温度。The DNA of the present invention has sufficient sequence identity with the nucleic acid molecule provided in the application, and hybridization can be achieved under strict hybridization conditions (low salt washing solution: about 0.2% SSC, reaction temperature of 50-65 ° C). Common hybridization techniques). The invention also encompasses nucleic acid molecules that are capable of hybridizing to one or more of the sequences in Table 2 or its complements. This nucleic acid molecule achieves hybridization under highly stringent conditions (Sambrook et al. Molecular Cloning: A Laboratory Manual, Most Recent Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). It is preferred to use a low salt wash (about 0.2% SSC) at a reaction temperature of about 50-65 °C.
疫苗vaccine
疫苗的制备众所周知。很典型的是，可注射的液体溶液或混悬液；易于在注射前溶解或悬浮在液体中的固态形式，可能通过乳化、蛋白脂质体化制备。免疫原性组合物常常和可药用的、与活性组合物兼容的辅料混合在一起。合适的辅料包括：水、生理盐水、葡萄糖、甘油、乙醇或其混合物。另外，如果需要，疫苗可以含有其它微量辅料，如浸润剂或乳化剂、pH缓冲液和/或增强疫苗效果的佐剂。佐剂的有效成分可能包括，但不限于：氢氧化铝，N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP)，N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine(CGP 11637，被称为nor-MDP)，N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A，被称为MTP-PE)和RIBI。RIBI是将3种细菌抽提组分：单磷酰脂质A、海藻糖二霉菌酸酯和细胞壁骨架(MPL+TDM+CWS)混合在2％三十碳六烯/Tween 80TM乳液中制成的佐剂。The preparation of vaccines is well known. Typically, injectable liquid solutions or suspensions; solid forms which are readily dissolved or suspended in the liquid prior to injection, may be prepared by emulsifying, proteolipidization. The immunogenic composition is often mixed with a pharmaceutically acceptable excipient that is compatible with the active composition. Suitable excipients include: water, physiological saline, dextrose, glycerol, ethanol or mixtures thereof. In addition, the vaccine may contain other minor excipients, such as infiltrants or emulsifiers, pH buffers, and/or adjuvants that enhance the effectiveness of the vaccine, if desired. The active ingredients of the adjuvant may include, but are not limited to, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine ( CGP 19835A, known as MTP-PE) and RIBI. RIBI is extracted three kinds of bacterial components: monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in a mixture of 2% squalene / Tween 80 TM manufactured by emulsion An adjuvant.
佐剂的效率通过免疫原性多肽直接诱导的抗体水平来评价，这种多肽含有结核分枝杆菌的免疫原性序列，与不同佐剂混合后免疫接种。通常疫苗的接种方式是皮下或肌肉注射，也有栓剂或口服配方。就栓剂而言，传统的粘合剂或载体包括诸如聚乙二醇、甘油三酯，这类栓剂是含有0.5％到10％(1％到2％较好)活性成分的混合物。口服配方包括正常使用的辅料，象药用级别的甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁之类。这些成分与10％-95％(25％-70％较好)活性组分构成溶液、悬浊液、片剂、药丸、胶囊，缓释剂或粉剂等形式。The efficiency of the adjuvant is assessed by the level of antibody directly induced by the immunogenic polypeptide containing the immunogenic sequence of M. tuberculosis, immunized with a mixture of different adjuvants. Usually the vaccine is vaccinated either subcutaneously or intramuscularly, as well as suppositories or oral formulations. In the case of suppositories, conventional binders or carriers include, for example, polyethylene glycol, triglycerides, and such suppositories are mixtures containing from 0.5% to 10% (1% to 2% by weight) of the active ingredient. Oral formulations include excipients that are normally used, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These ingredients are in the form of solutions, suspensions, tablets, pills, capsules, sustained release agents or powders with from 10% to 95% (25% to 70% by weight) of the active ingredient.
根据预防性和/或治疗性疫苗的使用目的，给予适当的疫苗免疫剂量。Appropriate vaccine immunization doses are administered depending on the purpose of the prophylactic and/or therapeutic vaccine.
疫苗可以以单剂量方案给予，或优选以多剂量方案给予。多剂量方案是这样的方案，其中初次接种疫苗过程可用在1-10个单独的剂量，接着在维持和或加强免疫应答所需的随后时间间隔给予其他剂量，例如在1-4个月给予第二剂量，如果需要，几个月后给予随后的剂量。给药方案至少部分由个体决定，并且有赖于从业人员的判断。The vaccine can be administered in a single dose regimen, or preferably in a multiple dose regimen. A multi-dose regimen is one in which the initial vaccination procedure can be used in 1-10 separate doses, followed by administration of other doses at subsequent intervals required to maintain and or boost the immune response, for example, at 1-4 months. Two doses, if needed, are given a subsequent dose a few months later. The dosage regimen is at least partially determined by the individual and depends on the judgment of the practitioner.
另外，疫苗可以与其他的诸如免疫球蛋白类免疫调节剂协同使用，本发明的一个从属部分也是一个由本疫苗与其他疫苗，尤其是BCG或重组BCG混合组成的多价疫苗配方。Alternatively, the vaccine may be used in conjunction with other immunoglobulin-like immunomodulators, and a sub-part of the invention is also a multivalent vaccine formulation consisting of a mixture of the vaccine and other vaccines, particularly BCG or recombinant BCG.
