CN116802197A - Pharmaceutical composition for preventing or treating tuberculosis - Google Patents
Pharmaceutical composition for preventing or treating tuberculosis Download PDFInfo
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- CN116802197A CN116802197A CN202180092760.8A CN202180092760A CN116802197A CN 116802197 A CN116802197 A CN 116802197A CN 202180092760 A CN202180092760 A CN 202180092760A CN 116802197 A CN116802197 A CN 116802197A
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- A61K2039/55527—Interleukins
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Abstract
The present invention relates to a fusion protein comprising: a fusion polypeptide derived from mycobacterium tuberculosis (Mycoba terium tuberculosis); a polynucleotide encoding the above protein; a recombinant vector comprising the polynucleotide; and a pharmaceutical composition for preventing or treating tuberculosis, comprising the above fusion protein, polynucleotide or recombinant vector.
Description
Technical Field
The present invention relates to a fusion protein comprising: a fusion polypeptide derived from mycobacterium tuberculosis (Mycoba terium tuberculosis); a polynucleotide encoding the above protein; a recombinant vector comprising the polynucleotide; and a pharmaceutical composition for preventing or treating tuberculosis, comprising the above fusion protein, polynucleotide or recombinant vector.
Background
Tuberculosis is an infectious disease caused by mycobacterium tuberculosis (Mycobacterium Tuberculosis), and when an infectious tuberculosis patient coughs or sneezes, the mycobacterium tuberculosis is discharged into the air, and surrounding people may inhale the droplets to be infected. In general, although not every infected person is a tuberculosis patient, even if the infected person is infected with mycobacterium tuberculosis, the infection is not affected in the normal immune system state, and the infection can also live in normal life. Therefore, there is a need to develop a vaccine for preventing or treating tuberculosis to protect people not infected with tuberculosis or people infected with tuberculosis.
BCG vaccine (bacillus Calmette-Gurin, BCG) is an attenuated live vaccine for weakening bovine type mycobacterium tuberculosis (Mycobacterium bovis), is the most commonly used tuberculosis vaccine worldwide, and can effectively protect children. However, it has a problem that the duration is limited to childhood, and the preventive effect on adult tuberculosis, which is a major form of tuberculosis, is in an unstable state of 0 to 80%. Thus, new tuberculosis vaccines are being developed in countries around the world that can replace or supplement existing tuberculosis vaccines.
The developed novel tuberculosis vaccine comprises: protein subunit vaccines that utilize tuberculosis antigens; expressing a tuberculosis antigen inducing defensive immunity in a DN A vaccine or a viral vector vaccine in a plasmid DNA or a viral vector; recombinant BCG vaccine expressing a tuberculosis antigen inducing defensive immunity on BCG vaccine of tuberculosis antigen; a gene recombinant tuberculosis vaccine for removing pathogenic genes of tuberculosis bacteria, etc. To date, DNA vaccines have been tried more frequently as novel tuberculosis preventive vaccines, and their efficacy has been demonstrated in animal models. In addition, many tuberculosis antigens have been developed using DNA vaccine technology.
DNA vaccines have proven to be very effective in establishing cellular immune responses including cytotoxic T lymphocytes (cytotoxic Tlymphocyte, CTLs) and Th1 responses, which are known to be critical for obtaining effective antibacterial immunity (anti-mycobacteria immunity) in mice and humans (Bonato VL et al, effect Immun,1998, 66:169-175). In addition, efforts are underway to develop antigens because of the importance of selecting appropriate antigens that are critical to the efficacy of the defense against tuberculosis in terms of introducing effective immunological techniques.
For this reason, the present inventors developed a vaccine for preventing or treating tuberculosis using an antigen having a defensive efficacy against tuberculosis, and completed the present invention.
Disclosure of Invention
Technical problem
It is an object of the present invention to provide a fusion protein comprising a fusion polypeptide derived from Mycobacterium tuberculosis.
It is still another object of the present invention to provide a polynucleotide for encoding the above fusion protein.
It is another object of the present invention to provide a recombinant vector comprising the above polynucleotide.
It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating tuberculosis comprising the above fusion protein.
It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating tuberculosis comprising the above polynucleotide.
It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating tuberculosis comprising the above recombinant vector.
It is a further object of the present invention to provide a method for preventing or treating tuberculosis, comprising the step of administering a therapeutically effective amount of the above fusion protein to a subject.
