CN106606774B - Tuberculosis immunodiagnosis molecular marker and application thereof in preparation of vaccine - Google Patents

Abstract

The invention belongs to the field of immunology and molecular biology, and relates to a tuberculosis immunodiagnosis molecular marker and application thereof in preparation of a vaccine. In particular to the application of the protein with the amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2 in the preparation of anti-tuberculosis drugs or drugs for inhibiting mycobacterium tuberculosis, or in the preparation of drugs for detecting tuberculosis or drugs for detecting mycobacterium tuberculosis. As an antigen, the protein shown by SEQ ID NO. 1 or SEQ ID NO. 2 has good sensitivity and specificity, the reaction intensity is obviously higher than that of a positive control 38kDa, and the protein has the potential of being applied to the preparation of anti-tuberculosis drugs such as anti-tuberculosis vaccines.

Description

Tuberculosis immunodiagnosis molecular marker and application thereof in preparation of vaccine

Technical Field

The invention belongs to the field of immunology and molecular biology, and relates to a tuberculosis immunodiagnosis molecular marker and application thereof in preparation of a vaccine.

Background

Mycobacterium tuberculosis is one of the most threatening pathogens to human health, and tuberculosis is the major infectious disease among adults in the world today and has posed a serious challenge to international public health. After infecting human body, tubercle bacillus is in latent state or persistent infection state, and can escape from host defense function in infected host cell to propagate in large amount and reach a delicate balance with host, and the infected person may attack at any time in life. Typically, 10% of infected individuals will develop active tuberculosis. An untreated active tuberculosis patient can infect 10-15 people a year.

In the last fifty years, the control theory and technology, the clinical diagnosis and treatment level and the research of tuberculosis have great progress. Due to the widespread application of effective chemotherapeutic drugs (isoniazid, rifampicin, pyrazinyl ammonium, streptomycin, ethambutol, etc.) and the worldwide popularization of bcg, the incidence of tuberculosis has once been reduced, so that people optimistically believe that tuberculosis will be completely eradicated by humans as with smallpox. However, in the mid eighties, due to an excessively optimistic estimated situation, investment is reduced, mechanisms are reduced, treatment and management of tuberculosis are relaxed, tuberculosis is burned again in the death and recrudescence of many countries, and the incidence rate is increased again all over the world. In 1993, the WHO announced that tuberculosis is in a global emergency state and called to rapidly take action to fight against the tuberculosis crisis, and the statement that the tuberculosis is published in the history of the WHO is the first time. Drug-resistant and multi-drug resistant tuberculosis have also become a significant threat in current tuberculosis control efforts.

Prevention of tuberculosis, the WHO has included Mycobacterium bovis Calmette-Guerin (BCG) in the scope of expanded immunization programs. However, the reported immune protective effects against tuberculosis vary widely from 0-80%. In addition, with the prevalence of HIV infection, AIDS patients have been reported to be vaccinated with BCG to cause systemic disseminated infection.

The current tuberculosis virus diagnosis methods are as follows: firstly, sputum bacteria examination: the sputum smear bacteria examination and the sputum tubercle bacillus examination are simple and easy to implement, the accuracy is higher, and the tuberculosis can be accurately diagnosed by examining the tubercle bacillus in the sputum. Generally, three sputum specimens, i.e., night sputum, early morning sputum and immediate sputum, should be examined for the first diagnosis. Although it is a 'gold index' for diagnosing pulmonary tuberculosis, the diagnosis rate is low and the culture period is long. The result of the tubercle bacillus culture is high in reliability, and the tubercle bacillus drug sensitivity test can be carried out, but the application is limited after 6-8 weeks. (II) X-ray inspection: the chest X-line examination can discover tuberculosis at an early stage, can determine the position, the property and the range of a focus, can know the morbidity and can be used for judging the treatment effect, and is convenient to develop and easy to accept by patients. The CT of the chest can find small or hidden lesions and can make up the deficiency of the general X-ray examination. But is easily confused with other pulmonary diseases and requires the confirmation of a professional physician. (III) immunological diagnosis of pulmonary tuberculosis: 1. the tuberculin Pure Protein Derivative (PPD) test is commonly used, positive test is one of evidences of tubercle bacillus infection, but the false positive rate is high, and misdiagnosis is easy. 2. The positive detection of the antibody of tuberculosis in blood and sputum also helps diagnosis and is easy to have false positive rate. The BACTEC method for detecting the metabolites of Mycobacterium tuberculosis can isolate the mycobacteria generally within two weeks, but the amount of the bacteria can affect the days of positive result. 4. Polymerase Chain Reaction (PCR) has the advantage of 98-100% sensitivity and the disadvantage of poor specificity. (IV) other checks: can only be used as auxiliary diagnosis and can not be used as diagnosis basis. 1. The fiber bronchoscopy can directly or indirectly judge the pathological changes in the bronchus and the lung, has the functions of biopsy, lavage, video recording, picture taking in the trachea and the like, and is particularly useful for diagnosis and differential diagnosis. 2. Thoracoscopy and mediastinoscopy can be used to observe enlarged lymph nodes in the thoracic cavity and mediastinum, and biopsy can be taken to facilitate diagnosis and differential diagnosis. 3. Ultrasonic examination is mainly used for diagnosing and differentially diagnosing pleural effusion.

