CN110684116A

CN110684116A - Mycobacterium tuberculosis EEC fusion protein, preparation method and application thereof

Info

Publication number: CN110684116A
Application number: CN201910788337.6A
Authority: CN
Inventors: 张鹏飞
Original assignee: Beijing Grace Wah Yuen Biological Technology Co Ltd
Current assignee: Chengdu Keen Biotechnology Co ltd
Priority date: 2019-08-23
Filing date: 2019-08-23
Publication date: 2020-01-14
Anticipated expiration: 2039-08-23
Also published as: CN110684116B

Abstract

The invention discloses a mycobacterium tuberculosis EEC fusion protein, the amino acid sequence of which is SEQ ID NO.8 in a sequence table; the polynucleotide for coding the polypeptide is shown as SEQ ID NO.7 in a sequence table; vectors and host cells comprising the polynucleotides. The invention also relates to the preparation of the fusion protein and the application of the fusion protein in the auxiliary diagnosis of tuberculosis, the screening of tubercle bacillus infection and the development of tuberculosis vaccines.

Description

Mycobacterium tuberculosis EEC fusion protein, preparation method and application thereof

Technical Field

The invention relates to the field of biotechnology, and particularly relates to mycobacterium tuberculosis EEC fusion protein, a preparation method and application thereof.

Background

Tuberculosis remains a significant public health problem, and tuberculosis control has received high attention from the WHO and governments of various countries. However, non-tuberculous mycobacteria (NTM) infection and NTM disease caused by the infection are in an ascending trend, and in some low-tuberculosis incidence areas, the number of NTM cases is more than that of tuberculosis; NTM belongs to illegal reported diseases in China, and the specific infection condition of the NTM is unknown. 80% of tuberculosis in China is bacterial negative tuberculosis, the bacterial negative tuberculosis and NTM disease need to be distinguished, an immunological detection method for NTM infection and NTM disease is lacked at present, PPD-B (intracellular mycobacterial protein purified derivative) is not on the market at home.

The immunological diagnosis method is based on the immunological principle to diagnose the immune state of the body and is divided into immune serological diagnosis and immune cytological diagnosis. The tuberculosis immunocytological diagnosis methods include the traditional tuberculin test (TST) and the interferon release test (IGRA). TST refers to an intradermal test for diagnosing type IV hypersensitivity caused by mycobacterium tuberculosis infection by injecting tuberculin intradermally and according to the skin condition of the injection site. Tuberculin is a bacterial component of tubercle bacillus and includes Old Tuberculin (OT) and Pure Protein Derivative (PPD). OT is prone to side reactions because it is not purified. PPD is obtained by culturing tubercle bacillus or BCG for 8-10 weeks, sterilizing the culture at 121 deg.C for 30min, filtering, precipitating the filtrate with trichloroacetic acid and saturated ammonium sulfate, redissolving the precipitate, and dialyzing. PPD greatly reduces side reactions compared with OT, but has cross-reactivity with various mycobacterial antigens and the specificity needs to be improved.

The WHO recommended TST or IGRA for the first time in the annual report of tuberculosis in 2018 as a diagnostic method of latent tuberculosis infection (WHO, TB report, 2018). IGRA is the detection of secreted IFN-gamma by peripheral blood or peripheral blood mononuclear lymphocytes stimulated by Mycobacterium tuberculosis antigen in vitro. According to the IFN-7 detection method, the IGRA is divided into ELISA-IGRA and ELISPOT-IGRA, the ELISA-IGRA is in vitro stimulated culture of peripheral blood, and the IFN-gamma content of culture supernatant is detected by ELISA; a centrifuge, an incubator, an enzyme-linked immunosorbent assay, an ELISA-IFN-gamma kit and the like are required. ELISPOT-IGRA is that peripheral blood mononuclear lymphocytes are subjected to in vitro culture, and the number of cells secreting IFN-gamma is detected by an enzyme-linked spot method; the operation is complex, the cost is high, and a centrifuge, a cell counter, a CO2 incubator, a spot counter, an ELISPOT plate coated by IFN-gamma monoclonal antibody, lymphocyte separation liquid, cell culture liquid, serum, a centrifuge tube and the like are needed. The in vitro stimulating antigens involved in the IGRA test are all antigens of the RD1 region, and the RD1 encoding gene exists in the genome of tubercle bacillus and is absent in BCG and most NTM genomes, so that the IGRA specificity is higher than that of a PPD skin test. ELISPOT-IGRA is high in sensitivity but not suitable for large-scale screening, and ELISA-IGRA is suitable for large-scale screening, but gray zone data which cannot be identified in positive and negative exists. On the other hand, the IGRA detection cost is high, the requirements on equipment and experiment operators are high, and the method is not suitable for basic popularization in developing countries. TST only needs injection and measurement, and practitioners can master quickly after training, and the kit is suitable for screening at any time and on a large scale. The specificity of the TST diagnostic reagent is improved, and the TST is an effective, cheap and convenient immunological diagnostic method for screening the tubercle bacillus infection.