治疗性成分(药物组合物)Therapeutic ingredients (pharmaceutical compositions)
本发明中的治疗性成分(药物组合物)用于哺乳动物抗结核分枝杆菌、牛型分枝杆菌、非洲分枝杆菌、麻风分枝杆菌或溃疡分枝杆菌的治疗或预防，也用于治疗退行性疾病，异常生理状态，如恶性肿瘤。The therapeutic component (pharmaceutical composition) of the present invention is used for the treatment or prevention of a mammal against Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium leprae or Mycobacterium ulcerans, and is also used for Treatment of degenerative diseases, abnormal physiological conditions, such as malignant tumors.
治疗性组合物可以以片剂、喷雾、气管灌注或静脉注射方式用于人类或其他动物。Therapeutic compositions can be administered to humans or other animals in the form of tablets, sprays, tracheal perfusions or intravenous injections.
下面结合具体实例，进一步阐述本发明。应理解，这些实例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，所有的出版物(包括GenBank条目)，专利和专利申请、本领域技术人员对本发明各种改动或修改，这些等价形式同样落于本申请所请求保护范围。例如，本申请所指的蛋白，涵盖了常用的肽或多肽；同样，本申请涉及的基因，也涵盖常用的核苷酸或基因片段。The invention is further illustrated below in conjunction with specific examples. It is to be understood that the examples are only illustrative of the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the teachings of the present invention, all publications (including GenBank entries), patents and patent applications, and various alterations or modifications of the invention are apparent to those skilled in the art. The scope of protection requested. For example, the protein referred to in the present application encompasses commonly used peptides or polypeptides; likewise, the genes involved in the present application also cover commonly used nucleotides or gene fragments.
实施例Example
实施例1：选定抗原的克隆、表达和纯化Example 1: Cloning, expression and purification of selected antigens
本发明选定的抗原蛋白Rv0976c、Rv1255c、Rv3160c和Rv0792c的克隆、表达和纯化方案如图1所示。Rv0976c的开放阅读框通过使用上游PCR引物5’-TAGGATCCGTGCGTATCGGAAACTGCTCG-3’(SEQ ID No.15)和下游PCR引物5’-TAGAAGCTTTCACAAC AGGGTCTCCGG GATCT-3’(SEQ ID No.16)扩增。Rv1255c、Rv3160c和Rv0792c的PCR扩增引物分别是：5’-CATGGATCCATGGCGGGTACCGACTGGCTG-3’(上游，Rv1255c，SEQ ID No.17)，5’-CTGAAGCTTTCACTCGGGTCCAGGGTGAC-3’(下游，Rv1255c，SEQ ID No.18)；5’-CGTGGATCCATGCCGAGGCAGGCCGGCCGCTG-3’(上游，Rv3160c，SEQ ID No.19)，5’-GCAAAGCTTCTAGAGCCCGCGGTCGGGGGGTGCG-3’(下游，Rv3160c，SEQ ID No.20)；5’-TATGGATCCATGACATCTGTCAAGCTGGACC-3’(上游，Rv0792c，SEQ ID No.21)，5’-GCGAAGCTTTCATGCGAAATCTCGTTTCTCG-3’(下游，Rv0792c，SEQ ID No.22)。对照抗原Ag85A(Rv3804c)、PPE18(Rv1196)和HspX(Rv2031c)按同样的方法进行克隆、表达和纯化，所用的PCR引物分别为：5’-ATAATACTTAAGGCCGCCACCATGCAGCTTGTTGACAGGGTTCGTGGCGCC-3’(上游，Ag85A，SEQ ID No.23)，5’-ATAATTCTAGATCAATGGTGATGGTGATGGGCGCCCTGGGGCGCGGGCCCGGT-3’(下游，Ag85A，SEQ ID No.24)；5’-ATAATACTTAAGGCCGCCACCATGGTGGATTTCGGGGCGTTACCACCGGAG-3’(上游，PPE18，SEQ ID No.25)，5’-ATAATTCTAGATCAAT GGTGATGGTGATGGCCGGCCGCCGGAGAATGCGG-3’(下游，PPE18，SEQ ID No.26)，5’-ATATACTTAAGGCCGCCACCATGGCCACCACCCTTCCCGTTC-3’(上游，HspX，SEQ ID No.27)，5’-AATATTCTAGATCAATGGTGATGGTGATGATGGTTGG TGGACCGGATCTGAATGTGCTT-3’(下游，HspX，SEQ ID No.28)。The cloning, expression and purification schemes of the selected antigenic proteins Rv0976c, Rv1255c, Rv3160c and Rv0792c of the present invention are shown in FIG. The open reading frame of Rv0976c by using the upstream PCR primer 5'-TAGGATCCGTGCGTATCGGAAACTGCTCG-3' (SEQ ID No. 15) and the downstream PCR primer 5'-TAGAAGCTTTCACAAC AGGGTCTCCGG GATCT-3' (SEQ ID No. 16) was amplified. The PCR amplification primers for Rv1255c, Rv3160c and Rv0792c are: 5'-CATGGATCCATGGCGGGTACCGACTGGCTG-3' (upstream, Rv1255c, SEQ ID No. 17), 5'-CTGAAGCTTTCACTCGGGTCCAGGGTGAC-3' (downstream, Rv1255c, SEQ ID No. 