CN116041543A - Mycobacterium tuberculosis multi-antigen fusion protein, encoding gene and application thereof - Google Patents
Mycobacterium tuberculosis multi-antigen fusion protein, encoding gene and application thereof Download PDFInfo
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- CN116041543A CN116041543A CN202211567096.0A CN202211567096A CN116041543A CN 116041543 A CN116041543 A CN 116041543A CN 202211567096 A CN202211567096 A CN 202211567096A CN 116041543 A CN116041543 A CN 116041543A
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
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Abstract
The invention relates to the technical field of biological medicine, in particular to a mycobacterium tuberculosis multi-antigen fusion protein, and a coding gene and application thereof. The mycobacterium tuberculosis multi-antigen fusion protein comprises an Rv3875 protein antigen, an Rv2628 protein antigen and an Rv1886c protein antigen. The mouse immune test proves that the fusion protein antigen of three proteins of the mycobacterium tuberculosis provided by the invention has stronger immunogenicity, can stimulate organisms to generate effective humoral immunity and cellular immune response, and can be used as tuberculosis vaccine.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a mycobacterium tuberculosis multi-antigen fusion protein, and a coding gene and application thereof.
Background
Mycobacterium tuberculosis (Mycobacterium tuberculosis, mtb) is a pathogen causing tuberculosis, which is an intracellular parasitic bacterium that can survive in macrophages for years, posing a great threat to human health. With the increasing severity of drug-resistant tuberculosis and the aggravation of HIV-tuberculosis co-infection, the prevention and control of tuberculosis face great challenges. At present, the prevention and control of tuberculosis mainly relates to three aspects of prevention, diagnosis and treatment, wherein the prevention is particularly important, and the most effective prevention approach for infectious diseases is vaccination. Live attenuated mycobacterium bovis vaccine-BCG is the only vaccine currently used worldwide for the prevention of tuberculosis in humans. Although numerous studies have shown that vaccination with BCG is effective in preventing tuberculosis in children, it is also possible to provide tuberculosis protection for leprosy patients. However, the protective effect of BCG is limited in duration and can last for about 5-10 years. Meanwhile, BCG has a large difference in effect of preventing adult mycobacterium tuberculosis infection, from 0 to 80%, so that no protective effect is considered. In addition, inoculation of BCG is detrimental to detection of mycobacterium tuberculosis infection. Based on the problems of limited protection time, unstable protection effect and the like of BCG, research on more effective novel tuberculosis vaccines is urgent.
In recent years, research on novel tuberculosis vaccines has become a hotspot in the field of tuberculosis research, and 16 different candidate vaccines are counted to enter clinical trials at present, and the vaccine mainly comprises recombinant BCG, subunit vaccine, viral vector vaccine, DNA vaccine, RNA vaccine and the like. Subunit vaccines have become a research hotspot of novel tuberculosis vaccines because of the advantages of safety, controllable production quality, low cost, convenient transportation, easy mass production and the like. The candidate antigens of the current tuberculosis recombinant subunit vaccine are mainly secreted proteins and cell wall proteins, such as heat shock protein HspX, early secreted protein ESAT-6, filtrate protein CFP-10 and the like. ESAT-6 is a virulence factor of mycobacterium tuberculosis, can be used for regulating host immune response singly or in combination with CFP-10, and has been proved to be capable of stimulating the organism to generate stronger immune response and providing better protection effect when the vaccine contains ESAT-6. The Ag85 compound is a group of mycobacteria secretory proteins with stronger cellular immunity and humoral immunity activity, including Ag85A, ag B and Ag85C, and researches prove that the Ag85 compound can induce experimental animals to generate cellular immunity and specific antibodies and enhance anti-tuberculosis capability, so that the compound is also a current vaccine research hotspot.
At present, most of preventive vaccines entering a clinical test stage and a research stage are live vaccines or virus vector vaccines, the live vaccines have a certain risk for people with low immunity, the virus vector vaccines have higher cost, and the construction and purification processes are complex. In addition, although a plurality of antigens expressed in multiple stages are selected, expression and purification of at least two antigens are involved in the preparation, and most of the used adjuvants are non-single components, which results in complex preparation process. Chinese patent, application number CN201310273393.9, publication No. 2017-02-08, a combined adjuvant tuberculosis subunit vaccine, which discloses a vaccine containing Ag85b protein and ESAT6-CFP10 fusion protein as antigen components, and aluminum and polyIC as composite adjuvants. Chinese patent, application number CN201410194559.2, publication No. 2014-05-09, an enhanced tuberculosis subunit vaccine, discloses a vaccine comprising Ag85b protein, ESAT6-CFP10 fusion protein, aluminum adjuvant, BCG-CpG adjuvant, and PolyIC adjuvant. Chinese patent, application number CN201810902885.2, publication No. 2018-12-25, a vaccine for preventing tuberculosis, a combined medicament, a preparation method and application thereof, which discloses a vaccine for preventing tuberculosis containing a mycobacterium microorganism composite adjuvant, comprising Ag85b protein, ESAT6 CFP10 protein and a cow mycobacterium extract. Although the above 3 patents disclose ESAT-6 and Ag85B antigens, the antigen components thereof are a mixture, i.e., a mixture of multiple antigens, which involves the expression and purification of multiple antigens, increasing costs and workload; meanwhile, two or more than two adjuvants are used in the patent CN201310273393.9 and the patent CN201410194559.2, so that the vaccine has a complex formula and a long treatment period.
Disclosure of Invention
In view of the technical problems in the background art, the invention provides a mycobacterium tuberculosis multi-antigen fusion protein which is convenient to express and purify. The mycobacterium tuberculosis multi-antigen fusion protein has stronger immunogenicity, can stimulate organisms to generate effective humoral immunity and cellular immunity reaction, and can be used as tuberculosis vaccine.
The invention provides a mycobacterium tuberculosis multi-antigen fusion protein, which comprises an Rv3875 protein antigen, an Rv2628 protein antigen and an Rv1886c protein antigen.
Preferably, the Rv3875 protein antigen, the Rv2628 protein antigen and the Rv1886c protein antigen are serially connected in sequence.