The OT and PPD skin tests have not met the clinical diagnosis and epidemiological investigation needs of tuberculosis. The development trend of preparing recombinant mycobacterium tuberculosis protein or fusion protein as new generation tuberculin by gene engineering technology has been. A plurality of recombinant tubercle bacillus proteins or fusion proteins are used for TST diagnosis for preclinical or clinical research, and the recombinant tubercle bacillus protein 38KD is used for tuberculosis diagnosis, which can improve the specificity of diagnosis, but has certain cross with BCG inoculation (He XY, et., Scand J infection Dis, 2008; tubercle bacillus protein for tuberculosis diagnosis, patent number: ZL200410044568. X). The research finds that the encoding genes of proteins ESAT6, CFP10 and MPT64 in the region of mycobacterium tuberculosis RD1 are deleted in the genomes of BCG and most NTM, so that the proteins and the encoding genes thereof become the first choice antigens for tuberculosis diagnosis. Recombinant Mycobacterium tuberculosis ESAT6 produced skin tests in different Mycobacterium-sensitized guinea pig skin tests, ESAT6 produced skin tests in 4 Mycobacterium-strain-sensitized guinea pig skin tests, while no skin test reactions were produced in both BCG and Mycobacterium avium-sensitized guinea pig skin tests (Mordi J, et al., Osong Public Heanlth Res Perscope, 2015, 6: 34-38). Recombinant ESAT6 antigen, recombinant CFP10 antigen and mycobacterium tuberculosis fusion protein EC have been approved for clinical use (Bersgted W, et al, Plos One, 2010). The ESAT6 phase II clinical trial showed that the specificity of the skin test diagnosis could be improved, with ESAT6 diagnostic sensitivity of 98.15% and 93.02% specificity (24h skin test results), control PPD diagnostic sensitivity of 96.82% and specificity of 80.3% (72h skin test results) (Sun QF, et al, Med Sci Monit, 2013, 19: 969-. The phase EC I Clinical trial demonstrated safety and no side effects (Li F, et al, Clinical Vaccine Immunology, 2016, 23: 767-.

The literature reports that there are two ways to test tubercle bacillus skin, one is patch test and the other is the Mondu method. Patch test reports are few, only MPT64 protein is diagnosed by a Patch test method, the two reports have great difference, MPB64 diagnoses by the Patch test method with 98.1% sensitivity and 100% specificity (Nakamura RM, et al, Int JTubercul Lung Dis, 1998); yet another document reports that the MPT64 protein was diagnosed with 27% sensitivity and 74% specificity using the patch test method (Pope V, et al, Int J tubocul Lung Dis, 2018). The Patch test method has high requirements on the preparation process, and various factors influence the absorption of an individual to the antigen, so the result repeatability is poor. The MonDoss method adopts intradermal injection, is convenient and simple, and can ensure consistent dosage of individuals. Therefore, recombinant tubercle bacillus proteins are used as TST agents, preferably by intradermal injection (monture's method).

Early studies demonstrated that ESAT6 (abbreviation E), CFP10 (abbreviation C) are mycobacterium tuberculosis RD1 region proteins, single or fusion proteins thereof are used for TST diagnosis, synthetic polypeptides from both proteins are used for IGRA in vitro stimulation antigens, ELISPOT-IGRA diagnostic kit t.tb-SPOT is that ESAT6 and CFP10 polypeptides stimulate peripheral blood mononuclear cell diagnosis, respectively, it was found that CFP10 enhances the number of plaques, while ESAT6 positivity is higher in absolute value, most samples are positive for both antigenic polypeptides, while some samples are positive for stimulation of only one antigen, and both antigens can enhance diagnostic sensitivity (qi of jiang et al. ESAT6 and CFP10 are small molecular proteins, the former consists of 95 amino acids, and the latter consists of 100 amino acids. E and C protein coding genes are connected in series, and an escherichia coli expression system is constructed and adopted to produce fusion protein EEC so as to improve the sensitivity and specificity of TST diagnosis.

Disclosure of Invention

The invention relates to a mycobacterium tuberculosis EEC fusion protein, a preparation method and an application thereof, the protein has higher antigen immunogenicity, the fusion protein with low dose can establish immunological diagnosis (including TST, cytokine release and serological diagnosis) of tuberculosis and latent tuberculosis infection, and the fusion protein can also be used as an antigen component of a tuberculosis subunit vaccine:

the invention is realized by an EEC fusion protein, which comprises 2E proteins and 1C protein which are connected in sequence; the amino acid sequence of the E protein is shown as SEQ ID NO.1, the amino acid sequence of the C protein is shown as SEQ ID NO.3, or the amino acid sequence is obtained by substituting, deleting or inserting one or more amino acids in the amino acid sequences shown as SEQ ID NO.1 or SEQ ID NO. 3. The amino acid sequence shown in SEQ ID NO.5 is obtained by connecting 2 SEQ ID NO.1 and 1 amino acid sequence shown in SEQ ID NO.3 in series at the first position, and the amino acid sequence obtained by inserting 1 glycine between the first amino acid and the second amino acid in SEQ ID NO.5 is the amino acid sequence shown in SEQ ID NO. 8:

(a) comprises or consists of an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No.8 and still being immunogenic;

(b) one or more amino acids are substituted, deleted, inserted and/or added in the amino acid sequence shown in SEQ ID NO.8, and still have immunogenicity;

(c) is encoded by a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 7;

(d) is coded by a nucleotide sequence which substitutes, deletes, inserts and/or adds one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 7.

The invention also relates to a nucleic acid comprising or consisting of:

(a) the polynucleotide sequence shown in SEQ ID NO.6 or the complementary sequence thereof is obtained by connecting the nucleic acid sequences of 2E protein coding genes (shown as the sequence shown in SEQ ID NO.2) and 1C protein coding gene (shown as the sequence shown in SEQ ID NO.4) in series. The polynucleotide sequence shown in SEQ ID NO.6 is optimized by codons, NcoI enzyme cutting sites and bases for preventing frame shift are sequentially added at the 5 'end, and termination codons and HindIII enzyme cutting sites are sequentially added at the 3' end to form an EEC protein coding gene, namely the polynucleotide sequence shown in SEQ ID NO. 7. The NcoI enzyme cutting site is CCATGG, the base for preventing frame shift is GC, the termination code is TAA, and the HindIII enzyme cutting site is GGATCC. And the polynucleotide sequences shown in the sequence 6 and the sequence 7 are obtained by replacing, deleting or inserting one or more nucleotides to obtain the polynucleotide sequences which encode the same functional protein.

(b) A polynucleotide having a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 7;

(c) polynucleotide, the nucleotide sequence of which is substituted, deleted, inserted and/or added with one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 7;

(d) a polynucleotide encoding a fusion protein having an amino acid sequence with at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 8;

(e) a polynucleotide encoding a fusion protein in which one or more amino acids are substituted, deleted, inserted and/or added in the amino acid sequence shown in SEQ ID No. 8; or

(f) A polynucleotide which encodes SEQ ID No.8 but differs from SEQ ID No.7 by the degeneracy of the codons.