18) ; 5'-CGTGGATCCATGCCGAGGCAGGCCGGCCGCTG-3' (upstream, Rv3160c, SEQ ID No. 19), 5'-GCAAAGCTTCTAGAGCCCGCGGTCGGGGGGTGCG-3' (downstream, Rv3160c, SEQ ID No. 20); 5'-TATGGATCCATGACATCTGTCAAGCTGGACC-3' (upstream, Rv0792c , SEQ ID No. 21), 5'-GCGAAGCTTTCATGCGAAATCTCGTTTCTCG-3' (downstream, Rv0792c, SEQ ID No. 22). The control antigens Ag85A (Rv3804c), PPE18 (Rv1196) and HspX (Rv2031c) were cloned, expressed and purified in the same manner. The PCR primers used were: 5'-ATAATACTTAAGGCCGCCACCATGCAGCTTGTTGACAGGGTTCGTGGCGCC-3' (upstream, Ag85A, SEQ ID No.). 23), 5'-ATAATTCTAGATCAATGGTGATGGTGATGGGCGCCCTGGGGCGCGGGCCCGGT-3' (downstream, Ag85A, SEQ ID No. 24); 5'-ATAATACTTAAGGCCGCCACCATGGTGGATTTCGGGGCGTTACCACCGGAG-3' (upstream, PPE18, SEQ ID No. 25), 5'-ATAATTCTAGATCAAT GGTGATGGTGATGGCCGGCCGCCGGAGAATGCGG-3' ( Downstream, PPE18, SEQ ID No. 26), 5'-ATATACTTAAGGCCGCCACCATGGCCACCACCCTTCCCGTTC-3' (upstream, HspX, SEQ ID No. 27), 5'-AATATTCTAGATCAATGGTGATGGTGATGATGGTTGG TGGACCGGATCTGAATGTGCTT-3' (downstream, HspX, SEQ ID No. 28).
以结核分枝杆菌H37Rv(ATCC93009)基因组DNA为模板进行PCR反应(50μl体系)，其中含有模板DNA(10ng)、上下游引物各0.5μM、0.2mM dNTPs、1×反应缓冲液、1.25单位PrimeSTAR HS DNA多聚酶(Clontech)。循环条件：95℃变性5min；30个循环的变性(98℃，10sec)、退火(65℃，20sec)、延伸(72℃，2min)；最后72℃延伸5min，4℃冷却。PCR扩增产物经琼脂糖凝胶电泳后使用凝胶纯化试剂盒(Qiagen)纯化。37℃下用BamHI和HindIII酶切PCR纯化产物3h，酶切后的目的片段用凝胶纯化试剂盒(Qiagen)纯化。pET28a质粒(Novagen)用同样条件酶切回收。连接反应体系(共10μl)包括：2μl PCR片段，2μl pET28a片段，1μl 10×T4连接酶缓冲液，1μl DNA T4连接酶(NEB)。室温下连接3h，65℃下孵育20min终止反应。质粒-基因片段连接产物pET28a-Rv0976c、pET28a-Rv1255c、pET28a-Rv3160c和pET28a-Rv0792c分别转化进入E.coli DH5α。连接反应液与E.coli DH5α感受态细胞混合，涂布在含有卡那霉素(50μg/ml)的LB平板上，37℃过夜培养，随机挑选单克隆接种入LB液体培养基。用Qiagen Miniprep试剂盒从E.coli DH5α菌体中抽提重组质粒pET28a-Rv0976c、pET28a-Rv1255c、pET28a-Rv3160c和pET28a-Rv0792c，DNA测序验证***序列正确。PCR reaction using M. tuberculosis H37Rv (ATCC93009) genomic DNA as a template (50 μl system) containing template DNA (10 ng), upstream and downstream primers 0.5 μM each, 0.2 mM dNTPs, 1× reaction buffer, 1.25 units of PrimeSTAR HS DNA polymerase (Clontech). Cycling conditions: denaturation at 95 ° C for 5 min; denaturation at 30 cycles (98 ° C, 10 sec), annealing (65 ° C, 20 sec), extension (72 ° C, 2 min); last 72 ° C extension for 5 min, 4 ° C cooling. The PCR amplification product was subjected to agarose gel electrophoresis and purified using a gel purification kit (Qiagen). The product was purified by digestion with BamHI and HindIII at 37 ° C for 3 h, and the digested target fragment was purified using a gel purification kit (Qiagen). The pET28a plasmid (Novagen) was recovered by restriction enzyme digestion under the same conditions. The ligation reaction system (total 10 μl) included: 2 μl of PCR fragment, 2 μl of pET28a fragment, 1 μl of 10×T4 ligase buffer, and 1 μl of DNA T4 ligase (NEB). The reaction was stopped by ligation at room temperature for 3 h and incubation at 65 ° C for 20 min. The plasmid-gene fragment ligation products pET28a-Rv0976c, pET28a-Rv1255c, pET28a-Rv3160c and pET28a-Rv0792c were transformed into E. coli DH5α, respectively. The ligation reaction mixture was mixed with E. coli DH5α competent cells, plated on LB plates containing kanamycin (50 μg/ml), cultured at 37 ° C overnight, and randomly selected for inoculation into LB liquid medium. Recombinant plasmids pET28a-Rv0976c, pET28a-Rv1255c, pET28a-Rv3160c and pET28a-Rv0792c were extracted from E. coli DH5α cells using Qiagen Miniprep kit, and the insertion sequence was verified by DNA sequencing.