Therefore, means for rapidly diagnosing tuberculosis, efficiently preventing and controlling tuberculosis are urgently needed.

Disclosure of Invention

The present inventors have conducted intensive studies and creative efforts to obtain two isolated polypeptides (proteins) named Rv0237 and Rv1111c, respectively, based on mycobacterium tuberculosis H37 Rv. And the inventor surprisingly finds that the two polypeptides can be used as antigens, have higher reaction strength in humoral immune response and good sensitivity and specificity, and have the potential for preparing anti-tuberculosis medicaments, particularly anti-tuberculosis vaccines. The following invention is thus provided:

one aspect of the invention relates to the use of an (isolated) polypeptide in the preparation of a medicament against tuberculosis or a medicament for inhibiting mycobacterium tuberculosis, wherein the amino acid sequence of the polypeptide is shown as SEQ ID No. 1 or SEQ ID No. 2.

Amino acid sequence of Rv0237 protein: (359aa)

STTSGASPATPVAVPVPRSCAEPAGIPALLSPRDKLAQLLVVGVRDAADAQAVVTNYHVGGILIGSDTDLTIFDGALAEIVAGGGPLPLAVSVDEEGGRVSRLRSLIGGTGPSARELAQTRTVQQVRDLARDRGRQMRKLGITIDFAPVVDVTDAPDDTVIGDRSFGSDPATVTAYAGAYAQGLRDAGVLPVLKHFPGHGRGSGDSHNGGVTTPPLDDLVGDDLVPYRTLVTQAPVGVMVGHLQVPGLTGSEPASLSKAAVNLLRTGTGYGAPPFDGPVFSDDLSGMAAISDRFGVSEAVLRTLQAGADIALWVTTKEVPAVLDRLEQALRAGELPMSAVDRSVVRVATMKGPNPGCGR(SEQ ID NO:1)

Amino acid sequence of Rv1111c protein: (185aa)

TALFDSIARKLSSLMTGDSDDDGGRRSAQRPARTRSRHARPPSEDNREPIAERRSRRRPRPQNDPHPRRNAHERPAPRSSRFDSYRSYQPSEPSGPAEPVNRYERRGARYQPYARYEPTYEPQRRRARPSEPTNPTHHPISQVRYRGSATRDARRDNYREEQRFDRRDRSRAPRRPPAESWEYDV(SEQ ID NO:2)

The experimental result of the example 2 of the invention shows that the average reaction intensity of the three experimental results of Rv0237 and Rv1111c is more than 2, the repeatability is good, and the method can be applied to the preparation of anti-tuberculosis drugs or drugs for inhibiting mycobacterium tuberculosis, for example, the method can be used as a vaccine candidate

Another aspect of the invention relates to the use of an (isolated) polynucleotide, wherein the nucleic acid sequence of said polynucleotide is as shown in SEQ ID NO. 3 or SEQ ID NO. 4, for the manufacture of a medicament against tuberculosis or for the inhibition of Mycobacterium tuberculosis.

A nucleic acid sequence encoding Rv 0237: (1080bp)

TCGACGACCTCCGGCGCGTCGCCCGCAACTCCGGTAGCCGTTCCCGTGCCCCGGAGCTGCGCCGAGCCGGCGGGGATCCCGGCGCTGCTGTCCCCCCGTGACAAGCTGGCCCAGCTGCTGGTGGTCGGCGTGCGAGATGCTGCGGACGCCCAAGCCGTGGTCACCAACTACCACGTCGGCGGCATCCTCATCGGCAGCGACACCGACCTGACGATTTTTGACGGCGCGCTGGCCGAGATCGTTGCCGGCGGGGGTCCGCTGCCGCTGGCGGTGAGTGTCGACGAGGAAGGCGGGCGGGTGTCCCGGTTGAGGTCGCTGATCGGCGGTACGGGGCCGTCGGCCCGCGAACTGGCACAAACCCGAACCGTCCAGCAGGTGCGCGACTTGGCTCGAGACCGCGGCCGGCAGATGAGAAAGCTGGGTATCACCATCGACTTCGCCCCGGTGGTCGACGTCACCGACGCCCCGGATGACACGGTGATCGGGGACCGGTCGTTCGGCTCGGATCCGGCTACGGTCACCGCGTATGCCGGGGCGTACGCGCAGGGTCTGCGCGATGCCGGGGTGCTGCCGGTGCTCAAGCATTTCCCCGGTCACGGGCGTGGCTCGGGTGATTCGCACAACGGGGGTGTCACGACACCACCGCTTGATGACCTGGTGGGCGATGACCTGGTGCCCTACCGAACGCTGGTGACCCAGGCGCCGGTCGGTGTGATGGTGGGTCATCTGCAGGTTCCTGGGTTGACCGGCTCCGAGCCGGCCAGTCTGAGCAAGGCCGCGGTGAACCTGCTGCGCACCGGCACGGGATACGGCGCACCGCCGTTCGATGGTCCAGTGTTCAGCGACGACCTCTCTGGTATGGCCGCGATCTCAGACCGGTTTGGCGTCAGCGAGGCGGTGTTGCGCACCTTGCAAGCCGGTGCCGATATCGCACTGTGGGTTACCACCAAAGAGGTGCCCGCGGTGCTGGACCGCCTGGAACAGGCGCTGCGCGCCGGTGAATTGCCGATGTCGGCGGTCGACCGGTCGGTGGTGCGGGTGGCGACCATGAAGGGGCCCAACCCGGGGTGTGGCCGTTAG(SEQ ID NO:3)