It is a further object of the present invention to provide a method for preventing or treating tuberculosis, comprising the step of administering a therapeutically effective amount of the polynucleotide as described above to a subject.
It is still another object of the present invention to provide a method for preventing or treating tuberculosis, comprising the step of administering a therapeutically effective amount of the recombinant vector as described above to a subject.
It is a further object of the present invention to provide the use of the fusion protein as defined above for the preparation of a medicament for the prevention or treatment of tuberculosis.
It is a further object of the present invention to provide the use of the above polynucleotide for the preparation of a medicament for preventing or treating tuberculosis.
It is a further object of the present invention to provide the use of the recombinant vector described above in the preparation of a medicament for the prevention or treatment of tuberculosis.
Means for solving the problems
In order to achieve the above object, the present invention provides a fusion protein comprising a fusion polypeptide derived from Mycobacterium tuberculosis.
Also, the present invention provides polynucleotides for encoding the above fusion proteins.
The present invention also provides a recombinant vector comprising the polynucleotide.
Also, the present invention provides a pharmaceutical composition for preventing or treating tuberculosis, comprising the above fusion protein.
Also, the present invention provides a pharmaceutical composition for preventing or treating tuberculosis, comprising the above polynucleotide.
And, the present invention provides a pharmaceutical composition for preventing or treating tuberculosis comprising the above recombinant vector.
Also, the present invention provides a method for preventing or treating tuberculosis, comprising the step of administering a therapeutically effective amount of the above fusion protein to a subject.
Also, the present invention provides a method for preventing or treating tuberculosis, comprising the step of administering a therapeutically effective amount of the above polynucleotide to a subject.
Also, the present invention provides a method for preventing or treating tuberculosis, comprising the step of administering a therapeutically effective amount of the recombinant vector described above to a subject.
Furthermore, the present invention provides the use of the above fusion protein in the preparation of a medicament for preventing or treating tuberculosis.
And, the present invention provides the use of the above polynucleotide for the preparation of a medicament for preventing or treating tuberculosis.
Furthermore, the invention provides the use of the recombinant vector in preparing a medicament for preventing or treating tuberculosis.
ADVANTAGEOUS EFFECTS OF INVENTION
The fusion protein provided by the invention has the effects of increasing the specific immune response of a human body to tuberculosis antigens and remarkably reducing the quantity of tubercle bacillus in the lung, and can be effectively used for preventing or treating tuberculosis.
Drawings
FIG. 1 shows a gene construct for preparing a fusion protein.
FIG. 2 shows the results of specific immune responses to tuberculosis antigens as determined by IFN-. Gamma.ELISPOT analysis after administration of DNA vaccine to a mouse animal model.
FIG. 3 shows the results of determining the number of Mycobacterium tuberculosis in the lung and the results of lung histopathology after administration of DNA vaccine to a mouse animal model.
FIG. 4 shows the results of determining the number of Mycobacterium tuberculosis in the lung and the results of lung histopathology after administration of DNA vaccine to a mouse animal model.
FIG. 5 shows the results of determining the number of Mycobacterium tuberculosis in the lung after administration of DNA vaccine to a mouse animal model and reinfection with Mycobacterium tuberculosis.
Detailed Description
Best Mode for Carrying Out The Invention
The present invention provides fusion proteins comprising a fusion polypeptide derived from mycobacterium tuberculosis.
The fusion polypeptide may be selected from the group consisting of Ag85A, TB 10.4.4, mtb32b and PstS3, and preferably may comprise Ag85A, TB 10.4.4, mtb32b and PstS3. In an embodiment of the invention, the Ag85A may consist of the amino acid sequence of seq id No. 1; the above TB10.4 may consist of the amino acid sequence of SEQ ID NO. 2; the Mtb32b may consist of the amino acid sequence of seq id No. 3; the above PstS3 may consist of the amino acid sequence of SEQ ID No. 4.
The fusion polypeptide may further comprise a polypeptide selected from the group consisting of PPE39, glcB and RipA, and preferably may further comprise a polypeptide comprising PPE39, glcB and RipA. In one embodiment of the present invention, the PPE39 may consist of the amino acid sequence of SEQ ID No. 5; the above GlcB may consist of the amino acid sequence of seq id No. 6; the RipA may consist of the amino acid sequence of SEQ ID No. 7.
The fusion polypeptide may be linked by a 2A peptide (2A self-cleaning peptide) or a GS linker, which may preferably comprise (GS) n (in this case, n is an integer from 1 to 10). In one embodiment of the present invention, the GS linker may be composed of the amino acid sequence of SEQ ID NO. 8.