Preferably, the Rv3875 protein antigen, the Rv2628 protein antigen and the Rv1886c protein antigen are sequentially connected in series through a linker, wherein the linker comprises a flexible connecting arm, and the sequence of the flexible connecting arm comprises SEQ ID No.3.
Preferably, the amino acid sequence of the mycobacterium tuberculosis multi-antigen fusion protein comprises SEQ ID NO.2
Or the amino acid sequence shown in SEQ ID NO.2 has the same function by replacing, deleting or adding one or more amino acids.
The invention provides a coding gene of the mycobacterium tuberculosis multi-antigen fusion protein, wherein the coding gene comprises an Rv3875 gene, an Rv2628 gene and an Rv1886c gene; the Rv3875 gene, the Rv2628 gene and the Rv1886c gene are sequentially connected in series.
Preferably, the nucleotide sequence of the coding gene comprises SEQ ID NO.1;
or the nucleotide sequence with the same function and formed by replacing, deleting or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 1.
The invention provides a biological material for expressing the mycobacterium tuberculosis multi-antigen fusion protein, which comprises an expression vector and engineering bacteria, and comprises the coding gene.
Preferably, the base vector of the expression vector comprises one of pET43.1a, pET32a and pET28 a.
The invention provides a mycobacterium tuberculosis protein antigen mixture, which comprises an Rv3875 protein antigen, an Rv2628 protein antigen and an Rv1886c protein antigen;
or comprises Rv3875 protein antigen, rv2628 protein antigen, rv1886c protein antigen and the mycobacterium tuberculosis multi-antigen fusion protein.
The invention provides application of the mycobacterium tuberculosis multi-antigen fusion protein or the coding gene thereof, or the biological material, or the mycobacterium tuberculosis antigen protein mixture in preparing mycobacterium tuberculosis vaccines.
The invention provides a mycobacterium tuberculosis vaccine, the effective components of which comprise the mycobacterium tuberculosis multi-antigen fusion protein, or the encoding gene, or the mycobacterium tuberculosis multi-antigen fusion protein obtained by expression of the expression vector, or the mycobacterium tuberculosis multi-antigen fusion protein prepared by the engineering bacteria, or the mycobacterium tuberculosis protein antigen mixture.
Preferably, the effective component of the mycobacterium tuberculosis vaccine further comprises an aluminum salt adjuvant.
The invention also provides application of the mycobacterium tuberculosis multi-antigen fusion protein, or the coding gene, or the biological material in preparing mycobacterium tuberculosis detection reagents or medicines for treating mycobacterium tuberculosis diseases.
The beneficial effects are that:
the invention selects 3 antigens of mycobacterium tuberculosis, namely Rv3875 (ESAT-6), rv2628 and Rv1886c (Ag 85B), and forms a novel mycobacterium tuberculosis multi-antigen fusion protein. The mouse immunity test proves that the mycobacterium tuberculosis multi-antigen fusion protein has stronger immunogenicity, can stimulate organisms to generate effective humoral immunity and cellular immunity reaction, and especially plays an important role in clearing mycobacterium tuberculosis for Th-1 type immunity reaction, which indicates that the mycobacterium tuberculosis multi-antigen fusion protein provided by the invention can be used as tuberculosis vaccine. In vitro infection experiments prove that the mycobacterium tuberculosis multi-antigen fusion protein can effectively inhibit the growth and reproduction of mycobacterium tuberculosis H37Rv, and has better protection effect. Meanwhile, the mycobacterium tuberculosis multi-antigen fusion protein can play an important role in diagnosis of tuberculosis.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be described below.
FIG. 1 is a schematic diagram of the structure of ERA005f in the recombinant plasmid according to example 1 of the present invention;
FIG. 2 is a diagram showing SDS-PAGE results after protein renaturation in example 2 and example 3 of the present invention; FIG. 2A is a diagram showing the results of purification and identification of a multi-antigen fusion expression protein ERA005f, wherein M represents the standard molecular mass of the protein, and 1 represents ERA005f; FIG. 2B is a graph showing the results of expression purification and identification of 3 single antigens, wherein M represents a protein quality standard, 1 represents Rv3875,2 represents Rv2628, and 3 represents Rv1886c; FIG. 2C is a graph showing the results of expression and identification of a multi-antigen fusion expression protein ERA005f, wherein M represents a protein quality standard, 1 represents a whole-cell expression product, 2 represents a soluble expression protein, and 3 represents an inclusion body expression protein.
FIG. 3 shows IgG antibodies and IgG subtype antibody titers in serum after immunization of example 4 of the present invention; panel A of FIG. 3 shows IgG antibody titers (immune panel on the abscissa and log of antibody titers on the ordinate) 2 ) Panel B of FIG. 3 shows IgG subtype antibody titers (immune panel on the ordinate and log of antibody titers on the abscissa) 2 )；
FIG. 4 shows the expression level of Th-1 type cytokines in lymphocytes in spleen according to example 4 of the present invention, wherein FIG. 4A shows CM-GSF, FIG. 4B shows TNF- α, FIG. 4C shows IL-12, and FIG. 4D shows IFN- γ;
FIG. 5 shows the expression level of Th-2 type cytokines in lymphocytes in the spleen of example 4 of the present invention, wherein FIG. 5A shows IL-4 and FIG. 5B shows IL-10;
FIG. 6 shows the expression level of Th-17 type cytokines in lymphocytes in the spleen of example 4 of the present invention, wherein FIG. 6A shows IL-17 and FIG. 6B shows IL-6;
FIG. 7 is a graph of ELISPOT results for spleen lymphocytes according to example 4 of the present invention, wherein FIG. 7 shows IFN-. Gamma.in panel A and IL-4 in panel B;
FIG. 8 is a graph showing the results of in vitro toxicity test of the Mycobacterium tuberculosis growth inhibition test described in example 5 of the present invention.
FIG. 9 shows the secretion amount of Th1 type cytokines by spleen lymphocytes according to example 6 of the present invention, wherein FIG. 9A shows IFN-. Gamma.and FIG. 9B shows TNF-. Alpha.and FIG. 9C shows IL-12.