Nucleic acid sequence encoding Rv1111 c: (555bp)

ACGGCTCTGTTCGACAGCATCGCCAGGAAGCTCAGCTCGCTGATGACCGGCGATTCGGACGACGACGGCGGCCGGCGGTCGGCCCAGCGGCCTGCCCGTACTCGTTCCCGACACGCCCGACCCCCGTCGGAGGACAACCGCGAACCCATAGCCGAGCGTCGATCGCGCCGCCGCCCTAGGCCGCAGAATGATCCGCACCCGCGCCGCAATGCCCACGAACGGCCGGCACCGCGCAGCAGCCGCTTCGATTCTTACCGCAGCTACCAGCCGTCCGAACCCTCCGGGCCTGCCGAGCCCGTCAACCGGTACGAACGTCGGGGTGCTCGATACCAGCCCTATGCGCGCTACGAGCCCACCTACGAGCCCCAACGCCGACGCGCCCGTCCCAGCGAGCCAACCAACCCGACACACCATCCGATCTCGCAGGTCCGCTACCGCGGGTCAGCTACTCGCGACGCGCGGCGGGACAACTACCGCGAGGAGCAGCGGTTCGATCGGCGGGATCGTTCGCGGGCACCGCGCAGACCACCGGCTGAATCCTGGGAATACGACGTC(SEQ ID NO:4)

The invention also relates to the application of the recombinant vector in preparing the anti-tuberculosis drugs or drugs for inhibiting mycobacterium tuberculosis, wherein the recombinant vector contains the polynucleotide shown in SEQ ID NO. 3 or SEQ ID NO. 4.

The invention also relates to the application of the host cell in the preparation of anti-tuberculosis drugs or drugs for inhibiting mycobacterium tuberculosis, wherein the host cell contains a recombinant vector, and the recombinant vector contains the polynucleotide shown in SEQ ID NO. 3 or SEQ ID NO. 4.

The invention also relates to the application of the polypeptide in the preparation of drugs for diagnosing tuberculosis or drugs for detecting mycobacterium tuberculosis, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

The invention also relates to the application of the polynucleotide in preparing a medicine for diagnosing tuberculosis or a medicine for detecting mycobacterium tuberculosis, wherein the nucleic acid sequence of the polynucleotide is shown as SEQ ID NO. 3 or SEQ ID NO. 4.

The use according to any of the above, wherein the tuberculosis is pulmonary tuberculosis.

The use according to any one of the above, wherein the mycobacterium tuberculosis is mycobacterium tuberculosis H37 Rv.

The invention also relates to a pharmaceutical composition, which contains polypeptide with amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2, and pharmaceutically acceptable auxiliary materials.

Still another aspect of the invention relates to a vaccine composition comprising a polypeptide having an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2, and an adjuvant for vaccines, such as an adjuvant. In one embodiment of the invention, the vaccine composition is a vaccine formulation.

Yet another aspect of the invention relates to a method of preventing and/or treating tuberculosis, such as pulmonary tuberculosis, comprising the step of administering (e.g. vaccinating) a subject an effective amount of a polypeptide as set forth in SEQ ID NO 1 or SEQ ID NO 2, a pharmaceutical composition of the invention or a vaccine composition of the invention.

The dosage to be administered will depend on a number of factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the particular polypeptide employed, the route of administration and the number of administrations desired. The above-mentioned dosage may be administered in a single dosage form or divided into several, e.g. two, three or four dosage forms.