In the present invention, the above fusion protein may further comprise a signal peptide.
The term "signal peptide" or secretion signal sequence "in the present invention refers to a short peptide of about 20 to 30 amino acids in length, which exists at the N-terminus of a newly synthesized protein designated as secretion pathway (secretory pathway).
The signal peptide may be selected from the group consisting of a tissue-type plasminogen activator (tissue plaminoge n activator, tPA) signal peptide, a herpes simplex virus glycoprotein Ds (Herpes Simplex Virus glycoprotein Ds, HSV gDs) signal peptide, and a growth hormone (growth hormo) signal peptide, and preferably, a secretion signal sequence used in higher eukaryotic cells including mammals, and more preferably, a tPA sequence may be used.
In one embodiment of the present invention, the tPA sequence may consist of the amino acid sequence of seq id No. 9. Furthermore, the signal sequence of the present invention may be replaced with a codon having a high expression frequency in the host cell.
In the present invention, the above fusion protein further comprises an immunopotentiating peptide.
The term "immunopotentiating peptide" in the present invention refers to a peptide that increases an immune response by activating cells (e.g., dendritic cells, etc.) involved in the immune response.
The immunopotentiating peptide may be selected from the group consisting of CD40 ligand, FMS-like tyrosine kinase 3 ligand (FMS-like tyrosine kinase-3, flt 3) ligand, flagellin (flagellin) and OX40, preferably, but not limited thereto, flt3 ligand. In one embodiment of the present invention, the Flt3 ligand may consist of the amino acid sequence of SEQ ID NO. 10.
The present invention provides a fusion protein comprising: a signal peptide; immunopotentiating peptides of Flt3 ligands; and fusion polypeptides comprising Ag85A, TB10.4, mtb32b and PstS3.
In one embodiment of the present invention, the fusion protein may consist of the amino acid sequence of SEQ ID NO. 11.
Also, the present invention provides a fusion protein comprising: a signal peptide; immunopotentiating peptides of Flt3 ligands; and fusion polypeptides comprising PPE39, glcB and RipA.
In one embodiment of the present invention, the fusion protein may consist of the amino acid sequence of SEQ ID NO. 12.
Also, the present invention provides polynucleotides for encoding the above fusion proteins.
The polynucleotides of the invention may be prepared by chemical synthesis or genetic engineering techniques. Chemical synthesis methods are known to those of ordinary skill in the art to which the present invention pertains, and any method may be used. Or by commission of commercial nucleic acid synthesis and purchase by suppliers. In the case of preparation by genetic engineering techniques, for example, it can be prepared by obtaining nucleic acid fragments encoding a fusion polypeptide, a signal peptide and an immunopotentiating peptide derived from Mycobacterium tuberculosis, which have been known in the past, respectively, and ligating these fragments in a manner suitable for the frame. Methods for obtaining such nucleic acid fragments are well known in the art to which the present invention pertains, and one of ordinary skill in the art can readily ligate using appropriate restriction enzymes. In particular embodiments of the invention, methods of preparation using chemical synthesis are disclosed.
The polynucleotide of the present invention may comprise a polynucleotide for encoding a fusion polypeptide comprising Ag85A, TB10.4, mtb32b and PstS3.
The Ag85A may consist of the nucleic acid sequence of SEQ ID NO. 13; the TB10.4 may consist of the nucleic acid sequence of SEQ ID NO. 14; the Mtb32b may be composed of the nucleic acid sequence of SEQ ID NO. 15; the above PstS3 may consist of the nucleic acid sequence of SEQ ID NO. 16. Ag85A, TB 10.4.4, mtb32b and PstS3 can be linked by a GS linker, which can preferably consist of a nucleic acid sequence selected from sequence numbers 20 to 23.
Also, the polynucleotide of the present invention may comprise a polynucleotide encoding a fusion polypeptide comprising PPE39, glcB and RipA.
The PPE39 may consist of the nucleic acid sequence of SEQ ID NO. 17; the above GlcB may consist of the nucleic acid sequence of seq id No. 18; the above RipA may consist of the nucleic acid sequence of SEQ ID NO. 19. PPE39, glcB and RipA may be linked by a GS linker, which may preferably consist of a nucleic acid sequence selected from sequence numbers 20 to 23.