Detailed Description
The invention provides a mycobacterium tuberculosis multi-antigen fusion protein, which comprises an Rv3875 protein antigen, an Rv2628 protein antigen and an Rv1886c protein antigen. In the present invention, the Rv3875 protein antigen, the Rv2628 protein antigen and the Rv1886c protein antigen are preferably serially connected by a linker, which preferably includes a flexible linker arm, the sequence of which preferably includes GGSGG (SEQ ID No. 3). For more convenient detection and purification, the present invention preferably adds 6His to the C-terminal end of the Mycobacterium tuberculosis multi-antigen fusion protein. In a more preferred embodiment of the present invention, the Mycobacterium tuberculosis multi-antigen fusion protein is ERA005f, the amino acid sequence of which comprises:
MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAGGSGGSTQRPRHSGIRAVGPYAWAGRCGRIGRWGVHQEAMMNLAIWHPRKVQSATIYQVTDRSHDGRTARVPGDEITSTVSGWLSELGTQSPLADELARAVRIGDWPAAYAIGEHLSVEIAVAVGGSGGTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGGGHHHHHH*(SEQ ID NO.2)。
because of the ubiquitous spatial conformation problem of artificially tandem protein fragments, the complete expression is not achieved, or the expected biological effect is not achieved, although Rv3875 (ESAT-6) and Rv1886c (Ag 85B) are currently known antigens with strong immunogenicity, rv2628 is an antigen with strong immunogenicity found in the earlier studies of the present invention. However, there is no report on the fusion of Rv3875 (ESAT-6), rv2628 and Rv1886c (Ag 85B) for tuberculosis vaccine development. According to the invention, the Rv3875 protein antigen, the Rv2628 protein antigen and the Rv1886c protein antigen are sequentially connected in series, and the mycobacterium tuberculosis multi-antigen fusion protein obtained by expression can remarkably improve the humoral immunity level and the cellular immunity level of mice compared with the three independent antigens and the mixture thereof; especially, the expression of Th-1 cytokines is obviously improved, and the expression plays an important role in the process of clearing mycobacterium tuberculosis; and can obviously inhibit the growth and proliferation of mycobacterium tuberculosis H37Rv in mouse macrophages.
The invention provides a coding gene of the mycobacterium tuberculosis multi-antigen fusion protein, which comprises an Rv3875 gene, an Rv2628 gene and an Rv1886c gene. In the present invention, the Rv3875 gene, rv2628 gene and Rv1886c gene are preferably serially connected. In a more preferred embodiment of the present invention, the nucleotide sequence of the coding gene comprises:
ATGACCGAACAGCAGTGGAATTTTGCCGGTATTGAAGCAGCCGCCAGTGCAATTCAGGGCAATGTTACCAGCATTCATAGCCTGCTGGATGAAGGCAAACAGAGCCTGACCAAACTGGCAGCCGCATGGGGTGGTAGCGGCAGCGAAGCATATCAGGGTGTGCAGCAGAAATGGGATGCCACCGCCACCGAACTGAATAATGCACTGCAGAATCTGGCCCGCACCATTAGTGAAGCCGGCCAGGCAATGGCAAGTACCGAAGGCAATGTTACAGGCATGTTTGCAGGCGGCAGCGGCGGTAGTACCCAGCGTCCTCGTCATAGTGGCATTCGCGCAGTTGGCCCGTATGCCTGGGCAGGTCGCTGTGGCCGCATTGGTCGCTGGGGCGTGCATCAGGAAGCAATGATGAATCTGGCCATTTGGCATCCGCGTAAAGTGCAGAGTGCAACCATCTATCAGGTGACCGATCGCAGTCATGATGGCCGTACCGCCCGTGTTCCGGGTGACGAAATTACCAGCACCGTGAGCGGCTGGCTGAGTGAACTGGGTACCCAGAGCCCGCTGGCAGATGAACTGGCCCGCGCCGTTCGTATTGGTGACTGGCCGGCAGCCTATGCAATTGGCGAACATCTGAGCGTTGAAATTGCCGTTGCAGTGGGTGGCAGTGGCGGCACCGATGTGAGTCGTAAAATTCGTGCATGGGGTCGTCGTCTGATGATTGGTACCGCCGCCGCAGTTGTTCTGCCGGGTCTGGTTGGCCTGGCCGGTGGTGCTGCAACCGCAGGTGCATTTTCTCGTCCGGGCCTGCCGGTTGAATATCTGCAGGTGCCGAGTCCGAGTATGGGCCGTGATATTAAGGTTCAGTTTCAGAGTGGTGGTAATAATAGCCCGGCCGTGTATCTGCTGGATGGTCTGCGCGCACAGGATGATTATAATGGTTGGGATATTAATACCCCGGCCTTTGAATGGTATTATCAGAGCGGTCTGAGCATTGTGATGCCGGTGGGCGGCCAGAGCAGTTTTTATAGTGATTGGTATAGTCCGGCATGTGGTAAAGCAGGTTGTCAGACCTATAAATGGGAAACCTTTCTGACCAGCGAACTGCCGCAGTGGCTGAGTGCAAATCGTGCCGTTAAACCGACCGGTAGTGCCGCCATTGGTCTGAGCATGGCCGGCAGTAGCGCCATGATTCTGGCCGCATATCATCCGCAGCAGTTTATCTATGCAGGCAGTCTGAGTGCCCTGCTGGATCCGAGCCAGGGCATGGGCCCGAGCCTGATTGGTCTGGCAATGGGTGACGCAGGCGGTTATAAAGCCGCAGATATGTGGGGCCCGAGTAGTGATCCGGCCTGGGAACGCAATGATCCGACCCAGCAGATTCCGAAACTGGTGGCAAATAATACCCGCCTGTGGGTGTATTGCGGCAATGGCACCCCGAATGAACTGGGTGGCGCCAATATTCCGGCCGAATTTCTGGAAAATTTTGTTCGCAGCAGCAATCTGAAATTTCAGGATGCCTATAATGCCGCAGGTGGTCATAATGCAGTTTTTAATTTTCCGCCGAATGGCACCCATAGCTGGGAATATTGGGGTGCCCAGCTGAATGCAATGAAAGGCGATCTGCAGAGTAGCCTGGGTGCCGGTGGCGGTCATCATCATCATCACCATTAA(SEQ ID NO.1)。
the invention obtains the gene sequences of Rv3875, rv2628 and Rv1886C from the genome of the mycobacterium tuberculosis H37Rv, adds a 6His tag at the C end after codon optimization, and synthesizes the sequence SEQ ID NO.1 according to Rv3875-linker-Rv2628-linker-Rv1886C-6His (flexible connecting arm 'GGSGG' is selected by linker); the codon-optimized SEQ ID NO.1 can obtain higher expression quantity in the expression process of the fusion protein.