The actual dosage level of each active ingredient (polypeptide) in the pharmaceutical composition can be varied so that the resulting amount of active ingredient is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. Dosage levels will be selected with regard to the activity of the particular polypeptide, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is common practice in the art to start doses from a level below that required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

The term "isolated" or "isolated" refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" or "isolated" does not exclude the presence of substances mixed artificially or synthetically or other impurities which do not affect the activity of the substance.

The term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

The term "host cell" refers to a cell which can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

The term "pharmaceutically acceptable adjuvant" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's pharmaceutical sciences. edited by genomic AR,19th ed. pennsylvania: mach Publishing Company,1995), and include, but are not limited to: pH regulator, surfactant, adjuvant, and ionic strength enhancer. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

The term "adjuvant" refers to a non-specific immunopotentiator which, when delivered to the body with or prior to an antigen, enhances the body's immune response to the antigen or alters the type of immune response. Adjuvants are of various types, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is currently the most commonly used adjuvant in animal testing. Aluminum hydroxide adjuvants are used more often in clinical trials.

The term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, an effective amount for preventing a disease (e.g., tuberculosis, particularly tuberculosis) refers to an amount sufficient to prevent, or delay the onset of a disease (e.g., tuberculosis, particularly tuberculosis); a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

The term "disease and/or disorder" refers to a physical condition of the subject that is associated with the disease and/or disorder of the present invention.

The term "subject" can refer to a patient or other animal, particularly a mammal, e.g., a human, dog, monkey, cow, horse, etc., that receives a pharmaceutical composition of the invention to treat, prevent, ameliorate, and/or alleviate a disease or disorder described herein.

The term "mycobacterium tuberculosis" includes, but is not limited to, for example, mycobacterium tuberculosis; the term "Mycobacterium tuberculosis" includes, but is not limited to, Mycobacterium tuberculosis H37 Rv.

The term "anti-tuberculosis" includes, but is not limited to, the prevention and/or treatment of tuberculosis.

The term "tuberculosis" includes, but is not limited to, diseases and/or conditions caused, for example, by infection with "tubercle bacillus", such as tuberculosis.

Advantageous effects of the invention

Humoral immunity experiments show that the protein shown by SEQ ID NO. 1 or SEQ ID NO. 2 has good sensitivity and specificity as antigen, the reaction intensity is obviously higher than that of a positive control 38kDa, and the protein has the potential of being applied to the preparation of anti-tuberculosis drugs such as anti-tuberculosis vaccines.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In examples 1 to 3 below, if not specifically mentioned, the protein samples used were Rv0237 protein and Rv1111c protein; it can be obtained by artificial chemical synthesis, or by expression and purification of corresponding coding nucleotide sequence through recombinant expression vector and recombinant host cell. Wherein, the nucleic acid sequence coding for Rv0237 and the nucleic acid sequence coding for Rv1111c can be cloned from Mycobacterium tuberculosis H37Rv or artificially synthesized by chemical synthesis.

Example 1: mass spectrometric identification experiment

1. Experimental materials and instruments

Target protein sample: the prepared Rv0237 protein (SEQ ID NO:1) and the Rv1111c protein (SEQ ID NO: 2).

The instrument comprises the following steps: ultimate 3000Nano-LC system (Dionex, USA), Linear ion trap tandem Mass spectrometer (LTQ), Orbitrap vector mass spectrometer (Thermoscientific, Germany).

Data processing software: MassLynx version 4.1(Dionex, USA), Xcalibur software version 2.2.6(Thermo), MASCOT version 2.2(Matrix Sciences, UK).

2. Experimental methods

(1) After SDS PAGE is carried out on the target protein, the film is washed for 15 minutes by deionized water; cutting off target fragments with the size of 1-2 mm; placing the mixture into a centrifuge tube with low protein adsorption, and using 100 mu L ddH₂O washing the gel particles in the dish and repeating the same, discarding the liquid, adding 40. mu.L of (ACN)/ddH₂O (50/50), discarding the liquid, and incubating for 15 minutes; discarding the liquid, adding 100 μ L100 mM ammonium bicarbonate, incubating for 15 min, adding 100 μ L acetonitrile, and incubating for 15 min; repeating the steps; the amount of gum required was taken out (10. mu.g each according to the estimate); adding 200 mu LACN for dehydration for 5 minutes, and discarding; the sample was dried under vacuum for 30 minutes and had to be very dry.

(2) Reduction of proteins and alkylation: to the sample was added 40. mu.L of 10mM dithiothreitol 1M stock solution (DTT, 1M)/100mM ammonium bicarbonate to completely immerse the micelle in the liquid, and the sample was taken out and allowed to cool in a 56 ℃ water bath for 45 minutes.

(3) mu.L of 55mM iodoacetamide (IAA1M stock solution)/100 mM ammonium bicarbonate was added immediately and incubated for 30 minutes at room temperature, protected from light.