The polynucleotide of the present invention may further comprise a polynucleotide encoding a signal peptide, wherein the signal peptide may be a tPA sequence, and the polynucleotide encoding the tPA sequence may be composed of a nucleic acid sequence of sequence No. 24 or 25.
The polynucleotide of the present invention may further comprise a polynucleotide encoding an immunopotentiating peptide, which may be a Flt3 ligand, and the polynucleotide encoding the Flt3 ligand may be composed of the nucleic acid sequence of SEQ ID NO. 26 or 27.
In one embodiment of the present invention, the polynucleotide may comprise: a polynucleotide for encoding a fusion polypeptide comprising Ag85A, TB10.4, mtb32b, and PstS 3; a polynucleotide encoding a signal peptide; a polynucleotide for encoding an immunopotentiating peptide. Preferably, the polynucleotide may consist of the nucleic acid sequence of SEQ ID NO. 28.
In one embodiment of the present invention, the polynucleotide may comprise: a polynucleotide encoding a fusion polypeptide comprising PPE39, glcB, and RipA; a polynucleotide encoding a signal peptide; polynucleotides encoding immunopotentiating peptides. Preferably, the polynucleotide may consist of the nucleic acid sequence of SEQ ID NO. 29.
The present invention also provides a recombinant vector comprising the polynucleotide.
The term "vector" according to the present invention refers to a genetic construct comprising an external DNA inserted into the genome encoding a polypeptide. The recombinant expression vector of the present invention is a vector in which a polynucleotide encoding a fusion protein comprising a fusion polypeptide derived from Mycobacterium tuberculosis, a signal peptide and an immunopotentiating peptide is inserted into the genome.
The vector may be a plasmid vector, a viral vector, a cosmid vector, or an artificial chromosome vector, and the viral vector may be a phage vector, an adenovirus vector, a retrovirus vector, or an adeno-associated virus vector.
The recombinant vector of the invention may comprise a nucleic acid capable of encoding the fusion protein of the invention in a form suitable for expression of the nucleic acid encoding the fusion protein in a host cell. That is, the recombinant vectors of the invention comprise one or more regulatory sequences selected according to the host cell used for expression, which are operably linked to the nucleic acid sequence to be expressed.
The term "operably linked" in the present invention refers to a desired nucleotide sequence (e.g., in an in vitro transcription/translation system or host cell) linked to the regulatory sequences described above in a manner that allows for its expression.
The term "regulatory sequence" in the present invention refers to promoters, enhancers and other regulatory elements (e.g., polyadenylation signals). Regulatory sequences include sequences that indicate expression of a desired nucleic acid in a number of host cells, as well as sequences that indicate that the desired nucleic acid is capable of expression only in a particular host cell (e.g., tissue-specific regulatory sequences). It will be appreciated by those of ordinary skill in the art that the design of the expression vector may vary depending on factors such as the choice of host cell to be transformed and the desired level of protein expression. The expression vectors of the invention may express the fusion proteins described above by introduction into a host cell.
Also, the present invention provides a pharmaceutical composition for preventing or treating tuberculosis, comprising the above fusion protein.
In one embodiment of the present invention, the composition may further comprise an immunoadjuvant (adjuvant) comprising interleukin-12 (IL-12), interleukin-21 (IL-21) and macrophage inflammatory protein-1α (MIP-1 a) proteins, preferably the immunoadjuvant may comprise an amino acid sequence of sequence No. 30 to 33. Wherein IL-12p35 consists of the amino acid sequence of SEQ ID NO. 30; IL-12p40 consists of the amino acid sequence of SEQ ID NO. 31; IL-21 consists of the amino acid sequence of SEQ ID NO. 32; MIP-1a (MIP-1 alpha) consists of the amino acid sequence of SEQ ID NO. 33.
The term "immunoadjuvant" according to the present invention refers to a substance added to a vaccine to stimulate the immune system response of an antigen.
Also, the present invention provides a pharmaceutical composition for preventing or treating tuberculosis, comprising the above polynucleotide or the above recombinant vector.
The above composition may further comprise a polynucleotide encoding an immunoadjuvant comprising IL-12, IL-21 and MIP-1a proteins, preferably a polynucleotide encoding an amino acid sequence of SEQ ID Nos. 30 to 33, more preferably a nucleic acid sequence of SEQ ID Nos. 34 to 37.
The pharmaceutical compositions of the present invention may also comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" in the context of the present invention means that it exhibits non-toxic properties to cells or humans exposed to the above-described compositions. And carriers known in the art to which the present invention pertains, such as buffers, preservatives, painless agents, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants and the like, may be used without limitation.