The invention also relates to a method for preparing a mycobacterium tuberculosis antigen as shown in SEQ ID NO.8 or a fusion protein with mycobacterium tuberculosis immunoreactivity, which comprises expressing a nucleic acid as shown in SEQ ID NO.7 in a suitable host cell.

The invention further relates to a nucleic acid construct or expression vector comprising the above polynucleotide, preferably pET28 a-EEC. The present invention further relates to a host cell comprising the above-described construct or expression vector, preferably a cell of the family Enterobacteriaceae, more preferably an E.coli cell, still more preferably an E.coli BL21(DE3) cell. In another aspect, the present invention also provides a method for preparing the above fusion protein or the above nucleic acid construct or expression vector pET28a-EEC, comprising using the polynucleotide, expression vector or host cell of the present invention.

More preferably, the above preparation method comprises the steps of:

1) synthesizing a nucleic acid shown as SEQ ID NO.7 by using a whole gene, connecting the synthesized whole gene to a vector, preferably a pET28a vector, and forming a vector-EEC recombinant plasmid;

2) transforming a suitable host cell, preferably escherichia coli TOP10, by using the obtained vector-EEC recombinant plasmid, screening, selecting a monoclonal colony for culturing, and carrying out double enzyme digestion and DNA sequencing identification on the recombinant plasmid;

3) transforming the qualified vector-EEC recombinant plasmid into a suitable host cell, preferably an Escherichia coli host cell, more preferably an Escherichia coli cell BL21(DE 3); selecting a monoclonal colony for culture and IPTG induced expression, and storing an expressed EEC protein colony to obtain an engineering bacterium;

4) IPTG inducing the expression engineering bacteria and collecting the engineering bacteria; and

5) the expressed fusion protein is purified and/or identified by extraction using conventional methods. Preferably, the extraction and purification comprises: breaking thallus, ammonium sulfate precipitating target protein, dissolving precipitate again, desalting, and purifying by ion chromatography and molecular sieve chromatography. Preferably, the identifying comprises: immunoblotting, TST, protein sequencing identification.

The invention further provides any one of the following applications of the mycobacterium tuberculosis fusion protein EEC or the polynucleic acid encoding the mycobacterium tuberculosis fusion protein EEC or the biological material containing the polynucleic acid:

(1) the application in preparing the mycobacterium tuberculosis fusion protein;

(2) the application in preparing a reagent or a kit for tuberculosis immunological diagnosis, tuberculosis auxiliary diagnosis or tubercle bacillus latent infection screening;

(3) the use in the manufacture of a vaccine for the prevention or treatment of tuberculosis.

Preferably, the immunological diagnosis comprises a TST, cytokine release assay and or serological diagnosis. More preferably, the cytokine in the desmocyte release assay is IFN-gamma and antibody IgG or IgM in serologically diagnosed serum.

The invention relates to a tuberculosis diagnostic kit, which comprises the fusion protein.

The invention also relates to the application of the fusion protein in the preparation of a tuberculosis diagnostic kit.

The invention also relates to the application of the fusion protein in preparing tuberculosis vaccines.

The invention also relates to an application of the kit, which is used for tuberculosis immunological diagnosis, including tuberculosis auxiliary diagnosis or screening latent infection of tubercle bacillus; immunological diagnostic methods include skin tests, cytokine release tests and serological diagnostics.

The present invention further provides a reagent or a kit for diagnosing mycobacterium tuberculosis infection, which comprises the mycobacterium tuberculosis fusion protein EEC. The reagent or kit may also contain other reagents required for diagnosis, including but not limited to TWEEN80, TWEEN20, phenol, PBS, RPMI1640, IFN- γ mab, fetal bovine serum, cell dot blot chromogen, etc.

When the fusion protein and the composition containing the fusion protein are used for skin test diagnosis of tuberculosis, the lower detection limit and the dosage are obviously reduced while higher specificity is ensured, and viable bacteria infection, dead bacteria sensitization and BCG inoculation of tubercle bacillus can be effectively distinguished. The mycobacterium tuberculosis fusion protein EEC provided by the invention is not limited to immunological diagnosis containing TST, and can also be used for preparing tuberculosis subunit vaccines. Compared with the preparation of a plurality of single antigens, the antigen extraction and purification process is reduced; the dosage is reduced, the production cost is reduced, and the method has good popularization and application values.

The term "Mycobacterium tuberculosis" as used herein may also be referred to simply as "Mycobacterium tuberculosis".

The term "fusion protein" as used herein refers to a region of one protein fused to the N-or C-terminus of a region of another protein. Fusion proteins are typically produced by fusing a polynucleotide encoding one protein to a polynucleotide encoding another protein. Techniques for producing fusion proteins are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame (in frame) and that expression of the fusion protein is under the control of the same promoter and terminator.

The term "epitope" as used herein, also called antigenic determinant, refers to a chemical group in an antigenic molecule that determines the specificity of an antigen, which is the basic unit capable of specifically binding to the T cell antigen receptor TCR or B cell antigen receptor BCR.

The term "immunogenicity" as used herein refers to the ability of the body to be stimulated to form specific antibodies or to sensitize lymphocytes. It also refers to the property of antigen to stimulate specific immune cells, activate, proliferate, differentiate, and finally produce immune effector antibodies and sensitized lymphocytes.

The term "host cell" as used herein includes any cell type that is susceptible to transformation, transfection, transduction, and the like using a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.

The term "expression vector" as used herein is defined as a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide of the present invention linked to other nucleotides for expression thereof. The term "construct" or "nucleic acid construct" refers to a nucleic acid molecule, either single-or double-stranded, that is isolated from a naturally occurring gene or that is modified to contain a non-naturally occurring gene, the construct may contain the regulatory sequences required for expression of the coding sequence of the invention.

The term "sequence identity" or "identity" as used herein is used to describe the relatedness of two nucleotide or amino acid sequences, expressed as a percentage, using conventional algorithms and penalties rules for sequence alignment as known in the art.