The invention provides a biological material for expressing the mycobacterium tuberculosis multi-antigen fusion protein, which comprises an expression vector and engineering bacteria, and comprises the coding gene. In the present invention, the base vector of the expression vector preferably includes one of pet43.1a, pET32a and pET28 a. In a preferred embodiment of the invention, the expression vector comprises pET43.1a-ERA005f, said pET43.1a-ERA005f being obtained by ligating a nucleotide sequence comprising said coding gene into the pET43.1a vector. In a more preferred embodiment of the invention, the invention inserts SEQ ID NO.1 into the expression vector pET43.1a by NdeI and XhoI to give a recombinant plasmid pET43.1a-ERA005f.
In the invention, the engineering bacteria comprise the coding gene. In a more preferred embodiment of the invention, the pET43.1a-ERA005f recombinant plasmid is transferred into competent cells of escherichia coli BL21 (DE 3) to obtain ERA005f engineering bacteria, and a large amount of inclusion body fusion proteins ERA005f can be obtained after IPTG induction of the ERA005f engineering bacteria at 37 ℃, and ERA005f proteins with activity can be obtained after purification and renaturation.
The invention provides a mycobacterium tuberculosis protein antigen mixture, which comprises an Rv3875 protein antigen, an Rv2628 protein antigen and an Rv1886c protein antigen. Based on the previous research, the invention finally obtains the mycobacterium tuberculosis protein antigen mixture comprising the 3 protein antigens through heuristic screening. Compared with the three independent antigens, the mycobacterium tuberculosis protein antigen mixture has high immunity intensity and can better help organisms resist the infection of mycobacterium tuberculosis.
The invention provides a mycobacterium tuberculosis vaccine, the effective components of which comprise the mycobacterium tuberculosis multi-antigen fusion protein, or the encoding gene, or the mycobacterium tuberculosis multi-antigen fusion protein obtained by expression of the expression vector, or the mycobacterium tuberculosis multi-antigen fusion protein prepared by the engineering bacteria, or the mycobacterium tuberculosis protein antigen mixture.
In the present invention, the active ingredient of the mycobacterium tuberculosis vaccine preferably further comprises an aluminum salt adjuvant; the aluminum salt adjuvant preferably comprises an aluminum hydroxide adjuvant.
The invention breaks through the mode of adopting single antigen as a candidate vaccine in the traditional subunit vaccine, adopts a multi-antigen co-expression mode to screen a novel tuberculosis vaccine, and simultaneously selects protein antigens expressed in different growth periods of mycobacterium tuberculosis, thereby improving the immunogenicity of multi-antigen fusion proteins. Under the condition that aluminum salt is used as an adjuvant, the immunogenicity of ERA005f protein is obviously higher than that of a single antigen mixture, and the expression of Th-1 cytokines such as IFN-gamma, GM-CSF, TNF-alpha, IL-12 and the like is obviously improved. In addition, the present invention demonstrates that: ERA005f also significantly increased the expression of the Th-17 type cytokines IL-17 and IL-6 compared to the single antigen mixture. Th-1 and Th-17 cytokines play an important role in anti-tuberculosis infection, and are also markers of organism generation of protective immune response against mycobacterium tuberculosis. Meanwhile, the in-vitro toxicity attack experiment result proves that the growth and proliferation of the mycobacterium tuberculosis H37Rv can be effectively inhibited after the ERA005f is immunized.
The invention also provides application of the mycobacterium tuberculosis multi-antigen fusion protein, or the coding gene, or the expression vector, or the engineering bacterium, or the mycobacterium tuberculosis protein antigen mixture in preparation of a mycobacterium tuberculosis detection reagent or a medicament for treating mycobacterium tuberculosis diseases.
The fusion protein provided by the invention can improve Th1 cell immunity of organisms, so that the antigen related in the invention can help tuberculosis patients to improve specific immune response aiming at mycobacterium tuberculosis, and has an important effect on clearing mycobacterium tuberculosis.
Mycobacterium tuberculosis is an intracellular parasitic bacterium that, after invasion into the body, mainly causes cellular immune responses. Namely, the clearing effect of the organism on the mycobacterium tuberculosis is mainly realized by secreting cytokines to participate in the processes of immunoregulation or opsonophagocytosis and the like, so that the range and the intensity of the cytokine expression profile are important to the effect of immune protection in the whole immune reaction process. Current studies have found that cytokines that are secreted mainly after Mycobacterium tuberculosis infects humans include IFN-r, IL-1 alpha, IL-1 beta, IL-18, TNF-alpha, IL-2, IL-4, IL-6, IL-17, and the like. The gamma interferon release test is a high-sensitivity and high-specificity detection method for diagnosing tuberculosis infection, which is currently accepted; t lymphocytes stimulated by specific antigens of mycobacterium tuberculosis release gamma interferon, and whether the infection is caused is judged by detecting the release intensity of the gamma interferon. After antigen stimulation provided by the invention, the capacity and strength of the sample for releasing gamma interferon are obviously higher than those of a control group. Therefore, the antigen provided by the invention has the potential of being used as a detection reagent. Similarly, the detection of the secretion levels of various cytokines, such as IL-2, IFN-gamma, IL-4, IL-6, IL-12, IL-17, etc., has been currently included in the evaluation of molecular markers for tuberculosis infection detection. After antigen stimulation provided by the invention, the secretion level of the cytokines is obviously changed compared with that of a control group, so that the cytokines can be used as potential molecular detection markers. The secretion level of protective cytokines is the most direct manifestation of cellular immunity; after the antigen is immunized, the level of the cytokine secretion and the type of the cytokine secretion are obviously increased or not lower than those of a control group. Research results show that when the organism is applied to medicaments prepared by the corresponding antigens, the organism can secrete cytokines with higher intensity and wider protectiveness, or assist an immune system, regulate an immune network and secretion of the cytokines, and help tuberculosis infected patients establish immune responses to pathogen infection. Therefore, the biological agent prepared based on the antigen provided by the invention has the potential of being a medicine for treating tuberculosis infection.