(4) Washing: discarding the solution in the micelle, adding 40 μ L of 100mM ammonium bicarbonate, and incubating for 15 min; adding 40 mu L of acetonitrile, and incubating for 15 minutes; discarding the liquid, adding 100 μ L ACN, drying for 5min, and discarding; the sample was dried in vacuo for 30 minutes.

(5) Trypsin digestion protein: mu.L of trypsin solution (in sufficient quantity, 1:10 mass ratio of trypsin to protein) was added and incubated at 4 ℃ for 45 minutes (ice bath). Incubation at 37 ℃ overnight (12-16 hours);

(6) protein extraction: sucking the supernatant, putting the supernatant into a low-adsorption protein centrifuge tube, adding 20 mu L of 25mM ammonium bicarbonate on the film, and incubating for 15 minutes; adding equal amount of CAN to make the solution Ambic/ACN at a ratio of 1:1, and incubating for 15 minutes; repeating the steps; sucking the supernatant, storing, adding 20 μ L of 5% formic acid to the film, and incubating for 15 min; adding equal amount of ACN, and incubating for 15 min; sucking the supernatant, storing, sucking the supernatant and storing; 0.1M DTT was added to give a final concentration of 1mM protein. Thoroughly drying at 30 deg.C for about 1 hr; redissolved with 5% formic acid to a final concentration of 500 fmol/. mu.L,

(7) desalting: salt removal with ZipTip c18 (www.merckmillipore.com, Germany) was performed as described.

(8) Loading: sample loading volume 10. mu.L plus 6. mu.L to separation column C18precolumn (Symmetry C18, 5. mu.m, 180. mu. m X mm), elution with 5% acetonitrile (containing 0.05% formic acid at a flow rate of 10. mu.L/min for 5 minutes, loading to C-18 analytical column (Waters acquisition UPLC BEH130C18, 1.7. mu.m, 100. mu.59100 mm) thermostatted 35 ℃ elution salt gradient from 5% to 35%, analysis of the positively charged peptides by electrospray, spray voltage of 2.0 kV. by Orbitrap (m/z 445.120025) in real time MS data (m/z 300. about.1,800, R. 60,000at m/z 400.) raw data by XCalibur software 2.2.6 (calibration modification data 2.2.2.6; calibration modification data of peptide fragment to protein search library 2, modification data of 2.5. mu.5. mu.10 mm; mutation of detection parameters of electrophoresis and variation of detection method [ C-32 ],247), deamidated (nq); (ii) a preferrosor ion masstolerance of + -50 ppm; fragment mass tolerance: + -0.6 Da.high peptide concentration (p <0.01) and the yarn positive rate (FPR) wave controlled at under 1%, missedCleavages permitted: 2.

3. Results of the experiment

See tables 1 and 2 below.

Table 1: mass spectrum identification protein information table

Note: PSMs: peptide spectra (number of hits)

Table 2: identification of peptide information Table after protein Secondary Mass Spectrometry

Note: PSMs: peptide spectra (number of hits)

The experimental results in table 1 show that each protein in table 1 is a unique protein, and compared with the NCBI original sequence, the protein score value and the coverage rate are very high, and the identification standard is met.

Table 2 identifies amino acid sequences and scores for twenty peptides of each protein identified, and for more than 2 peptides identified as being consistent as the protein criteria, and for others indicating the correctness of the peptides and the respective criteria (generally more than two peptides identified need not be mass-mapped). Therefore, the prepared protein is determined to be the H37Rv standard strain protein.

Example 2: humoral immunity experiment

The humoral immunity antigen can combine with the specificity generated in the body to prevent the humoral immunity antigen from infecting normal cells, and can combine with macrophages to ensure that the macrophages phagocytose antigens to achieve the aim of sterilization, so that the protein with high humoral immunity reaction strength can also stimulate the body to generate antibodies to achieve the immune protection effect.

1. Experimental Material

Protein sample: rv0237 protein and Rv1111c protein. The positive control was 38kDa (Immuno diagnostics Inc. USA).

The main reagents are as follows: a murine secondary antibody high-throughput kit, PS-MK15 Wes-Mouse 12-230 kDa master kit with split buffer for 200 data (Immuno Diagnostics Inc. USA). Streptomycin labeled goat anti-human IgG, Thermo Scientific, Germany).

2. Principles and methods of experimentation

Western Blot method was used. The Western Blot technique is a classical protein detection technique, and the conventional method is to transfer proteins which are separated by polyacrylamide gel or other gel or electrophoresis to nitrocellulose filter, and the protein components fixed on the filter still retain the antigen activity and the ability of specific binding with other macromolecules, so that the protein components can be bound with specific antibodies or nucleic acids, and after the first antibody is bound with specific antigens on the membrane, the protein components are detected by using a labeled secondary antibody (isotopic or non-isotopic enzyme). The existing new instrument protein simple (please see http:// www.proteinsimple.com /) can completely replace the traditional method, and the reagents and antigens used are all carried out in trace amount, and a special kit is provided, so that the time is saved, the experimental steps are standardized, and the quantification is accurate.