The term "substitution" as used herein means that a nucleotide/amino acid occupying a certain position is replaced with a different nucleotide/amino acid; "deletion" means the removal of the nucleotide/amino acid occupying a position; "insertion" means the addition of nucleotides/amino acids in the middle of a sequence, next to and immediately after the nucleotide/amino acid occupying a position; "addition" means the addition of nucleotides/amino acids at both ends of the sequence.

The term "kit" used in the present invention refers to a kit prepared by using the protein of the present invention to complete tuberculosis detection diagnosis and tuberculosis infection screening. The kit is used for diagnosing tuberculosis and screening tuberculosis infection. The kit comprises a container containing the fusion protein of the present invention. The fusion proteins of the invention may be packaged in any convenient and suitable packaging means. The kit of the present invention may further comprise other containers, which may contain standards, antibodies or labeled antibodies, enzymes, substrates or buffers, etc., for detection, respectively. In the kit, a label and a package insert are included to provide instructions for use of the kit. Other materials may also be included to suit the needs of the user, such as microtiter plates and the like.

The term "tuberculosis" in the present invention refers to a chronic infectious disease caused by infection with mycobacterium tuberculosis. The term "tuberculosis" refers to an infectious disease caused by infection of the lung by Mycobacterium tuberculosis. The term "extrapulmonary tuberculosis" refers to tuberculosis occurring in various parts outside the lung due to pulmonary lesions spreading to various organs of the human body through the blood or lymphatic system.

The term "vaccine" of the present invention refers to a preventive and therapeutic biological preparation for vaccination of a subject for the purpose of preventing, controlling the occurrence and/or prevalence of infectious diseases, and specifically includes preparations for prevention, diagnosis and treatment prepared from microorganisms or their toxins, enzymes, human or animal sera, cells, and the like.

The term "codon optimization" as used herein refers to the composition of codons for every three adjacent nucleotides in messenger RNA and the determination of one amino acid; there are 64 genetic codons, the most frequently used one is called the optimal codon, the less frequently used one is called the rare or low-availability codon, the gene cooperation can be realized by using the preferred codon and avoiding the low-availability codon, and the redesign of the gene is called codon optimization.

Drawings

FIG. 1 shows SDS-polyacrylamide gel electrophoresis patterns of total mycoprotein obtained by shake-bed culture of recombinant engineering bacteria in different culture media;

lane 1: protein standard molecular weight: 98 KD; 66.2 KD; 45 KD; 31 KD; 20 KD; 14.4KD,

Lane 2: tryptone prepared culture medium without adding inducer and kanamycin,

Lane 3: preparing culture medium of tryptone without adding kanamycin and IPTG inducer,

Lane 4: preparing culture medium of tryptone without adding kanamycin and lactose inducer,

Lane 5: preparing culture medium of tryptone, adding kanamycin and not adding inducer,

Lane 6: preparing culture medium with tryptone, adding kanamycin and IPTG inducer,

Lane 7: preparing culture medium of tryptone, adding kanamycin and lactose inducer,

Lane 8: preparing culture medium of pea peptone without adding kanamycin and inducer,

Lane 9: preparing culture medium of pea peptone without adding kanamycin and IPTG inducer,

Lane 10: the pea peptone preparation culture medium is not added with kanamycin and lactose inducer;

FIG. 1 shows that engineering bacteria cultured in tryptone medium require inducer IPTG and lactose to express EEC; when the strain is cultured in a culture medium prepared from pea peptone, the EEC can be expressed without adding inductive agents IPTG and lactose. Kanamycin does not influence the expression of EEC by engineering bacteria.

FIG. 2EEC expression pattern identification in E.coli;

Lane 2: IPTG induced engineering bacteria holosomatic protein,

Lane 3: lane 2 bacterial cell-breaking supernatant,

Lane 4: lane 2 bacterial disruption and precipitation

FIG. 2 shows that the engineering bacteria express the fusion protein under IPTG induction and express in a soluble form.

FIG. 3 Western blot profile; wherein:

lane 1: hybridizable protein marker (only 25KD protein band is shown),

Lane 2: non-induced full thallus in tryptone culture medium,

Lane 3: tryptone prepared culture medium IPTG induced whole thallus,

Lane 4: lactose induced full thallus in tryptone culture medium,

Lane 5: preparing culture medium of plant source peptone without inducing whole thallus,

Lane 6: preparing culture medium IPTG with plant source peptone to induce complete thallus,

Lane 7: preparing a culture medium lactose by using plant source peptone to induce the whole thallus;

FIG. 3 shows that the host bacteria expresses EEC and the anti-E protein monoclonal antibody has positive reaction when tryptone is prepared into a culture medium and an inducer IPTG or lactose is added; the host bacteria can express EEC and show positive reaction with the E-resistant monoclonal antibody by adding and not adding IPTG or lactose into the plant source peptone culture medium.

FIG. 4 shows the EEC-containing protein band cleavage sequencing of the bacterial proteins of engineering bacteria cultured in pea peptone medium;

wherein: FIG. 4a is an electropherogram before gel cutting, and FIG. 4b is an electropherogram after gel cutting;

lane 1: protein standard molecular weight,

Lane 2: bacteria without inducer,

Lane 3: a thallus of an IPTG inducer;

FIG. 4 shows protein sequencing by cutting gel containing EEC protein band in pea peptone preparation medium with and without induction culture of thalli to express EEC protein.