The invention adopts a prokaryotic expression system to express the protein, is suitable for large-scale commercial production, and has lower cost. The detection reagent based on the invention can be widely used in the related fields of auxiliary diagnosis, epidemiological monitoring, infection screening and the like of tuberculosis, and provides a new thought for developing new anti-tuberculosis vaccines.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention. Unless otherwise indicated, all the experimental procedures used in the examples were conventional; the materials, reagents and the like used are all commercially available.
Example 1 construction of fusion protein ERA005f
The gene sequences of Rv3875, rv2628 and Rv1886c were downloaded at NCBI and codons were optimized as e.coli preferred codons. After editing the sequence according to Rv3875-linker-Rv2628-linker-Rv1886c-6His (linker is GGSGG), the gene was synthesized by general organism (Anhui) Co., ltd and the target gene was inserted into expression vector pET43.1a, designated pET43.1a-ERA005f. The structure of ERA005f in recombinant plasmid is shown in scheme 1. After synthesis, the gene is sequenced, and after successful gene sequencing, the gene is used for subsequent experiments.
Example 2 expression of fusion protein ERA005f
1. Transformation of fusion protein ERA005f
The synthesized recombinant plasmid pET43.1a-ERA005f dry powder is centrifuged at 12000rpm/min for 2min, 50 μl of deionized water is added for dissolution, 5 μl of the recombinant plasmid is added into competent cells (Beijing holo-gold organism) of escherichia coli BL21 (DE 3), the mixture is placed on ice for 30min, heat shock is conducted at 42 ℃ for 90s, ice bath is conducted for 2min, 900 μl of LB liquid culture medium is added, shaking culture is conducted at 37 ℃ for 1h, centrifugation is conducted at 4000rpm for 1min, 600 μl of supernatant is discarded, then gentle blowing and mixing are conducted, 200 μl of bacterial liquid is coated on an LB solid plate containing ampicillin, and culture is conducted at 37 ℃ for 16h. Single colonies were picked up in 5ml LB liquid medium containing ampicillin, cultured with shaking at 37℃for 12h and then sent to Beijing qing department of biological Co.Ltd for sequencing. The strain with successful sequencing is subjected to expansion culture to extract plasmid preservation at-70 ℃ for subsequent experiments.
2. Expression of fusion protein ERA005f
(1) Marking out BL21 (DE 3) engineering bacteria transformed and identified in the last step on an LB solid medium containing ampicillin, and inversely culturing at 37 ℃ for 16-20h;
(2) Picking single colony in 5ml LB liquid medium containing ampicillin, shaking and culturing at 37 ℃ and 180rpm/min for overnight;
(3) Adding the cultured overnight bacterial liquid into 300ml of LB liquid medium containing ampicillin (Beijing Soy Bao technology Co., ltd.) and culturing until the OD value of the bacterial liquid is 0.6-0.8, adding isopropyl-beta-D-thiopyran galactoside (IPTG) with the final concentration of 1mmol/L, and inducing and expressing for 4 hours at 37 ℃;
(4) Centrifuging the bacterial liquid at 4000rpm for 10min to collect bacterial cells, re-suspending the bacterial cells by using 10mmol/L Tris-HCl solution with pH of 8.0 and containing 0.5% Triton-X100, and performing ultrasonic crushing (220W, ultrasonic treatment for 15s at intervals of 20s,10 min);
(5) Taking 100 μl of post-ultrasound bacterial suspension, centrifuging at 12000rpm for 1min, taking out the supernatant, re-suspending the precipitate with 100 μl of deionized water, and performing SDS-PAGE electrophoresis to detect recombinant protein expression. The results are shown in figure 2, panel C; the results show that the protein is expressed mainly in inclusion body form.
Example 3 purification and renaturation of fusion protein ERA005f
1. Ni affinity chromatography purification
a. After ultrasonic crushing and centrifugation, the precipitate is resuspended by 6M guanidine hydrochloride (Beijing national medicine), and imidazole is added to a final concentration of 5mmol/L;
b. packing 25ml GE Healthcare streamline chelating Ni NTA (GE healthcare) into GE 26X 16 columns;
c. pumping 10mmol/L Tris-HCl with pH of 8.0 and 2-5 times of column volume into the packing until the pH of effluent liquid is 8.0;
d. 40ml of 1mmol/L NiSO were pumped into the packing 4 The solution was then pumped further with 10mmol/L Tris-HCl pH8.0 to an effluent pH of 8.0;
e. pumping base solution (6M guanidine hydrochloride+5 mmol/L imidazole) for zeroing;
f. pumping the treated sample into a filler, observing the change of an OD value, and collecting liquid;
g. pumping 8M urea and 5mmol/L imidazole until the OD value is unchanged;
h. eluting with 8M urea containing 30mmol/L, 60mmol/L and 300mmol/L imidazole respectively, and collecting the liquid of each peak value of the elution;
i. pure water was pumped into 3-5 column volumes and the collected samples were subjected to SDS-PAGE to identify ERA005f protein eluting predominantly at 60mmol/L imidazole, but containing a band.
2. DEAE anion exchange purification
a. The DEAE Stremline chromatography packing is filled, and 10mmol/L of Tris-HCl (pH8.0) is pumped into 3-5 column volumes;
b. 40ml of 0.2mol/L NaOH solution was pumped in, followed by further pumping in 10mmol/L Tris-HCl pH8.0 to effluent pH=8.0;
c. pumping 8mol/L urea until the value of the protein detector is unchanged, and zeroing;
d. pumping Ni for affinity chromatography to collect a sample, observing the change of OD value and collecting the sample;
e. elution with different concentrations of NaCl (100 mmol/L, 200mmol/L, 400 mmol/L)
f. The eluted peaks were collected and subjected to SDS-PAGE electrophoresis. The detection results are shown in FIG. 2. FIG. 2A shows the results of expression purification and identification of the multi-antigen fusion expression protein ERA005f. The fusion protein with higher purity is obtained by the invention, and can be used for subsequent experiments.