为了获得重组蛋白，将重组质粒pET28a-Rv0976c、pET28a-Rv1255c、pET28a-Rv3160c和pET28a-Rv0792c分别转化入E.coli BL21，涂布在含有卡那霉素(50μg/ml)的LB平板上，37℃过夜培养，随机挑选单克隆接种入LB液体培养基，扩培至1L。26℃培养3h后加入1mM IPTG， 26℃过夜诱导培养。4℃下12,000rpm离心10min收集菌体，重悬于BugBuster(Novagen)蛋白抽提液中。To obtain the recombinant protein, the recombinant plasmids pET28a-Rv0976c, pET28a-Rv1255c, pET28a-Rv3160c and pET28a-Rv0792c were separately transformed into E. coli BL21 and plated on LB plates containing kanamycin (50 μg/ml), 37 Incubate overnight at °C, randomly select the monoclonal inoculation into LB liquid medium, and expand to 1L. After incubation at 26 ° C for 3 h, 1 mM IPTG was added. The culture was induced overnight at 26 °C. The cells were collected by centrifugation at 12,000 rpm for 10 min at 4 ° C and resuspended in BugBuster (Novagen) protein extract.
为了纯化重组蛋白，上述E.coli裂解液在4℃下12,000rpm离心20min收集上清。吸取上清和Ni-NTA His·
Figure PCTCN2015091068-appb-000007
Resin介质(Novagen)加入到空色谱柱中，按照操作手册(Novagen公司，Ni-NTA His·
Figure PCTCN2015091068-appb-000008
Resin介质使用手册)的流程纯化蛋白。蛋白纯度经聚丙烯酰胺凝胶电泳判断，浓度用BCA实验(Sigma)确定。Rv0976c的表达纯化如图2所示，Rv1255c、Rv3160c和Rv0792c用同样方法纯化。经鉴定，所得的纯化蛋白的序列正确。To purify the recombinant protein, the above E. coli lysate was centrifuged at 12,000 rpm for 20 min at 4 ° C to collect the supernatant. Aspirate supernatant and Ni-NTA His·
Figure PCTCN2015091068-appb-000007
Resin media (Novagen) is added to the empty column, according to the operating manual (Novagen, Ni-NTA His·
Figure PCTCN2015091068-appb-000008
Resin Media User Manual) Process Purification of Proteins. Protein purity was determined by polyacrylamide gel electrophoresis and the concentration was determined by BCA assay (Sigma). Expression purification of Rv0976c is shown in Figure 2, and Rv1255c, Rv3160c and Rv0792c were purified in the same manner. The sequence of the purified protein obtained was identified to be correct.
实施例2、Rv0976c、Rv1255c、Rv3160c和Rv0792c蛋白的免疫原性Example 2. Immunogenicity of Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins
将50μl实施例1的纯化蛋白(10μg)和50μl Freund’s不完全佐剂(Sigma)充分混合，皮下注射免疫C57BL/6小鼠(上海斯莱克公司，SPF级，6-8周雌性)(每组4只)。隔周免疫一次，共免疫3次以评价这些抗原的免疫原性。首次免疫8周后处死小鼠，分离脾脏。获得脾脏淋巴细胞后，将1×106个淋巴细胞加入至24孔细胞培养板中，分别用Rv0976c、Rv1255c、Rv3160c和Rv0792c抗原刺激，刺激浓度分别为5μg/ml、10μg/ml；以PBS代替抗原作为无抗原阴性对照。37℃、5％CO2、100％饱和湿度下培养60小时。50 μl of the purified protein of Example 1 (10 μg) and 50 μl of Freund's incomplete adjuvant (Sigma) were thoroughly mixed, and subcutaneously injected with C57BL/6 mice (Shanghai Slack, SPF grade, 6-8 weeks female) (each group) 4)). Immunization was performed once every other week for 3 times to evaluate the immunogenicity of these antigens. After 8 weeks of the first immunization, the mice were sacrificed and the spleens were isolated. After obtaining spleen lymphocytes, 1×10 6 lymphocytes were added to 24-well cell culture plates and stimulated with Rv0976c, Rv1255c, Rv3160c and Rv0792c antigens respectively, and the stimulation concentrations were 5 μg/ml and 10 μg/ml, respectively; The antigen served as an antigen-free negative control. Incubate for 60 hours at 37 ° C, 5% CO 2 , 100% saturated humidity.
离心收集细胞培养上清，检测Th1型细胞因子IFN-γ、TNF-α和IL-2释放水平，使用OptEIATM预包被ELISA试剂盒(BD Biosciences)，按照试剂盒推荐的程序操作。纯化的Ag85A作为参照，不同抗原细胞因子表达结果根据Ag85A结果做均一化处理。结果显示Rv0976c、Rv1255c、Rv3160c和Rv0792c蛋白具有高度免疫原性，能够诱导接近于或强于Ag85A的Th1型细胞因子分泌水平(图3)。The cell culture supernatant was collected by centrifugation, and the levels of Th1 type cytokines IFN-γ, TNF-α, and IL-2 were measured, and the OptEIATM pre-coated ELISA kit (BD Biosciences) was used, and the procedure recommended by the kit was followed. Purified Ag85A was used as a reference, and the expression results of different antigenic cytokines were homogenized according to the results of Ag85A. The results showed that the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins were highly immunogenic and capable of inducing Th1 type cytokine secretion levels close to or stronger than Ag85A (Fig. 3).