FIG. 5 protein sequencing search results

Wherein: FIG. 5a is the result of the second lane of the cleavage protein sequencing search of FIG. 4 b; FIG. 5b shows the result of the sequencing search of the cleaved protein in the third lane of FIG. 4 b;

FIG. 6H37Rv mean diameter of skin reaction of live bacteria-sensitized guinea pigs;

FIG. 7H37Ra mean diameter of skin reaction in live bacteria-sensitized guinea pigs.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The first embodiment is as follows: recombinant plasmid PET28a-EEC construction for expression of protein EEC: according to the genome of tubercle bacillus H37Rv (http:// genetic. pateur. fr/Tubercullist /), the amino acid sequence of E protein and the base sequence of coding gene (shown in SEQ ID NO.1 and SEQ ID NO.2) and the amino acid sequence of C protein and the base sequence of coding gene (shown in SEQ ID NO.3 and SEQ ID NO.4) are obtained, the amino acid sequence of EEC protein and the base sequence of coding gene (shown in SEQ ID NO.5 and SEQ ID NO.6) are obtained in series, the amino acid sequence of SEQ ID NO.6 is entrusted to Shanghai bioengineering company, NcoI enzyme cutting site (CCATGG) and base GC are added in sequence at the 5 'end, and termination codes TAA and HindIII enzyme cutting site (AAGCTT) are added in sequence at the 3' end. Designing an NcoI enzyme cutting site and a HindIII enzyme cutting site to clone a synthesized whole gene into a vector, wherein ATGG is left after CCATGG is subjected to NcoI, genetic codons are triplet codes, 2 basic groups are required to be added for preventing frameshift, and 2 triplet codes are formed by the ATGG and the genetic codons; theoretically, the code of the triplet formed by adding two bases and the original base G can be translated into any one of 20 amino acids, the base added in the invention is GC, and GGC codes glycine. Any amino acid. The polynucleotide (shown in SEQ ID NO.7) with the changed base sequence shown in SEQ ID NO.6 is subjected to codon optimization, the whole gene is synthesized into the polynucleotide (shown in SEQ ID NO.7), the synthesized gene is cloned to an expression vector PET28a, and the correct recombinant plasmid PET28a-EEC is proved by screening enzyme digestion and sequencing.

Example two: the EEC protein (the amino acid sequence is shown as SEQ ID NO. 8) is expressed and identified in Escherichia coli: the recombinant plasmid PET28a-EEC transforms competent host bacterium Escherichia coli BL21(DE3), spreads on a Kannan resistant LB agar plate, cultures overnight at 37 ℃, selects a single colony for induction expression, and the bacterium expressing the target protein is called PET28a-EEC/BL21(DE3) engineering bacterium. The target protein EEC is expressed by engineering bacteria pET28a-EEC/BL21(DE3) cultured in culture medium prepared from tryptone under the induction of IPTG. Pea peptone prepared culture medium cultured pET28a-EEC/BL21(DE3) engineering bacteria all express target protein EEC in culture with or without an inducer; the EEC protein is expressed in soluble form in E.coli.

The electrophoresis results of the whole thallus protein of the engineering bacteria cultured by the culture medium prepared by the plant source peptone and the animal source peptone are shown in figure 1, and the expression form results of the target protein are shown in figure 2. The molecular weight of the E.coli expressed EEC protein shown in FIG. 1 and FIG. 2 is close to 31KD, and the molecular weight is close to 30.6KD calculated by the amino acid sequence shown in SEQ ID NO.5 (expasy-computer pI/MWtool, https:// web. expasy. org/computer _ pI /). Coli expressing EEC reacted with ESAT6 monoclonal antibody (fig. 3). Sequencing proves that the EEC expressed by the pea peptone culture medium with and without the inducer is the same protein. The bacterial protein electrophoresis pattern of pea peptone medium culture engineering bacteria (PET28a-EEC/BL21(DE3)) is shown in FIG. 4, wherein FIG. 4a is the electrophoresis pattern before gel cutting, and FIG. 4b is the electrophoresis pattern after gel cutting; sequencing results retrieval as shown in figure 5, the sequencing amino acid sequence is in the italicized font portion of the following sequence:

sequencing results of uninduced thallus cutting protein band

IPTG induced thallus cutting protein band sequencing result

Example three: EEC protein purification strategy: the method for purifying the EEC fusion protein comprises the following steps:

step one, crushing thalli: high-pressure homogenization 600-800Bar is circulated for 3 times;

step two, centrifugally collecting supernatant: centrifuging at 12000-15000rpm at 4 deg.C for 30min-1 hr;

step three, supernatant sulfuric acid precipitation: precipitating the supernatant with 8-10% ammonium sulfate with 30-35% ammonium sulfate, and collecting the precipitate;

step four, desalting: g-25 gel or ultrafiltration;

step five, anion chromatography;

collecting pipes containing target protein, combining, and precipitating with ammonium sulfate or ultrafiltering and concentrating;

seventhly, performing molecular sieve Superdex chromatography, collecting chromatographic peaks, detecting the purity by protein electrophoresis andor HPLC, and collecting and combining the tubes with the purity of more than 90%;

and step eight, combining protein dialysis and changing liquid to prepare stock solution.

Example four: skin test result of EEC skin test for guinea pig infected by H37Rv live bacteria

White SPF-grade guinea pigs which were healthy and not tested at all and which were negative for the TB-PPD skin test, weigh 300g to 500 g. Removing frozen Mycobacterium tuberculosis H37Rv (viable bacteria content of 7 × 10)⁵CFU/ml) strain 1, naturally dissolving at room temperature, and diluting with normal saline to 1 × 10⁴CFU/ml; 0.5ml of diluted bacteria solution is injected into the hind leg groin of each guinea pig subcutaneously, and the guinea pigs are sensitized for 5-6 weeks before skin test. Guinea pigs were depilated bilaterally and injected intradermally with 0.2ml of each dilution of EEC and TB-PPD and EC standards. Measuring the longitudinal diameter and the transverse diameter (mm) of the red swelling and/or induration of the injection part respectively in 24 hours and 48 hours by a double-blind method, and taking the mean value of the longitudinal diameter and the transverse diameter as the skin test reaction diameter of the injection sample at the point; the mean values of the skin test reaction diameters of 4 guinea pigs are shown in FIG. 7, and the ratio of the mean value of the EEC skin test reaction diameters to the mean values of the TB-PPD and EC standard skin test reaction diameters is shown in Table 1.