3. Protein renaturation
And (3) loading the purified ERA005f protein into a treated dialysis bag, and dialyzing and renaturating by using a gradient urea concentration reduction method. The renaturated sample is filtered and sterilized by a 0.22 mu m filter membrane, and then the protein concentration is measured by a BCA method, and the protein concentration is split-packed and stored at-20 ℃ for subsequent experiments. In the present invention, 3 antigens of Rv3875, rv2628 and Rv1886c (which can be autonomously synthesized according to the gene sequences downloaded on NCBI) were also used, and the purified samples were detected by SDS-PAGE electrophoresis, as shown in fig. 2. FIG. 2-B shows the results of expression purification assays for 3 single antigens (M: protein quality standard, 1: rv3875,2: rv2628,3: rv1886 c).
As can be seen from FIG. 2, the purified protein sizes of ERA005f, rv3875, rv2628 and Rv1886c were 57.8kDa, 30kDa, 33kDa and 56kDa, respectively, consistent with the expected molecular weight sizes. The results show that the invention obtains high-purity ERA005f protein and 3 corresponding single antigen proteins.
EXAMPLE 4 evaluation of immunogenicity of ERA005f protein
The invention utilizes SPF-grade BABL/c mice to evaluate the immunogenicity of ERA005f protein, adopts a subcutaneous multipoint immunization mode in experiments, separates spleen lymphocytes of the mice after immunization to detect the expression level of multiple cytokines and evaluates the stimulated T cell reaction type in ELISPOT experiments, and simultaneously detects the antibody titer in serum to evaluate the humoral immunity level.
1. Immunization of animals
SPF class 6-8 week old BABL/c female mice (Si Bei Fu (Beijing) biotechnology Co., ltd.) were selected for the experiment, 20 mice were randomly divided into 4 groups of 5 mice each, which were PBS control group, individual adjuvant group (aluminum hydroxide adjuvant), ERA005f protein (50. Mu.g) mixed aluminum hydroxide adjuvant group (antigen to adjuvant volume ratio 3:1), rv3875, rv2628 and Rv1886c mixture (1:1 in terms of molar mass ratio, total 50. Mu.g) mixed adjuvant group, and specific groups are shown in Table 1.
Table 1 animal immunization groups
The invention adopts subcutaneous multipoint immunization mode to immunize 3 times, the interval period is 10 days, and the mice are sacrificed one week after the 3 rd immunization is finished to collect samples.
2. Sample collection
Blood is collected by orbital blood collection before primary immunization and before mice sacrifice, standing for 2h at 37 ℃, centrifuging for 10min at 4000rpm, separating serum, packaging, and storing at-20 ℃ for standby. Meanwhile, spleens of immunized mice were collected for subsequent experiments.
3. Serum antibody titer detection
Specific IgG, igG1 and IgG2a titers were measured in mouse serum:
a. coating an ELISA plate with ERA005f protein and ESAT-6, rv2628 and Ag85B mixed antigen respectively, diluting the antigen to 2 mug/ml, adding 100 mug/ml per hole, sealing the film, and then coating at 4 ℃ overnight;
b. pouring out the coating liquid, washing the PBST plate 5 times, adding 100 μl of sealing liquid (PBST+0.5% skimmed milk powder) into each well, and incubating at 37deg.C for 2 hr;
c. discarding the sealing liquid, washing the plate for 5 times by using PBST, and removing the residual liquid after the last time;
d. 100 μl of diluted serum (serum dilution was diluted by a multiple of 2) was added to each well and incubated at 37deg.C for 2h;
e. the samples were discarded, the plates were washed 6 times with PBST and the residual liquid was removed clean;
f. mu.l of HRP-labeled goat anti-mouse IgG, igG1 and IgG2a antibody (1:10000 dilution, antibodies from Boolon technologies Co., st. Of Suzhou) were added to each well and incubated at 37℃for 1h;
g. washing the plate 6 times and removing residual liquid;
h. adding 50 mu l of TMB color development solution A and 50 mu l B solution into each hole, vibrating for 10s by a vibrator, uniformly mixing, and incubating for 15min at 37 ℃;
i. the reaction was stopped by adding 50. Mu.l of 2M H2SO 4;
j. detecting a 450nm light absorption value by an enzyme label instrument, and recording data;
k. antibody titer value discrimination method: a certain dilution sample OD/negative control OD is more than or equal to 2.1, and the dilution factor is the corresponding antibody titer of the sample when the next dilution factor OD/negative control OD is less than 2.1 (note that the dilution factor is calculated according to 0.05 when the negative sample OD is less than 0.05);
4. cellular immune response detection
(1) Isolation of spleen lymphocytes from mice (spleen was taken one week after the mice were sacrificed after the last immunization)
a. Anaesthetizing the mice with pentobarbital sodium until death, soaking the mice with 75% medical alcohol for sterilization, dissecting the mice, taking out spleen, putting the spleen into RPMI1640, and separating lymphocytes within 1h;
b. the mouse lymphocyte separation of Beijing daceae biotechnology limited company is utilized to separate lymphocytes, and the specific steps are carried out according to the specification;
c. lymphocyte concentration was determined after lymphocyte isolation and adjusted to a concentration of 1-2X 10-6 cells/ml for subsequent experiments.