The composition of the present invention can be formulated into non-oral preparations such as oral preparations or injections, for example, granules, powders, solutions, tablets, capsules, dry syrups, and the like. Preferably, the composition of the present invention may be in the form of a solution or an injection, etc., but is not limited thereto.
In the pharmaceutical compositions of the present invention, the pharmaceutically effective amount is administered. The term "administration" according to the present invention means that a predetermined substance is introduced into a subject in an appropriate manner, and the administration route of the above-mentioned composition may be by any conventional route as long as it is capable of reaching a desired tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration may be used, but are not limited thereto.
The term "subject" refers to all animals including humans, such as rats, mice, and livestock, among others. Preferably, it may be a mammal, including a human.
The term "pharmaceutically effective amount" as defined above refers to an amount sufficient to treat a disease without causing side effects in a reasonable benefit/risk ratio applicable to medical treatment, and the effective amount level can be readily determined by one of ordinary skill in the art based on such conditions as sex, age, weight, health, disease type, severity, pharmaceutical activity, sensitivity to drugs, method of administration, time of administration, route and rate of administration, period of treatment, elements combining or containing simultaneous drugs, and other elements well known in the medical arts of the patient. At the time of administration, the administration may be carried out once daily or in several times according to the recommended doses described above.
Also, the present invention provides a method for preventing or treating tuberculosis, comprising the step of administering a therapeutically effective amount of the above fusion protein to a subject.
Also, the present invention provides a method for preventing or treating tuberculosis, comprising the step of administering a therapeutically effective amount of the above polynucleotide or the above recombinant vector to a subject.
Preferably, the above therapeutically effective amount may be variously applied according to the type and extent of the reaction to be achieved, the specific composition of other formulation or the like according to circumstances, the age, weight, general health, sex and diet of the subject, administration time, administration route and secretion rate of the composition, treatment period, drug used together or simultaneously with the specific composition, and the like, and similar factors widely applied in the medical field. Thus, preferably, an effective amount of a composition suitable for the purposes of the present invention can be determined based on the factors described above.
The subject may be applied to any mammal, including not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs, cats, and the like.
Furthermore, the present invention provides the use of the above fusion protein in the preparation of a medicament for preventing or treating tuberculosis.
Furthermore, the present invention provides the use of the above polynucleotide or the above recombinant vector for the preparation of a medicament for preventing or treating tuberculosis.
Embodiments of the invention
Hereinafter, the present invention will be described in more detail with reference to examples. These examples are intended to illustrate the present invention in more detail, but the scope of the present invention is not limited to these examples only.
EXAMPLE 1 preparation of fusion proteins
The fusion protein of the present invention comprises a fusion polypeptide derived from Mycobacterium tuberculosis, a signal peptide and an immunopotentiator peptide (immunity enhancer peptide). The above fusion polypeptide derived from Mycobacterium tuberculosis contains Ag85A, TB 10.4.4, mtb32b and PstS3, and each polypeptide (antigen) is prepared by 2A peptide ligation. And, the tPA peptide in the signal peptide and the Flt3L ligand in the immunopotentiating peptide are linked to the N-terminal of the fusion polypeptide.
The fusion polypeptide derived from Mycobacterium tuberculosis was prepared by including a polypeptide of PPE39, glcB and/or RipA in addition to Ag85A, TB 10.4.10.4, mt b32b and PstS3. Each polypeptide (antigen) was prepared by ligation via a 2A peptide or GS linker, and was also prepared at the N-terminus of the fusion polypeptide as a Flt3L ligand in the tPA peptide and the immunopotentiating peptide in the signal peptide.
Hereinafter, the fusion polypeptide of Ag85A, TB 10.4.4, mtb32B and PstS3 was linked by a 2A peptide, the fusion protein in which HA tag was added to the C-terminus was designated "GX-70", the fusion protein in which Ag85A, TB 10.4.4, mtb32B, PPE39 and PstS3 was linked by a 2A peptide was designated "GX-171A", the fusion protein in which Ag85A, TB 10.4.4, mtb32B, ripA and PstS3 was linked by a 2A peptide was designated "GX-171B", the fusion protein in which Ag85A, TB 10.4.4, mtb32B and PstS3 was linked by a GS linker was designated "GX-170A", and the fusion protein in which PPE39, glcB and RipA were linked by a GS linker was designated "GX-170B" (FIG. 1).