38kDa (Rv0934, GenBank accession: NC-000962.2, gi:15608080) is the most commonly used, the earliest developed commercial diagnostic kit antigen. The kit is very effective in diagnosing active tuberculosis, and has the defects of great sensitivity fluctuation for different people, and poor effect on smear negative tuberculosis patients and tuberculosis patients with combined HIV infection. It is a phosphate transport protein, belongs to membrane-bound lipoprotein, is 10 times higher than BCG (Mycobacterium bovis BCG, Bacillus Calmette-Guerin), and is also one of the most commonly used tuberculosis diagnosis antigens at present. When the reagent is used for tuberculosis serological detection, Wilkinson and the like use 38kD antigen to detect the serum of a tuberculosis patient, and the sensitivity and the specificity are 65.6 percent and 95.8 percent respectively. Therefore, the experiment uses 38kDa as a positive control to detect the antigen humoral immunity effect of the expressed antigen.

The experimental method is carried out according to the method given by the kit specification, and comprises the following specific steps:

(1) sample preparation

The target protein sample and the positive control 38kDa (Immuno Diagnostics Inc. USA), respectively, were added to the 5 Xprotein loading buffer as 500 ng/. mu.l diluted protein sample.

Preparing a primary antibody:

A. selecting 90 ESAT-6 positive pulmonary tuberculosis patients for clinical examination, extracting anticoagulation blood, centrifuging, collecting supernatant, and mixing 200 microliters per patient. The prepared E.coli powder was added to 100. mu.l of plasma in a ratio of 100. mu.g of E.coli powder, mixed well, and incubated overnight at 4 ℃. Centrifuging to collect supernatant, and removing impure protein in serum by using a magnetic bead adsorption method. Diluting with antibody diluent at a ratio of 1:100 times of the same volume as the original blood plasma for later use.

Wherein, the used Escherichia coli powder is prepared by the following method:

inoculating E.coli BL21(DE3) to 100mL LB culture medium, culturing overnight at 37 ℃ at 220rpm, and collecting the thallus the next day; resuspending E.coli BL21(DE3) thallus in 10mL PBS, adding 40mL precooled acetone, and fully mixing at 4 ℃; centrifuging at 12000rpm at 4 deg.C for 10min, collecting thallus, and washing twice with acetone; and taking out the thalli, and placing the thalli on clean paper for natural air drying for later use.

B. Magnetic bead IgG adsorption treatment method

The magnetic bead processing method comprises the following steps:

250μL ddH₂o Wash 50. mu.L of magnetic beads, 500. mu.L of IgG Binding buffer (20mM Na)₃PO₄pH7.5) was equilibrated with 50. mu.L of magnetic beads.

Magnetic bead treatment of serum:

treated magnetic beads (50. mu.L) were mixed with 500. mu.L of serum, incubated at room temperature for 30min, then placed on a magnetic stand, and the supernatant discarded.

mu.L of the magnetic beads was eluted with 250. mu.L of 0.1M Gly-HCl buffer pH 2.7, and the supernatant was collected, separated from IgG and magnetic beads, and collected.

Adding 60 μ L Tris-HCl pH 9.0 neutral buffer solution into the collected supernatant to neutralize to pH 7.0, subpackaging at-80 deg.C, and storing for humoral immunity experiment.

Preparing a secondary antibody: the anti-human IgG of the sheep is marked by streptomycin, and diluted by 1:200 times by using an antibody diluent.

(2) Sample loading

According to the requirements of the assay plate (25x 6). Antigen (5 μ l) was added to each well in row a, the first well in row a being the system control and the other wells being the sample wells. Mu.l of 38kDa positive control was added to the sample wells in one well, 5. mu.l of protein sample was added to the other wells, 10. mu.l of primary antibody diluent was added to the B well, 10. mu.l of primary antibody was added to the C well, 10. mu.l of diluted primary antibody was added to the C well, antibody diluent was added to the first well, secondary antibody (10. mu.l) was added to the D well, streptomycin peroxidase was added to the first well, 10. mu.l of system luminescent peroxide mixture was added to line E, and blank line F. Add 500. mu.l of three lines (3X5) of wash solution to wash away unbound primary and secondary antibodies, centrifuge for 10min to remove air bubbles, and hold for loading.

(3) Upper machine

Opening the glass software v2.7, opening an wes interface, and adopting an instrument default setting program as a setting method: the capillary electrophoresis time is 25min, the voltage during electrophoresis is 375V, the antibody dilution time is 5min, the primary antibody incubation time and the secondary antibody incubation time are both 30min, and the plate washing time system is default. Opening the carpillary cartridge support frame, placing the apillary cartridge, tearing off the sealing film of the assay cartridge, and closing the door. Clicking the start button starts running the machine. The machine will run automatically and perform picture collection and reaction intensity value taking.