The results in FIG. 6 show that: the skin tests of TB-PPD, EC standard, EEC-A and EEC-B with different concentrations in H37Rv live bacteria sensitized guinea pigs all showed positive reactions at 24 hours and 48 hours. 24 hours, TB-PPD and 10 μ g/ml EC standards skin test reaction mean diameter below 10 μ g/ml EEC-B (P ═ 0.031 and P ═ 0.036); at 48 hours, the 10. mu.g/ml EEC-A skin test reaction mean diameter was less than 10. mu.g/ml EEC-B (P ═ 0.028), and the TB-PPD skin test reaction diameter was slightly less than 10. mu.g/ml EEC-B (P ═ 0.077).

Table 1 the results show that: the EEC and different concentrations of EEC which peak at different times of molecular sieve chromatography are close to the ratio of TB-PPD and 10 mu g/ml EC standard. All ratios are in the range of 0.9-1.3.

And (4) conclusion: skin test of 10 mu g/ml EEC-B in H37Rv live bacteria-sensitized guinea pig has better skin test reaction effect. The ratio of the average diameter of skin test reaction of 5 mu g/ml EEC in H37Rv live bacteria sensitized guinea pig to the average diameter of skin test reaction of TB-PPD meets the requirement of TB-PPD titer evaluation in the three departments of Chinese pharmacopoeia.

Example five: skin test result of EEC skin test for guinea pig infected by H37Ra live bacteria

White SPF-grade guinea pigs which were healthy and not tested at all and which were negative for the TB-PPD skin test, weigh 300g to 500 g. The H37Ra cells eluted on the slant were weighed by centrifugation and made into a 50mg/ml bacterial solution with physiological saline for injection. Subcutaneously injecting 0.5ml of bacterial liquid into the inguinal region of the hind leg of each guinea pig, sensitizing the guinea pigs for 5-6 weeks, performing skin test, removing hairs from vertebral columns of the guinea pigs on two sides, and intradermally injecting 0.2ml of EEC, TB-PPD with different dilution concentrations and EC standard substances with different dilution concentrations; the longitudinal diameter and the transverse diameter (mm) of the red swelling and/or induration of the injection part are measured 24 and 48 hours after the skin test by a double blind method, and the average value of the longitudinal diameter and the transverse diameter is used as the skin test reaction diameter of the injection sample at the point. The average of the cumulative values for 24 hours, 48 hours, 24 hours and 48 hours for 3 guinea pigs is shown in fig. 7; the ratio to TB-PPD and the ratio to EC standard are shown in Table 2.

FIG. 7 shows: after 24 hours, skin tests of guinea pigs sensitized by EEC, EC standard substances and TB-PPD at different concentrations in H37Ra live bacteria show positive reactions; the average diameter of TB-PPD skin test reaction is lower than 5 mug/ml EC standard (P0.021) and 1 mug/ml, 5 mug/ml and 10 mug/ml EEC (P0.05, P0.001 and P0.026), the average diameter of 1 mug/ml and 10 mug/ml EC standard skin test reaction is lower than 5 mug/ml EEC (P0.011 and P0.006), and the average diameter of 5 mug/ml EEC skin test reaction is slightly higher than 1 mug/ml EEC (P0.076). 48 hours, except that the skin test reaction diameter of the EC standard substance of 1 mu g/ml shows a negative reaction, the skin test of guinea pigs sensitized by live bacteria H37Ra with different concentrations of EEC, EC standard substances of different concentrations and TB-PPD shows a positive reaction; TB-PPD, 10 μ g/ml EC standards skin test mean diameter below 10 μ g/ml EEC (P ═ 0.029 and P ═ 0.055) and 5 μ g/ml EEC (P ═ 0.034 and P ═ 0.047), 5 μ g/ml EEC skin test mean diameter above 1 μ g/ml EEC (P ═ 0.047).

Table 2 the results show: at 24 hours, the ratios of the average diameter of the EEC skin test reaction of 5 mu g/ml EC, 5 mu g/ml EEC and 10 mu g/ml to the average diameter of the TB-PPD skin test reaction are all more than 1.3, and the ratios of the average diameter of the EEC skin test reaction of 5 mu g/ml EEC to the average diameter of the EC skin test reaction of 1 mu g/ml and 10 mu g/ml EC are all more than 1.3. And in 48 hours, the ratios of the mean EEC skin test reaction diameters of 5 mu g/ml and 10 mu g/ml to the mean TB-PPD skin test reaction diameter are both more than 1.3, and the ratios of the mean EEC skin test reaction diameters of 5 mu g/ml and 10 mu g/ml to the mean EC skin test reaction diameters of 1 mu g/ml, 5 mu g/ml and 10 mu g/ml are both more than 1.2.

And (4) conclusion: skin tests of 5 mu g/ml EEC in H37 Ra-sensitized guinea pigs show that the skin test reaction effect is better.

Example six: EEC skin test results of H37Rv dead bacteria-sensitized guinea pig

White SPF-grade guinea pigs which were healthy and not tested at all and which were negative for the TB-PPD skin test, weigh 300g to 500 g. The tubercle branch sterilizing liquid (H37Rv) with the concentration of 200mg/ml and Freund's incomplete adjuvant with the same volume are emulsified by a medical three-way pipe, and 0.1ml is injected into the inguinal region of two hind legs of a guinea pig subcutaneously. Carrying out skin test after 5-6 weeks of guinea pig sensitization, unhairing the vertebral column of the guinea pig bilaterally, and injecting 0.2ml of EEC each diluted concentration sample, TB-PPD and EC standard substance intradermally; the longitudinal diameter and the transverse diameter (mm) of the red swelling and/or induration of the injection part are measured 24 and 48 hours after the skin test by a double blind method, and the average value of the longitudinal diameter and the transverse diameter is used as the skin test reaction diameter of the injection sample at the point. The cumulative skin test reactions at 24 hours, 48 hours, 24 hours and 48 hours are shown in table 3.