The above candidate fusion proteins were prepared by synthesizing pGX27 (see Korean patent No. 1442254) having the gene sequence of each corresponding fusion protein inserted into a high expression vector.
EXAMPLE 2 preparation of immunoadjuvant
The immunoadjuvant of the present invention comprises IL-12, IL-21 and MIP-1a proteins. The N-terminus of IL-12 was linked to CMV promoter (pCMV), the N-terminus of IL-21 was linked to RSV promoter (pRSV), and the N-terminus of MIP-1a was linked to EF1a promoter (pEF 1 a). The immunoadjuvant comprising IL-12, IL-21 and MIP-1A proteins is hereinafter referred to as "BD-121A" (FIG. 1).
Prepared by synthesizing a cassette for expressing each corresponding fusion protein and inserting it into pGX27 (refer to korean patent No. 1442254) of a high expression vector.
EXAMPLE 3 immunopotentiating efficacy analysis of compositions comprising fusion proteins and immunoadjuvants
The present inventors have conducted experiments to confirm whether or not a composition comprising the fusion protein and an immunoadjuvant has an immunopotentiating effect. Briefly, use is made of(SL VAXiGEN, korea) in vivo electroporation apparatus PBS (dosage form control) or DNA vaccine was administered to thigh muscle of C57BL/6 mice 2 times every 2 weeks. Spleens were removed 2 weeks after final administration and specific immune responses to tuberculosis antigens were determined using IFN-gamma ELISPOT analysis. Information for PBS or DNA vaccine dosing groups are as follows: group 1, pbs (dosage form control); group 2, GX-70 μg; group 3, GX-70 μg+BD-121A8 μg; group 4, GX-171A8 μg; group 5, GX-171A8 μg+BD-121A8 μg; group 6, GX-171B 8 μg; and group 7, GX-171B 8 μg+BD-121A8 μg.
As a result, it was confirmed that tuberculosis specific immune responses were induced in tuberculosis candidate substances GX-70, GX-171A and GX-171B, and in particular, in groups 3, 5 and 7, in which the immunoadjuvant BD-121A was added to GX-70, GX-171A and GX-171B, the tuberculosis specific immune responses were further increased (FIG. 2).
EXAMPLE 4 analysis of the prevention efficacy of tuberculosis
The present inventors conducted experimentsTo confirm whether the composition comprising the fusion protein and the immunoadjuvant has the effect of preventing tuberculosis. Briefly, use is made of(SL VAXiGE N, korea) in vivo electroporation apparatus PBS (dosage form control) or DNA vaccine was administered on thigh muscle of C57BL/6 mice 3 times every 3 weeks. In the positive control BCG group, C57BL/6 mice were administered 1 time at week 3. After 3 weeks of final administration, mycobacterium tuberculosis was released into the air, and after 3 weeks, the number of Mycobacterium tuberculosis in the lung was measured, and the defensive efficacy against Mycobacterium tuberculosis was confirmed. Information for PBS or DNA vaccine dosing groups are as follows: group 1, pbs (dosage form control); group 2, GX-70 μg; group 3, GX-171A 4 μg+BD-121A4 μg; group 4, GX-171B 4 μg+BD-121A4 μg; and group 5, bcg (positive control group).
The results confirmed that the number of Mycobacterium tuberculosis was lower in the administration group of all vaccine candidate substances than in the dosage form control group, and the degree of inflammation was also suppressed in the lung histopathological results (FIG. 3). However, the differences in defensive efficacy between individual vaccine candidate substances have not been confirmed.
To this end, the present inventors conducted further experiments to evaluate the efficacy of preventing tuberculosis by administering additionally prepared GX-170A and GX-170B in combination. In short, using OrbiJector(SL VAXiGEN, korea) in vivo electroporation apparatus PBS (dosage form control) or DNA vaccine was administered on thigh muscle of C57BL/6 mice 3 times every 3 weeks. In the BCG group of the positive control group, the C57BL/6 mice were administered once at week 3. After 3 weeks of final administration, mycobacterium tuberculosis was released into the air, and after 3 weeks, the number of Mycobacterium tuberculosis in the lung was measured, confirming the preventive effect on Mycobacterium tuberculosis. Information for PBS or DNA vaccine dosing groups are as follows: group 1, pbs (dosage form control); group 2, GX-70 μg; group 3, GX-70 μg+BD-121A8 μg; group 4, GX-171A8 μg+BD-121A8 μg; group 5, GX-170A 8. Mu.g+GX-170B 8. Mu.g+BD-121A 4. Mu.gg; and group 6, bcg (positive control group).