And adjusting the standard molecular weight of the system, then adjusting the exposure time under an analysis menu under the edit, and copy selecting a picture format to save the picture under the edit. After the program is set, click and save the whole running file under the file. The peak area of the reaction intensity may be output, and then the peak area ratio of the target protein and the positive control may be calculated as the reaction intensity. The positive control 38kDa reaction intensity was considered as 1.

The experiment was repeated 2 more times after completion.

3. Results of the experiment

As shown in table 3 below.

Table 3: results of reaction intensity

As can be seen from Table 3, the average reaction intensity of the results of three experiments of Rv0237 and Rv1111c is more than 2, and the repeatability is good, so that the vaccine can be used as a vaccine candidate.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> institute of pathogenic biology of Chinese academy of medical sciences

<120> tuberculosis immunodiagnosis molecular marker and application thereof in preparing vaccine

<130>1602

<160>4

<170>PatentIn version 3.2

<210>1

<211>359

<212>PRT

<213>Mycobacterium tuberculosis

<400>1

Ser Thr Thr Ser Gly Ala Ser Pro Ala Thr Pro Val Ala Val Pro Val

1 5 10 15

Pro Arg Ser Cys Ala Glu Pro Ala Gly Ile Pro Ala Leu Leu Ser Pro

20 25 30

Arg Asp Lys Leu Ala Gln Leu Leu Val Val Gly Val Arg Asp Ala Ala

35 40 45

Asp Ala Gln Ala Val Val Thr Asn Tyr His Val Gly Gly Ile Leu Ile

50 55 60

Gly Ser Asp Thr Asp Leu Thr Ile Phe Asp Gly Ala Leu Ala Glu Ile

65 70 75 80

Val Ala Gly Gly Gly Pro Leu Pro Leu Ala Val Ser Val Asp Glu Glu

85 90 95

Gly Gly Arg Val SerArg Leu Arg Ser Leu Ile Gly Gly Thr Gly Pro

100 105 110

Ser Ala Arg Glu Leu Ala Gln Thr Arg Thr Val Gln Gln Val Arg Asp

115 120 125

Leu Ala Arg Asp Arg Gly Arg Gln Met Arg Lys Leu Gly Ile Thr Ile

130 135 140

Asp Phe Ala Pro Val Val Asp Val Thr Asp Ala Pro Asp Asp Thr Val

145 150 155 160

Ile Gly Asp Arg Ser Phe Gly Ser Asp Pro Ala Thr Val Thr Ala Tyr

165 170 175

Ala Gly Ala Tyr Ala Gln Gly Leu Arg Asp Ala Gly Val Leu Pro Val

180 185 190

Leu Lys His Phe Pro Gly His Gly Arg Gly Ser Gly Asp Ser His Asn

195 200 205

Gly Gly Val Thr Thr Pro Pro Leu Asp Asp Leu Val Gly Asp Asp Leu

210 215 220

Val Pro Tyr Arg Thr Leu Val Thr Gln Ala Pro Val Gly Val Met Val

225 230 235 240

Gly His Leu Gln Val Pro Gly Leu Thr Gly Ser Glu Pro Ala Ser Leu

245 250 255

Ser Lys Ala Ala Val Asn LeuLeu Arg Thr Gly Thr Gly Tyr Gly Ala

260 265 270

Pro Pro Phe Asp Gly Pro Val Phe Ser Asp Asp Leu Ser Gly Met Ala

275 280 285

Ala Ile Ser Asp Arg Phe Gly Val Ser Glu Ala Val Leu Arg Thr Leu

290 295 300

Gln Ala Gly Ala Asp Ile Ala Leu Trp Val Thr Thr Lys Glu Val Pro

305 310 315 320

Ala Val Leu Asp Arg Leu Glu Gln Ala Leu Arg Ala Gly Glu Leu Pro

325 330 335

Met Ser Ala Val Asp Arg Ser Val Val Arg Val Ala Thr Met Lys Gly

340 345 350

Pro Asn Pro Gly Cys Gly Arg

355

<210>2

<211>185

<212>PRT

<213>Mycobacterium tuberculosis

<400>2

Thr Ala Leu Phe Asp Ser Ile Ala Arg Lys Leu Ser Ser Leu Met Thr

1 5 10 15

Gly Asp Ser Asp Asp Asp Gly Gly Arg Arg Ser Ala Gln Arg Pro Ala

20 25 30