Table 3 the results show: skin tests of H37Rv dead bacteria-sensitized guinea pigs show negative reactions in EEC and EC standard skin tests, and positive reactions in TB-PPD skin tests. The differences were not significant in comparison of the average diameters of the TB-PPD skin tests of guinea pigs sensitized with H37Rv live bacteria (fig. 6) and dead bacteria (table 3) at 24 hours and 48 hours (24 hours for comparison, t ═ 2.29, P ═ 0.071; 48 hours for comparison, t ═ 1.45, P ═ 0.21). Thus, the EEC and EC skin tests were able to discriminate live and dead H37Rv infections, whereas TB-PPD was not.

Example seven: test result of EEC skin test of dead bacteria-sensitized guinea pig H37Ra

White SPF-grade guinea pigs which were healthy and not tested at all and which were negative for the TB-PPD skin test, weigh 300g to 500 g. Separately, 0.2ml of 50mg/ml inactivated Mycobacterium tuberculosis H37Ra was injected subcutaneously into the inguinal region of the guinea pig. Carrying out skin test after 5-6 weeks of guinea pig sensitization, unhairing the vertebral column of the guinea pig bilaterally, and injecting 0.2ml of EEC each diluted concentration sample, TB-PPD and EC standard substance intradermally; the longitudinal diameter and the transverse diameter (mm) of the red swelling and/or induration of the injection part are measured 24 and 48 hours after the skin test by a double blind method, and the average value of the longitudinal diameter and the transverse diameter is used as the skin test reaction diameter of the injection sample at the point. The cumulative skin test reactions at 24 hours, 48 hours, 24 hours and 48 hours are shown in table 4.

Table 4 the results show: the skin test of H37Rv dead bacteria-sensitized guinea pigs shows negative reactions in EEC and EC standard skin tests, and basically shows positive reaction in TB-PPD skin test. Comparison of the skin test with live H37Ra (FIG. 7) and dead (Table 4) sensitized guinea pig TB-PPD showed no statistical difference (24 hours, t-1.49, P-0.20; 48 hours, t-0.72, P-0.51); it is shown that EEC and EC skin tests can distinguish between live and dead H37Ra infections, while TB-PPD cannot distinguish between live and dead H37Ra infections.

Example eight: BCG sensitized guinea pig EEC skin test

White SPF-grade guinea pigs that were healthy and not tested at all and that were negative for TB-PPD skin test weigh 300g-500 g. Washing BCG culture bacteria from the culture medium with sterilized normal saline, centrifuging at 6000r/min for 30min, weighing, and diluting with normal saline to obtain 50mg/ml bacteria solution. 0.2ml of 50mg/ml BCG solution was injected subcutaneously into the inguinal region of guinea pig. Carrying out skin test after 5-6 weeks of guinea pig sensitization, unhairing the vertebral column of the guinea pig bilaterally, and injecting 0.2ml of EEC each diluted concentration sample, TB-PPD and EC standard substance intradermally; the longitudinal diameter and the transverse diameter (mm) of the red swelling and/or induration of the injection part are measured 24 and 48 hours after the skin test by a double blind method, and the average value of the longitudinal diameter and the transverse diameter is used as the skin test reaction diameter of the injection sample at the point. The skin test reactions at 24 hours, 48 hours and 24 hours and 48 hours are summarized in Table 5.

Table 5 the results show: skin tests of BCG-sensitized guinea pigs show negative reactions to EEC and EC standards, and positive reactions to TB-PPD and BCG-PPD skin tests. The results indicate that the EEC and EC skin tests were able to discriminate between H37Rv infection and BCG vaccination, whereas TB-PPD and BCG-PPD were not able to discriminate between H37Rv infection and BCG vaccination.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

SEQUENCE LISTING

<110> Beijing Enyuanhua Biotech Co., Ltd

<120> Mycobacterium tuberculosis EEC fusion protein, preparation method and application thereof