为了评价B细胞反应，收集小鼠血清，用ELISA法检测抗原特异性抗体水平。血清经系列稀释后(1:400到1:51200)加入96孔板，孔中预包被Rv0976c、Rv1255c、Rv3160c和Rv0792c蛋白。抗体亚型的ELISA检测使用HRP标记的抗鼠IgG1(优宁维公司，sc2969)或IgG2c(优宁维公司，ab97255)二抗。结果显示Rv0976c、Rv1255c、Rv3160c和Rv0792c蛋白在免疫小鼠体内诱导出高水平的抗原特异性抗体，显示出强B细胞反应能力(图4)。To evaluate the B cell response, mouse sera were collected and antigen-specific antibody levels were measured by ELISA. The serum was serially diluted (1:400 to 1:51200) and added to a 96-well plate pre-coated with Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins. ELISA assays for antibody subtypes were performed using HRP-labeled anti-mouse IgG1 (Youinwei, sc2969) or IgG2c (Youinwei, ab97255) secondary antibody. The results showed that the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins induced high levels of antigen-specific antibodies in the immunized mice, showing strong B cell reactivity (Fig. 4).
实施例3、Rv0976c、Rv1255c、Rv3160c和Rv0792c的保护效力Example 3, Protective efficacy of Rv0976c, Rv1255c, Rv3160c and Rv0792c
1、候选基因克隆进入哺乳动物表达载体1. Candidate gene clones enter mammalian expression vector
将Rv0976c、Rv1255c、Rv3160c和Rv0792c的编码基因克隆至哺乳动物表达载体pVAX1或pcDNA3.1的方案如图5所示。Rv0792c克隆进入pVAX1的引物是：5’-TAGAATTCGCCACCATGGGCATGCGTATCGGAAACTG-3’(上游，SEQ ID No.29)和5’-TAACTGCAGCTAGTGATGGTGATGGTGATGCAACAGGGTCTCCG-3’(下游，SEQ ID No.30)。PCR的体系与反应条件同实施例1。将PCR产物用EcoRI和PstI酶切消化，连接入同样 酶切消化的pVAX1质粒(Invitrogen公司，货号：V260-20)产生pVAX1-Rv0976c。以同样的方式构建质粒pVAX-Ag85A和pVAX-PPE18。The scheme for cloning the gene encoding Rv0976c, Rv1255c, Rv3160c and Rv0792c into the mammalian expression vector pVAX1 or pcDNA3.1 is shown in FIG. The primers for the Rv0792c clone into pVAX1 were: 5'-TAGAATTCGCCACCATGGGCATGCGTATCGGAAACTG-3' (upstream, SEQ ID No. 29) and 5'-TAACTGCAGCTAGTGATGGTGATGGTGATGCAACAGGGTCTCCG-3' (downstream, SEQ ID No. 30). The system and reaction conditions of the PCR were the same as in Example 1. The PCR product was digested with EcoRI and PstI, and ligated into the same The digested pVAX1 plasmid (Invitrogen, Cat. No. V260-20) produced pVAX1-Rv0976c. Plasmids pVAX-Ag85A and pVAX-PPE18 were constructed in the same manner.
将Rv1255c、Rv3160c和Rv0792c克隆进入pcDNA3.1，对应的PCR引物是：5’-ATATACTTAAGGCCGCCACCATGGCGGGTACCGACTGGCTGTC-3’(上游，Rv1255c，SEQ ID No.31)，5’-AATATTCTAGATCAATGGTGATGGTGATGATGCTCGGGTCCAGGGTGACCGGC-3’(下游，Rv1255c，SEQ ID No.32)；5’-ATATACTTAAGGCCGCCACCATGCCGAGGCAGGCCGGCCG-3’(上游，Rv3160c，SEQ ID No.33)，5’-AATATCTCGAGTCAATGGTGATGGTGATGATGGAGCCCGCGGTCGGGGGGTG-3’(下游，Rv3160c，SEQ ID No.34)；5’-ATATACTTAAGGCCGCCACCATGACATCTGTCAAGCTGGACCTGGAC-3’(上游，Rv0792c，SEQ ID No.35)，5’-AATATTCTAGATCAATGGTGATGGTGATGATGTGCGAAATCTCGTTTCTCGATAATTCCGGC-3’(下游，Rv0792c，SEQ ID No.36)。PCR体系和反应条件同实施例1。将PCR产物用AflII和XbaI酶切消化，连接入同样酶切消化的pcDNA3.1质粒(Invitrogen公司，货号：V790-20)，产生pcDNA-Rv1255c、pcDNA-Rv3160c和pcDNA-Rv0792c。以同样的方式构建质粒pcDNA-Ag85A和pcDNA-HspX。Rv1255c, Rv3160c and Rv0792c were cloned into pcDNA3.1, and the corresponding PCR primers were: 5'-ATATACTTAAGGCCGCCACCATGGCGGGTACCGACTGGCTGTC-3' (upstream, Rv1255c, SEQ ID No. 31), 5'-AATATTCTAGATCAATGGTGATGGTGATGATGCTCGGGTCCAGGGTGACCGGC-3' (downstream, Rv1255c, SEQ ID No. 32); 5'-ATATACTTAAGGCCGCCACCATGCCGAGGCAGGCCGGCCG-3' (upstream, Rv3160c, SEQ ID No. 33), 5'-AATATCTCGAGTCAATGGTGATGGTGATGATGGAGCCCGCGGTCGGGGGGTG-3' (downstream, Rv3160c, SEQ ID No. 34); 5'-ATATACTTAAGGCCGCCACCATGACATCTGTCAAGCTGGACCTGGAC-3 '(Upstream, Rv0792c, SEQ ID No. 35), 5'-AATATTCTAGATCAATGGTGATGGTGATGATGTGCGAAATCTCGTTTCTCGATAATTCCGGC-3' (downstream, Rv0792c, SEQ ID No. 36). The PCR system and reaction conditions were the same as in Example 1. The PCR product was digested with AflII and XbaI, and ligated into the same digested pcDNA3.1 plasmid (Invitrogen, Cat. No. V790-20) to produce pcDNA-Rv1255c, pcDNA-Rv3160c and pcDNA-Rv0792c. The plasmids pcDNA-Ag85A and pcDNA-HspX were constructed in the same manner.