(2) ELISPOT detection of IL-4 and IFN-gamma secretion in T lymphocytes
IL-4 and IFN-gamma kits were pre-coated using mouse ELISPOT from Daidae Biotechnology Inc., the specific steps are as follows:
a. opening the kit in a biosafety cabinet, adding 200 μl of RPMI-1640 or serum-free culture medium into each hole, standing at room temperature for 5-10min, and deducting;
b. adding 100 μl/well (1-2×10ζ5 cells) of cell suspension, adding 5 μl positive stimulator Canavalia protein as positive control, adding no stimulator as negative control, and adding 10 μl/10 μg of immunized antigen as experimental group;
c. placing at 37 deg.C, 5% CO 2 Culturing in an incubator for 18h;
d. pouring the culture medium, adding 200 μl/hole of deionized water which is cooled, and standing at 4deg.C for 10min;
e. pouring liquid, washing for 5 times by using 1 Xwash buffer 200 μl/hole, and cleaning the liquid by beating after the last washing is finished;
f. 100 μl of diluted biotin-labeled antibody was added to each well at 37deg.C for 1h;
g. pouring the liquid in the holes, washing for 5 times by using 200 μl/hole of a 1 xwash buffer, and cleaning the liquid by beating after the last washing is finished;
h. 100 μl of diluted enzyme-labeled avidin is added into each well, and the temperature is 37 ℃ for 1h;
i. pouring the liquid in the holes, washing for 5 times by using 200 μl/hole of a 1 xwash buffer, and cleaning the liquid by beating after the last washing is finished;
j. adding the AEC color developing solution into each experimental hole, 100 μl/hole, and standing at room temperature in dark for 20-30min (constantly observing change, and stopping dyeing when more spots appear in the holes);
k. pouring the liquid in the holes, uncovering the plate base, repeatedly flushing the front and the back with deionized water, airing the plate base in a ventilation place, and counting spots by using an automatic plate reader after the film is aired, wherein the result is shown in fig. 7. According to fig. 7: ERA005f protein and ESAT-6, rv2628 and Ag85B mixtures all increased T lymphocytes secreting IFN-gamma and IL-4, and ERA005f was superior to the antigen mixture; meanwhile, the partial results also show that after the two vaccine preparations are immunized, the cell number of the Th1 cell factor secreting IFN-gamma is obviously higher than that of the cell secreting the Th2 cell factor IL-4, which indicates that both preparations tend to stimulate the Th1 cell immunity.
(3) Luminex multi-factor detection
The detection is carried out by adopting a luminex liquid chip of Shanghai you Ning vitamin Co., ltd, and the specific operation steps are as follows:
a. the isolated lymphocytes were added to 96-well cell culture plates at 100. Mu.l per well (2X 10. Sup.5cells), 50. Mu.l of stimulating antigen (10. Mu.g, RPMI-1640 dilution), 37℃and 5% CO 2 Culturing in an incubator for 18h;
b. collecting a sample, centrifuging at 4000rpm for 2min, and collecting a supernatant;
c. preparing the reagent, taking the reagent kit out of the refrigerator, and balancing the reagent kit at room temperature for 30min;
standard preparation: the standard substances are 1 bottle in total, diluted according to the dilution mode provided by COA, the diluted solution is RD6-52 fully and uniformly mixed, and then the mixture is kept stand for 15-20min at room temperature, 100ul of each standard substance is taken into the same EP tube, and the volume of 1ml is complemented by RD6-52 to be used as the highest concentration of a standard curve. Sequentially carrying out 3-time dilution, and adding 7 standard substances and 1 Blank;
preparing loads: fully mixing the premixed beads mixture, sucking corresponding volumes according to a proportion, sucking the volume of 500ul beads from 96 holes, adding 5ml of diluent RD1W, and fully mixing for later use;
preparation of detection antibody: fully mixing and premixing the detection antibody mixture, sucking corresponding volumes according to a proportion, sucking the volume of 500ul detection antibody from 96 holes, adding 5ml of diluent RD1W, fully mixing and waiting for use;
PE-streptavidin preparation: fully mixing PE-streptavidin reagent, sucking corresponding volume according to proportion, sucking volume of 220ul PE-streptavidin of 96 holes, adding 5.35ml cleaning liquid, fully mixing for standby;
preparing a cleaning solution: 20ml 25 x rinse+480 ml deionized water;
d. after the beads are resuspended, 50ul of diluted beads are added to each well, and 50ul of standard substances and samples are respectively and correspondingly added to each well according to the arrangement before the experiment, and the shaking table is kept at room temperature for 2 hours;
e. placing the microplate on a magnetic rack for at least 1min to ensure that the beads are adsorbed, cleaning with a cleaning solution for 3 times per 100ul of wells;
f. 50ul of the diluted biotin-labeled detection antibody complex was added to each well, and the same was carried out at room temperature for 1 hour;
g. repeating e;
h. 50ul of diluted streptavidin-labeled PE was added to each well, again at room temperature for 0.5h;
i. repeating e;
j. the beads were resuspended with 100ul of wash solution, incubated for 2min, and checked on the machine.
5. Statistical analysis
Statistical analysis of the results was performed using a one-way anova with a Tukey multiple comparison test in GraphPad Prim 5.0, with P <0.05 being statistically significant as the difference in results.
6. Analysis of results
(1) ERA005f significantly increased antigen-specific IgG antibody titers in mice
The serum one week after 3 immunizations was assayed for antibodies by ELISA, as shown in FIG. 3. According to the illustration in fig. 3: the ERA005f and the mixture of 3 antigens in combination with the aluminum hydroxide adjuvant enhanced specific IgG antibody titers in the serum of immunized mice, and the 3 antigen mixed group was significantly higher than ERA005f group (P <0.0001, fig. 3A). In addition, ERA005f and 3 antigen mixtures combined with aluminum hydroxide adjuvant groups all increased the ratio of IgG1/IgG2a, and ERA005f combined with aluminum hydroxide adjuvant group had the highest ratio (FIG. 3B), indicating that the fusion protein combined with aluminum hydroxide adjuvant can promote Th-1 type immune response.
(2) ERA005f promotes secretion of Th-1 and Th-17 cytokines
Spleens of mice were collected one week after 3 immunizations and lymphocytes were isolated and secretion of IFN-gamma and IL-4 by lymphocytes after immunization antigen stimulation was detected using ELISPot technology. The results are shown in FIG. 4. According to fig. 4: ERA005f promoted secretion of IFN-gamma as well as IL-4, and IFN-gamma was secreted at higher levels than IL-4, compared to the single antigen mixture, indicating that ERA005f tended to promote Th-1 type immune responses.