<130>20190505

<160>7

<170>PatentIn version 3.3

<210>1

<211>95

<212>PRT

<213> Artificial Synthesis

<400>1

Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser

1 5 10 15

Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly

20 25 30

Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser

35 40 45

Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu

50 55 60

Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly

65 70 75 80

Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala

85 90 95

<210>2

<211>288

<212>DNA

<213> Artificial Synthesis

<400>2

atgacagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga 60

aatgtcacgt ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca 120

gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc 180

acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt 240

caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcatag 288

<210>3

<211>100

<212>PRT

<213> Artificial Synthesis

<400>3

Met Ala Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly

1 5 10 15

Asn Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val

20 25 30

Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly

35 40 45

Thr Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys

50 55 60

Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly

65 70 75 80

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

85 90 95

Gln Met Gly Phe

100

<210>4

<211>303

<212>DNA

<213> Artificial Synthesis

<400>4

atggcagaga tgaagaccga tgccgctacc ctcgcgcagg aggcaggtaa tttcgagcgg 60

atctccggcg acctgaaaac ccagatcgac caggtggagt cgacggcagg ttcgttgcag 120

ggccagtggc gcggcgcggc ggggacggcc gcccaggccg cggtggtgcg cttccaagaa 180

gcagccaata agcagaagca ggaactcgac gagatctcga cgaatattcg tcaggccggc 240

gtccaatact cgagggccga cgaggagcag cagcaggcgc tgtcctcgca aatgggcttc 300

tga 303

<210>5

<211>290

<212>PRT

<213> Artificial Synthesis

<400>5

Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser

1 5 10 15

Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly

20 25 30

Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser

35 40 45

Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu

50 55 60

Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly

65 70 75 80

Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Met

85 90 95

Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala

100 105 110

Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly Lys

115 120 125

Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser Glu

130 135 140

Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu Leu

145 150 155 160

Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly Gln

165 170 175

Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Met Ala

180 185 190

Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly Asn Phe

195 200 205

Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val Glu Ser

210 215 220

Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly Thr Ala

225 230 235 240

Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys Gln Lys

245 250 255

Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly Val Gln

260 265 270

Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser Gln Met

275 280 285

Gly Phe

290

<210>6

<211>870

<212>DNA

<213> Artificial Synthesis

<400>6

atgacagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga 60

aatgtcacgt ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca 120

gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc 180

acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt 240

caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcaatgac agagcagcag 300

tggaatttcg cgggtatcga ggccgcggca agcgcaatcc agggaaatgt cacgtccatt 360

cattccctcc ttgacgaggg gaagcagtcc ctgaccaagc tcgcagcggc ctggggcggt 420

agcggttcgg aggcgtacca gggtgtccag caaaaatggg acgccacggc taccgagctg 480

aacaacgcgc tgcagaacct ggcgcggacg atcagcgaag ccggtcaggc aatggcttcg 540

accgaaggca acgtcactgg gatgttcgca atggcagaga tgaagaccga tgccgctacc 600

ctcgcgcagg aggcaggtaa tttcgagcgg atctccggcg acctgaaaac ccagatcgac 660

caggtggagt cgacggcagg ttcgttgcag ggccagtggc gcggcgcggc ggggacggcc 720

gcccaggccg cggtggtgcg cttccaagaa gcagccaata agcagaagca ggaactcgac 780

gagatctcga cgaatattcg tcaggccggc gtccaatact cgagggccga cgaggagcag 840

cagcaggcgc tgtcctcgca aatgggcttc 870

<210>7

<211>884

<212>DNA

<213> Artificial Synthesis

<400>7

ccatgggcac cgaacagcag tggaacttcg caggcattga agcggcggct tctgcaatcc 60

agggtaacgt tacctctatt cattctctgt tagatgaagg taaacagagc ctgaccaaac 120

tggctgctgc atggggtggt agcggtagtg aagcgtatca gggtgttcag caaaaatggg 180

acgcaactgc aactgaactg aacaatgcac ttcagaacct ggctcgtacc atctctgaag 240

caggccaggc tatggcgagc accgaaggta atgtgactgg tatgttcgca atgactgaac 300

aacagtggaa ttttgcgggt atcgaagcag ctgcatctgc aattcagggt aacgtgacct 360

ctatccactc tctgctcgat gaaggtaaac agtctttaac taaactggcc gcagcatggg 420

gtggttctgg ttctgaagca taccagggtg tgcagcagaa atgggatgct actgctaccg 480

aattaaacaa cgcgttacaa aacctggcgc gtactatttc tgaagcaggt caggctatgg 540

cttctactga aggtaacgta acgggtatgt tcgcgatggc tgaaatgaaa actgatgcgg 600

ctaccctggc tcaagaagct ggtaactttg aacgtattag cggtgacctg aaaactcaga 660

ttgatcaagt tgaatctacc gctggttctc tgcaaggtca gtggcgtggt gctgctggta 720

ccgctgctca ggctgcagtt gttcgctttc aagaagcggc gaacaaacag aaacaggaac 780

tggatgaaat cagcaccaac atccgtcagg ctggtgtgca gtatagccgt gctgatgaag 840

aacagcagca ggcactgtct tctcagatgg gtttctaagg atcc 884

<210>8

<211>884

<212>PRT

<213> Artificial Synthesis

<400>8

Met Gly Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala

1 5 10 15

Ser Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu

20 25 30

Gly Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly

35 40 45

Ser Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr

50 55 60

Glu Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala

65 70 75 80

Gly Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala

85 90 95

Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser

100 105 110

Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly

115 120 125

Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser

130 135 140

Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu

145 150 155 160

Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly

165 170 175

Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Met

180 185 190

Ala Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly Asn

195 200 205

Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val Glu

210 215 220

Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly Thr

225 230 235 240

Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys Gln

245 250 255

Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly Val

260 265 270

Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser Gln

275 280 285

Met Gly Phe

290

Claims

1. A Mycobacterium tuberculosis EEC fusion protein, which is characterized in that: has an amino acid sequence shown as SEQ ID NO. 8.

2. The Mycobacterium tuberculosis EEC fusion protein of claim 1, wherein: the SEQ ID NO.8 is an amino acid sequence obtained by inserting 1 glycine between the first amino acid and the second amino acid of SEQ ID NO. 5; the SEQ ID NO.5 is 2 amino acid sequences shown in SEQ ID NO.1 and 1 amino acid sequence shown in SEQ ID NO.3 which are connected in sequence.

3. A Mycobacterium tuberculosis EEC fusion protein according to claim 1 or 2, wherein: the polynucleotide sequence for coding the fusion protein is shown as SEQ ID NO. 7.

4. The Mycobacterium tuberculosis EEC fusion protein of claim 3, wherein the polynucleotide vector is PET28a-c, PET15b or PET30a plasmid, more preferably PET28a plasmid.

5. The Mycobacterium tuberculosis EEC fusion protein of claim 4, wherein: the host cell of the vector is a prokaryotic host cell; preferably Escherichia coli, more preferably Escherichia coli BL21(DE 3).

6. A method for preparing an EEC fusion protein of Mycobacterium tuberculosis according to any one of claims 1-5, characterized in that: expressing the polynucleotide of the EEC fusion protein in a suitable host cell, comprising the steps of:

(1) obtaining a polynucleotide;

(2) introducing the polynucleotide into a vector to construct a recombinant vector;

(3) introducing the vector obtained in step (2) into a host cell;

(4) culturing the host cell under conditions conducive to expression of the polynucleotide; and

(5) harvesting the host cells, disrupting the host cells, and purifying the protein.

7. A tuberculosis diagnostic kit of Mycobacterium tuberculosis fusion protein EEC as described in any one of claims 1-5.

8. Tuberculosis diagnostic kit according to claim 7, characterized in that: the diagnosis is that the EEC protein is used for immunological diagnosis of tuberculosis latent infection and tuberculosis patients, and the immunological diagnosis comprises skin test, cytokine release test and serological diagnosis.

9. Use of a mycobacterium tuberculosis EEC fusion protein according to any one of claims 1-5, wherein: is applied to the preparation of vaccines for preventing or treating tuberculosis.

10. The use of the EEC fusion protein of Mycobacterium tuberculosis as claimed in claim 9, wherein: the vaccine can be used for preventing people without tubercle bacillus infection, and treating tuberculosis infection people, tuberculosis patients with drug resistance or chronic tuberculosis patients.