2、Rv0976c的保护效力2. Protection effectiveness of Rv0976c
用100μg/鼠的pVAX-Rv0796c、pVAX-Ag85A或pVAX-PPE18，隔周免疫BALB/c小鼠(华阜康公司，SPF级，6-8周雌性，每组6只)，共免疫3次。第一次免疫8周后，以6×105CFU/鼠的剂量尾静脉感染结核分枝杆菌H37Rv菌株(购自中国微生物菌种保藏中心，ATCC93009，上海市肺科医院保存、培养)。感染5周后，处死小鼠，脏器匀浆液经系列稀释后，涂布7H11琼脂平板(添加OADC，即Middlebrook OADC，混合营养添加剂，10％的比例添加)，计数肺、脾脏器荷菌数量。结果显示Rv0976c的抗结核分枝杆菌保护效果强于Ag85A，与PPE18接近：接种Rv0976c DNA疫苗的小鼠在感染结核分枝杆菌5周后的肺、脾脏器中结核分枝杆菌数量明显低于阴性对照(pVAX)或者接种Ag85ADNA疫苗的小鼠中结核分枝杆菌数量，与接种PPE18DNA疫苗的小鼠中结核分枝杆菌数量相近(图6)。BALB/c mice (Hua Fukang, SPF grade, 6-8 weeks female, 6 rats per group) were immunized every other week with 100 μg/mouse of pVAX-Rv0796c, pVAX-Ag85A or pVAX-PPE18. . Eight weeks after the first immunization, M. tuberculosis H37Rv strain (purchased from the China Microbial Culture Collection, ATCC93009, Shanghai Pulmonary Hospital, cultured and cultured) was injected into the tail vein at a dose of 6×10 5 CFU/mouse. After 5 weeks of infection, the mice were sacrificed, and the organ homogenate was serially diluted, and then coated with 7H11 agar plates (adding OADC, namely Middlebrook OADC, mixed nutrient additive, 10% ratio), and counting the number of lung and spleen bacteria. . The results showed that the protective effect of Rv0976c against M. tuberculosis was stronger than that of Ag85A, which was close to that of PPE18: the number of M. tuberculosis in the lung and spleen of mice infected with Rv0976c DNA vaccine was significantly lower than that after 5 weeks of infection with M. tuberculosis. The number of M. tuberculosis in the control (pVAX) or mice inoculated with the Ag85A DNA vaccine was similar to the number of M. tuberculosis in the mice inoculated with the PPE18 DNA vaccine (Fig. 6).
3、Rv1255c、Rv3160c和Rv0792c的保护效力。3. The protective efficacy of Rv1255c, Rv3160c and Rv0792c.
用100μg/鼠的pcDNA-Rv1255c、pcDNA-Rv3160c、pcDNA-Rv0792c、pcDNA-Ag85A或pcDNA-HspX，隔周免疫BALB/c小鼠(华阜康公司，SPF级，6-8周雌性，每组6只)，共免疫3次。第一次免疫8周后，以气溶胶形式感染结核分枝杆菌H37Rv菌株(100CFU/肺)。感染9周后，处死小鼠，脏器匀浆液经系列稀释后，涂布7H11琼脂平板(添加OADC，同上)，计数肺、脾脏器荷菌数量。结果证明Rv1255c、Rv3160c和Rv0792c具有与Ag85A或HspX相同或接近的保护效果(图7)。 BALB/c mice were immunized every week with 100 μg/mouse of pcDNA-Rv1255c, pcDNA-Rv3160c, pcDNA-Rv0792c, pcDNA-Ag85A or pcDNA-HspX (Hua Fukang, SPF grade, 6-8 weeks female, each group) 6), a total of 3 immunizations. After 8 weeks of the first immunization, M. tuberculosis H37Rv strain (100 CFU/lung) was infected as an aerosol. After 9 weeks of infection, the mice were sacrificed, and the organ homogenate was serially diluted, and then coated on a 7H11 agar plate (adding OADC, ibid.), and the number of lung and spleen organs was counted. The results confirmed that Rv1255c, Rv3160c and Rv0792c have the same or similar protective effects as Ag85A or HspX (Fig. 7).