Arg Thr Arg Ser Arg His Ala Arg Pro Pro Ser Glu Asp Asn Arg Glu

35 40 45

Pro Ile Ala Glu Arg Arg Ser Arg Arg Arg Pro Arg Pro Gln Asn Asp

50 55 60

Pro His Pro Arg Arg Asn Ala His Glu Arg Pro Ala Pro Arg Ser Ser

65 70 75 80

Arg Phe Asp Ser Tyr Arg Ser Tyr Gln Pro Ser Glu Pro Ser Gly Pro

85 90 95

Ala Glu Pro Val Asn Arg Tyr Glu Arg Arg Gly Ala Arg Tyr Gln Pro

100 105 110

Tyr Ala Arg Tyr Glu Pro Thr Tyr Glu Pro Gln Arg Arg Arg Ala Arg

115 120 125

Pro Ser Glu Pro Thr Asn Pro Thr His His Pro Ile Ser Gln Val Arg

130 135 140

Tyr Arg Gly Ser Ala Thr Arg Asp Ala Arg Arg Asp Asn Tyr Arg Glu

145 150 155 160

Glu Gln Arg Phe Asp Arg Arg Asp Arg Ser Arg Ala Pro Arg Arg Pro

165 170 175

Pro Ala Glu Ser Trp Glu Tyr Asp Val

180 185

<210>3

<211>1080

<212>DNA

<213>Mycobacterium tuberculosis

<400>3

tcgacgacct ccggcgcgtc gcccgcaact ccggtagccg ttcccgtgcc ccggagctgc 60

gccgagccgg cggggatccc ggcgctgctg tccccccgtg acaagctggc ccagctgctg 120

gtggtcggcg tgcgagatgc tgcggacgcc caagccgtgg tcaccaacta ccacgtcggc 180

ggcatcctca tcggcagcga caccgacctg acgatttttg acggcgcgct ggccgagatc 240

gttgccggcg ggggtccgct gccgctggcg gtgagtgtcg acgaggaagg cgggcgggtg 300

tcccggttga ggtcgctgat cggcggtacg gggccgtcgg cccgcgaact ggcacaaacc 360

cgaaccgtcc agcaggtgcg cgacttggct cgagaccgcg gccggcagat gagaaagctg 420

ggtatcacca tcgacttcgc cccggtggtc gacgtcaccg acgccccgga tgacacggtg 480

atcggggacc ggtcgttcgg ctcggatccg gctacggtca ccgcgtatgc cggggcgtac 540

gcgcagggtc tgcgcgatgc cggggtgctg ccggtgctca agcatttccc cggtcacggg 600

cgtggctcgg gtgattcgca caacgggggt gtcacgacac caccgcttga tgacctggtg 660

ggcgatgacc tggtgcccta ccgaacgctg gtgacccagg cgccggtcgg tgtgatggtg 720

ggtcatctgc aggttcctgg gttgaccggc tccgagccgg ccagtctgag caaggccgcg 780

gtgaacctgc tgcgcaccgg cacgggatac ggcgcaccgc cgttcgatgg tccagtgttc 840

agcgacgacc tctctggtat ggccgcgatc tcagaccggt ttggcgtcag cgaggcggtg 900

ttgcgcacct tgcaagccgg tgccgatatc gcactgtggg ttaccaccaa agaggtgccc 960

gcggtgctgg accgcctgga acaggcgctg cgcgccggtg aattgccgat gtcggcggtc 1020

gaccggtcgg tggtgcgggt ggcgaccatg aaggggccca acccggggtg tggccgttag 1080

<210>4

<211>555

<212>DNA

<213>Mycobacterium tuberculosis

<400>4

acggctctgt tcgacagcat cgccaggaag ctcagctcgc tgatgaccgg cgattcggac 60

gacgacggcg gccggcggtc ggcccagcgg cctgcccgta ctcgttcccg acacgcccga 120

cccccgtcgg aggacaaccg cgaacccata gccgagcgtc gatcgcgccg ccgccctagg 180

ccgcagaatg atccgcaccc gcgccgcaat gcccacgaac ggccggcacc gcgcagcagc 240

cgcttcgatt cttaccgcag ctaccagccg tccgaaccct ccgggcctgc cgagcccgtc 300

aaccggtacg aacgtcgggg tgctcgatac cagccctatg cgcgctacga gcccacctac 360

gagccccaac gccgacgcgc ccgtcccagc gagccaacca acccgacaca ccatccgatc 420

tcgcaggtcc gctaccgcgg gtcagctact cgcgacgcgc ggcgggacaa ctaccgcgag 480

gagcagcggt tcgatcggcg ggatcgttcg cgggcaccgc gcagaccacc ggctgaatcc 540

tgggaatacg acgtc 555

Claims (3)

1. The application of the polypeptide in preparing a medicine for diagnosing tuberculosis or a medicine for detecting mycobacterium tuberculosis, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 2.

2. The use according to claim 1, wherein the tuberculosis is pulmonary tuberculosis.

3. The use according to claim 1, wherein the mycobacterium tuberculosis is mycobacterium tuberculosis H37 Rv.