In addition, the invention also uses a luminex liquid chip to detect Th-1 cytokines (GM-CSF, IFN-alpha, IL-12, IFN-gamma), th-2 cytokines (IL-4 and IL-10) and Th-17 cytokines (IL-6 and IL-17), and the results are shown in figures 4, 5 and 6 respectively. The results show that: of the Th-1 cytokines, ERA005f in combination with aluminium hydroxide adjuvant significantly increased the expression of the other 3 cytokines in addition to GM-CSF, with statistical differences (P < 0.05), especially TNF- α and IFN- γ 3-fold compared to the single antigen cocktail group (FIG. 4). There was no significant difference between the Th-like cytokines IL-4 and IL-10 that stimulated lymphocyte secretion in the body by ERA005f group compared to the single antigen mixed group (FIG. 5). After the mice were immunized subcutaneously with ERA005f in combination with aluminum hydroxide adjuvant, the expression of IL-17 and IL-6, especially IL-6, was significantly increased up to 5000pg/ml, 2-fold more than that of the single antigen mixed group (2200 pg/ml) (FIG. 6).
The above results confirm that: the ERA005f multi-antigen fusion protein can obviously improve Th-1 type and Th-17 type immune responses, promote secretion of Th-1 type and Th-17 type cytokines, and especially Th-1 type cytokines.
EXAMPLE 5 Mycobacterium tuberculosis growth inhibition assay (Mycobacterial Growth Inhibition Assays, MGIAs)
In order to evaluate the ability of ERA005f protein as a tuberculosis vaccine to resist to mycobacterium tuberculosis infection, spleen lymphocytes of immunized mice are co-cultured with mycobacterium tuberculosis:
adding cells into 24-well plate, 200ul (2×10ζ5 cells) per well, adding 50CFU of Mycobacterium tuberculosis H37Rv, mixing, and mixing at 37deg.C with 5% CO 2 Culturing for 96h. The supernatant was discarded, and after 500ul of pre-chilled deionized water was added for 10min, centrifuged at 4000rpm for 5min, the supernatant was collected. After 10-fold gradient dilution of the supernatant, 50ul of stock solution, 10-fold dilution and 100-fold dilution were each applied to a 7H10 solid plate. The growth of single colonies in the plates was observed and recorded at 37℃for about 3 weeks, and the results are shown in FIG. 8. According to fig. 8: ERA005f and the single antigen mixture combined with the aluminum hydroxide adjuvant all obviously inhibit the growth and reproduction of the H37Rv of the mycobacterium tuberculosis, and have statistical differences (P<0.001 But there was no statistical difference between the two groups (P>0.5). The in vitro toxicity attack experiment result shows that: both ERA005f and the single antigen mixture in combination with aluminium hydroxide adjuvants can provide an effective protective effect against mycobacterium tuberculosis.
Example 6 comparison of expression of ERA005f protein, antigen mixture and ESAT-6, rv2628 and Ag85B Th 1-like cytokines
The invention is to verify that ERA005 and the mixed immune effect of three antigens are better than that of a single antigen, the mice are immunized respectively, and the spleen lymphocytes are separated to compare the expression of Th1 cytokines (IFN-gamma, TNF-alpha and IL-12), and the immune mode and period are the same as in example 4. The results are shown in FIG. 9.
The results show that the ERA005f and the three antigen mixtures stimulate IFN-gamma, TNF-alpha and IL-12 secreted by the organism to be higher than ESAT-6, rv2628 and Ag85B single antigen immune groups, and the ERA005f and the three antigen mixtures stimulate Th1 cell immunity of the organism to be stronger than single antigen immunity, so that the organism can be better helped to resist the infection of mycobacterium tuberculosis.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (10)
1. The mycobacterium tuberculosis multi-antigen fusion protein is characterized by comprising an Rv3875 protein antigen, an Rv2628 protein antigen and an Rv1886c protein antigen.
2. The mycobacterium tuberculosis multi-antigen fusion protein of claim 1, wherein the amino acid sequence of the mycobacterium tuberculosis multi-antigen fusion protein comprises SEQ ID No.2;
or the amino acid sequence shown in SEQ ID NO.2 has the same function by replacing, deleting or adding one or more amino acids.
3. A gene encoding a mycobacterium tuberculosis multi-antigen fusion protein according to claim 1, wherein the encoding gene comprises an Rv3875 gene, an Rv2628 gene and an Rv1886c gene; the Rv3875 gene, the Rv2628 gene and the Rv1886c gene are sequentially connected in series.
4. A coding gene according to claim 3, wherein the nucleotide sequence of the coding gene comprises SEQ ID No.1;
or the nucleotide sequence with the same function and formed by replacing, deleting or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 1.
5. A biological material for expressing the mycobacterium tuberculosis multi-antigen fusion protein according to claim 1, the biological material comprising an expression vector and an engineering bacterium, wherein the biological material comprises the encoding gene according to claim 3 or 4.
6. The mycobacterium tuberculosis protein antigen mixture is characterized by comprising an Rv3875 protein antigen, an Rv2628 protein antigen and an Rv1886c protein antigen;
or comprises an Rv3875 protein antigen, an Rv2628 protein antigen, an Rv1886c protein antigen and a mycobacterium tuberculosis multi-antigen fusion protein according to claim 1 or 2.
7. Use of a mycobacterium tuberculosis multi-antigen fusion protein or a coding gene thereof according to claim 1 or 2, or a biomaterial according to claim 5, or a mycobacterium tuberculosis antigen protein mixture according to claim 6, in the preparation of a mycobacterium tuberculosis vaccine.
8. A mycobacterium tuberculosis vaccine, characterized in that the effective component of the mycobacterium tuberculosis vaccine comprises the mycobacterium tuberculosis multi-antigen fusion protein of claim 1 or 2, or the encoding gene of claim 3 or 4, or the biological material of claim 5, or the mycobacterium tuberculosis protein antigen mixture of claim 6.
9. The mycobacterium tuberculosis vaccine of claim 8, wherein the active component of the mycobacterium tuberculosis vaccine further comprises an aluminum salt adjuvant.
10. Use of a mycobacterium tuberculosis multi-antigen fusion protein according to claim 1 or 2, or a coding gene according to claim 3 or 4, or a biological material according to claim 5, or a mycobacterium tuberculosis protein antigen mixture according to claim 6, for the preparation of a mycobacterium tuberculosis detection reagent or a medicament for the treatment of a mycobacterium tuberculosis disease.
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