实施例4、用选定抗原对活动性结核、潜伏结核感染者、健康对照者进行血清学检测Example 4: Serological detection of active tuberculosis, latent tuberculosis, and healthy controls with selected antigens
为了评价Rv0976c、Rv1255c、Rv3160c和Rv0792c是否能够与人血清中的特异性抗体反应，继而区分健康人群和不同疾病人群，本发明将实施例1纯化的Rv0976c、Rv1255c、Rv3160c和Rv0792c蛋白与来自不同人群的血清进行ELISA反应。实验包含3组人群样本：活动性结核组(20个病人)，结核潜伏感染组(25个样本)，健康对照组(24个个体)。活动性结核组包括11名痰涂片阳性患者和9名X线检查具有临床症状的患者。结核潜伏感者是指那些与结核病人有长期、连续密切接触(2-8年，平均4.3年)但未显示活动性结核临床症状的人群。健康个体是指不存在结核病人密切接触史，没有结核患病史或临床结核病症状的人群。所有个体来自于同一地理区域并有BCG接种史。In order to evaluate whether Rv0976c, Rv1255c, Rv3160c and Rv0792c can react with specific antibodies in human serum, and then distinguish between healthy people and different disease populations, the present invention purified the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins of Example 1 from different populations. The serum was subjected to an ELISA reaction. The experiment included three groups of population samples: active tuberculosis group (20 patients), tuberculosis latent infection group (25 samples), and healthy control group (24 individuals). The active tuberculosis group included 11 sputum-positive patients and 9 patients with clinical signs of X-ray examination. Tuberculosis latent sensation refers to those who have long-term, continuous close contact with TB patients (2-8 years, mean 4.3 years) but do not show the clinical symptoms of active tuberculosis. A healthy individual is a population that does not have a history of close contact with TB patients, no history of tuberculosis or clinical TB symptoms. All individuals were from the same geographical area and had a history of BCG vaccination.
在96孔板中分别加入每一种纯化的抗原(Rv0976c、Rv1255c、Rv3160c和Rv0792c)，0.25μg/孔；将分别来自活动性结核、潜伏结核感染者、健康对照者实验组人群的血清按1：100稀释，加入至抗原包被的孔内，抗原特异性抗体水平用标准ELISA方法检测，结果如图8所示。Each purified antigen (Rv0976c, Rv1255c, Rv3160c, and Rv0792c) was added to a 96-well plate at 0.25 μg/well; the serum from the experimental group of active tuberculosis, latent tuberculosis, and healthy controls was 1 : 100 dilution, added to the antigen-coated wells, antigen-specific antibody levels were detected by standard ELISA methods, and the results are shown in FIG.
实验结果显示，Rv0976c、Rv1255c、Rv3160c和Rv0792c与不同疾病模式的人群的血清反应能力存在差异，特别的是，源于活动性结核和潜伏感染人群的血清，特异性识别Rv0976c、Rv1255c、Rv3160c抗原的抗体水平显著高于健康人群血清(图8中B、C、D)。潜伏感染人群血清中特异性结合Rv0792c的抗体水平显著高于活动性结核和健康对照组(图8中A)。这些结果表明，Rv0976c、Rv1255c、Rv3160c和Rv0792c是具有开发前景的诊断候选抗原，可用于活动性结核、结核潜伏感染和健康个体间的鉴别诊断。 The results showed that Rv0976c, Rv1255c, Rv3160c and Rv0792c differed from those of different disease patterns in the serum. In particular, serum derived from active tuberculosis and latent infections specifically recognized Rv0976c, Rv1255c, and Rv3160c antigens. Antibody levels were significantly higher than in healthy populations (B, C, D in Figure 8). The level of antibodies that specifically bind Rv0792c in the serum of latently infected populations was significantly higher than that of active tuberculosis and healthy controls (Figure 8 A). These results indicate that Rv0976c, Rv1255c, Rv3160c and Rv0792c are promising diagnostic candidate antigens for the differential diagnosis of active tuberculosis, latent tuberculosis infection and healthy individuals.

Claims (12)

  1. 一种免疫原性成分，其包括选自以下的一种或多种多肽或其免疫性片段作为免疫原性成分：An immunogenic component comprising one or more polypeptides selected from the group consisting of or an immunological fragment thereof as an immunogenic component:
    (a)由SEQ ID No.1、SEQ ID No.2、SEQ ID No.3或SEQ ID No.4所示的氨基酸序列组成的多肽或其免疫性片段；(a) a polypeptide consisting of the amino acid sequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4 or an immunological fragment thereof;
    (b)与(a)限定的氨基酸序列具有至少70％一致性且与(a)具有相同功能的由(a)衍生的多肽或其免疫性片段；(b) a polypeptide derived from (a) or an immunological fragment thereof having at least 70% identity with the defined amino acid sequence of (a) and having the same function as (a);
    所述免疫性片段例如T细胞表位。The immunological fragment is, for example, a T cell epitope.
  2. 一种免疫原性成分，其包括选自以下的一种或多种核酸分子作为免疫原性成分：An immunogenic component comprising one or more nucleic acid molecules selected from the group consisting of:
    (a)由SEQ ID No.5、SEQ ID No.6、SEQ ID No.7或SEQ ID No.8所示的核苷酸序列或其互补序列组成的核酸分子；(a) a nucleic acid molecule consisting of the nucleotide sequence shown in SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8 or a complement thereof;
    (b)由与(a)中核酸分子编码相同氨基酸序列的核苷酸序列或其互补序列组成的核酸分子；或者(b) a nucleic acid molecule consisting of a nucleotide sequence encoding the same amino acid sequence as the nucleic acid molecule of (a) or a complement thereof; or
    (c)在严格条件下与(a)或(b)限定的核酸分子杂交且具有至少10个核苷酸长度的核酸分子。(c) a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule as defined in (a) or (b) and which has a length of at least 10 nucleotides.
  3. 一种免疫原性组合物，其包含：权利要求1中的一种或多种多肽或其免疫性片段、和/或权利要求2中的一种或多种核酸分子作为免疫原性成分；所述免疫原性组合物还可进一步包含佐剂。An immunogenic composition comprising: one or more polypeptides of claim 1 or an immunological fragment thereof, and/or one or more nucleic acid molecules of claim 2 as an immunogenic component; The immunogenic composition may further comprise an adjuvant.
  4. 一种表达载体或非致病性微生物，其中整合有至少一个拷贝的包含权利要求2的核酸分子的DNA片段(例如，放在游离质粒中或整合进入微生物基因组)，且DNA片段能够以多肽形式在微生物中表达。An expression vector or a non-pathogenic microorganism in which at least one copy of a DNA fragment comprising the nucleic acid molecule of claim 2 is integrated (for example, placed in a free plasmid or integrated into a microbial genome), and the DNA fragment is capable of being in the form of a polypeptide Expressed in microorganisms.
  5. 根据权利要求4所述的表达载体或非致病性微生物，其中所述表达载体或非致病性微生物是牛痘、腺病毒、BCG或转化细胞。The expression vector or non-pathogenic microorganism according to claim 4, wherein the expression vector or the non-pathogenic microorganism is vaccinia, adenovirus, BCG or transformed cells.
  6. 权利要求1或2中的免疫原性成分、权利要求3所述的免疫原性组合物、或权利要求4所述的表达载体或非致病性微生物在制备用于抗结核分枝杆菌感染的药物中的应用。The immunogenic component according to claim 1 or 2, the immunogenic composition according to claim 3, or the expression vector or non-pathogenic microorganism of claim 4, for preparation for infection against Mycobacterium tuberculosis Application in medicine.
  7. 一种抗结核分枝杆菌感染的药物，该药物包含权利要求1或2所述的免疫原性成分、权利要求3所述的免疫原性组合物、或权利要求4所述的表达载体或非致病性微生物；优选地，所述药物为疫苗。A medicament for infection against Mycobacterium tuberculosis, comprising the immunogenic component according to claim 1 or 2, the immunogenic composition according to claim 3, or the expression vector of claim 4 or Pathogenic microorganism; preferably, the drug is a vaccine.
  8. 一种人类或其他动物优选哺乳类动物的抗结核的免疫方法，该方法包括施予个体权利要求1或2的免疫原性成分、权利要求3所述的免疫原性组合物、或权利要求4所述的表达载体或非致病性微生物。 An anti-tuberculosis immunization method for a human or other animal, preferably a mammal, comprising administering an individual of the immunogenic component of claim 1 or 2, the immunogenic composition of claim 3, or claim 4 Said expression vector or non-pathogenic microorganism.
  9. 根据权利要求8所述的方法，其中，通过皮内、皮下透皮、肌肉、或粘膜递送的方式施予个体权利要求1或2的免疫原性成分、权利要求3所述的免疫原性组合物、或权利要求4所述的表达载体或非致病性微生物。The method according to claim 8, wherein the individual immunogenic component of claim 1 or 2, the immunogenic combination of claim 3, is administered by intradermal, subcutaneous transdermal, intramuscular, or mucosal delivery. Or the expression vector or non-pathogenic microorganism of claim 4.
  10. 权利要求1或2的免疫原性成分、或权利要求3所述的免疫原性组合物在制备用于检测和/或诊断结核分枝杆菌感染的制剂中的应用。Use of the immunogenic component of claim 1 or 2, or the immunogenic composition of claim 3, in the preparation of a formulation for the detection and/or diagnosis of a Mycobacterium tuberculosis infection.
  11. 根据权利要求10所述的应用，其中，检测结核分枝杆菌感染包括检测机体针对结核分枝杆菌的抗体反应或细胞免疫反应。The use according to claim 10, wherein detecting the Mycobacterium tuberculosis infection comprises detecting an antibody response or a cellular immune response against the Mycobacterium tuberculosis.
  12. 一种用于结核样本的诊断试剂盒，其中包括权利要求1或2的免疫原性成分、或权利要求3所述的免疫原性组合物。 A diagnostic kit for a tuberculosis sample, comprising the immunogenic component of claim 1 or 2, or the immunogenic composition of claim 3.
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