The results confirmed that, among the administration groups of all vaccine candidate substances, there was a significant effect of preventing tuberculosis as compared with the dosage form control group (fig. 4). In the group to which GX-70 was administered, it was confirmed that the effect of preventing tuberculosis was obtained regardless of the addition or non-addition of an immunoadjuvant, as compared with the dosage form control group, and in the group to which GX-171A and BD-121A were administered in combination, it was confirmed that the effect was similar to that of the group to which GX-70 was administered (FIG. 4). In particular, it was confirmed that in the group to which GX-170A, GX-170B and BD-121A were administered in combination, the effect of preventing tuberculosis was more remarkable than in the group to which GX-70 was administered, and the same effect was observed in the pathological results of lung tissue (FIG. 4).
EXAMPLE 5 analysis of treatment efficacy of tuberculosis
The present inventors have conducted experiments to confirm whether or not a composition containing the fusion protein and an immunoadjuvant has an effect of treating tuberculosis. Briefly, after releasing mycobacterium tuberculosis into the air, after 4 weeks, agents INH (isoniazid) and PZA (pyrazinamid e) for tuberculosis were administered to C57BL/6 mice, and treated with drinking water for 14 weeks. During this treatment period, BCG group (positive control group) was administered 1 time every 8 weeks, and ori was used(SL VAXiGEN, korea) in vivo electroporation apparatus PBS (dosage form control) or DNA vaccine was administered on thigh muscle of C57BL/6 mice 3 times every 5, 8, 11 and 13 weeks. After confirming that no mycobacterium tuberculosis was detected at the end time point of the tuberculosis drug treatment, the mycobacterium tuberculosis was released into the air a second time at week 20, after which the number of reinfected mycobacterium tuberculosis was observed in the lungs. The information of the group to which the DNA vaccine was administered was as follows: group 1, naiv without tuberculosis agent administered; group 2, pbs (dosage form control); group 3, GX-70 μg; group 4, GX-170A8 μg; group 5, GX-170A8 μg+GX-170B8 μg; group 6, GX-70 μg+BD-121A8 μg; group 7, GX-170A8 μg+BD-121A8 μg; group 8, GX-170A8 μg+GX-170B8 μg+BD-121A8 μg.
The results confirm that in all groups, tuberculosis re-infection has a lower incidence compared to untreated groups after infection with tuberculosis (fig. 5). In the group to which GX-170A and GX-170B were administered in combination, the number of Mycobacterium tuberculosis tended to decrease, and in particular, in the group to which GX-170A, GX-170B and BD-121A were administered in combination, it was confirmed that the number of Mycobacterium tuberculosis was significantly lower than that in the dosage form control group (FIG. 5).
From the above results, the present inventors confirmed that there is an efficacy of treating tuberculosis when GX-170A and GX-170B are administered in combination, and confirmed that the efficacy of treating tuberculosis is further improved when an additional immunoadjuvant is administered in combination.
<110> Gernacini Co., ltd
Disease management hall
<120> pharmaceutical composition for preventing or treating tuberculosis
<130> PCT5040957
<150> KR 10-2020-0168502
<151> 2020-12-04
<160> 37
<170> KoPatentIn 3.0
<210> 1
<211> 295
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of Ag85A
<400> 1
Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro
1 5 10 15
Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Ala Asn
20 25 30
Ser Pro Ala Leu Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Phe
35 40 45
Ser Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gln Ser
50 55 60
Gly Leu Ser Val Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser
65 70 75 80
Asp Trp Tyr Gln Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys
85 90 95
Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gly Trp Leu Gln Ala Asn
100 105 110
Arg His Val Lys Pro Thr Gly Ser Ala Val Val Gly Leu Ser Met Ala
115 120 125
Ala Ser Ser Ala Leu Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Val
130 135 140
Tyr Ala Gly Ala Met Ser Gly Leu Leu Asp Pro Ser Gln Ala Met Gly
145 150 155 160
Pro Thr Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala
165 170 175
Ser Asp Met Trp Gly Pro Lys Glu Asp Pro Ala Trp Gln Arg Asn Asp
180 185 190
Pro Leu Leu Asn Val Gly Lys Leu Ile Ala Asn Asn Thr Arg Val Trp
195 200 205
Val Tyr Cys Gly Asn Gly Lys Pro Ser Asp Leu Gly Gly Asn Asn Leu
210 215 220
Pro Ala Lys Phe Leu Glu Gly Phe Val Arg Thr Ser Asn Ile Lys Phe
225 230 235 240
Gln Asp Ala Tyr Asn Ala Gly Gly Gly His Asn Gly Val Phe Asp Phe
245 250 255
Pro Asp Ser Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn
260 265 270
Ala Met Lys Pro Asp Leu Gln Arg Ala Leu Gly Ala Thr Pro Asn Thr
275 280 285
Gly Pro Ala Pro Gln Gly Ala
290 295
<210> 2
<211> 95
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of TB10.4
<400> 2
Ser Gln Ile Met Tyr Asn Tyr Pro Ala Met Leu Gly His Ala Gly Asp
1 5 10 15
Met Ala Gly Tyr Ala Gly Thr Leu Gln Ser Leu Gly Ala Glu Ile Ala
20 25 30
Val Glu Gln Ala Ala Leu Gln Ser Ala Trp Gln Gly Asp Thr Gly Ile
35 40 45
Thr Tyr Gln Ala Trp Gln Ala Gln Trp Asn Gln Ala Met Glu Asp Leu
50 55 60
Val Arg Ala Tyr His Ala Met Ser Ser Thr His Glu Ala Asn Thr Met
65 70 75 80
Ala Met Met Ala Arg Asp Thr Ala Glu Ala Ala Lys Trp Gly Gly
85 90 95
<210> 3
<211> 323
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of Mtb32d
<400> 3
Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro Ala Leu
1 5 10 15
Pro Leu Asp Pro Ser Ala Met Val Ala Gln Val Gly Pro Gln Val Val
20 25 30
Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn Ala Val Gly Ala Gly Thr
35 40 45
Gly Ile Val Ile Asp Pro Asn Gly Val Val Leu Thr Asn Asn Ala Val
50 55 60
Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe Ser Val Gly Ser Gly Gln
65 70 75 80
Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr Gln Asn Val Ala
85 90 95
Val Leu Gln Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala Ala Ile Gly
100 105 110
Gly Gly Val Ala Val Gly Glu Pro Val Val Ala Met Gly Asn Ser Gly
115 120 125
Gly Gln Gly Gly Thr Pro Arg Ala Val Pro Gly Arg Val Val Ala Leu
130 135 140
Gly Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu Glu Thr
145 150 155 160
Leu Asn Gly Leu Ile Gln Phe Asp Ala Ala Ile Gln Pro Gly Asp Ala
165 170 175
Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val Gly Met Asn Thr
180 185 190
Ala Ala Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly Gln Gly Phe Ala
195 200 205
Ile Pro Ile Gly Gln Ala Met Ala Ile Ala Gly Gln Ile Arg Ser Gly
210 215 220
Gly Gly Ser Pro Thr Val His Ile Gly Pro Thr Ala Phe Leu Gly Leu
225 230 235 240
Gly Val Val Asp Asn Asn Gly Asn Gly Ala Arg Val Gln Arg Val Val
245 250 255
Gly Ser Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly Asp Val Ile
260 265 270
Thr Ala Val Asp Gly Ala Pro Ile Asn Ser Ala Thr Ala Met Ala Asp
275 280 285
Ala Leu Asn Gly His His Pro Gly Asp Val Ile Ser Val Thr Trp Gln
290 295 300
Thr Lys Ser Gly Gly Thr Arg Thr Gly Asn Val Thr Leu Ala Glu Gly
305 310 315 320
Pro Pro Ala
<210> 4
<211> 347
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of PstS3
<400> 4
Cys Gly Asn Asp Asp Asn Val Thr Gly Gly Gly Ala Thr Thr Gly Gln
1 5 10 15
Ala Ser Ala Lys Val Asp Cys Gly Gly Lys Lys Thr Leu Lys Ala Ser
20 25 30
Gly Ser Thr Ala Gln Ala Asn Ala Met Thr Arg Phe Val Asn Val Phe
35 40 45
Glu Gln Ala Cys Pro Gly Gln Thr Leu Asn Tyr Thr Ala Asn Gly Ser
50 55 60
Gly Ala Gly Ile Ser Glu Phe Asn Gly Asn Gln Thr Asp Phe Gly Gly
65 70 75 80
Ser Asp Val Pro Leu Ser Lys Asp Glu Pro Ser Gly Ala Ala Arg Cys
85 90 95
Gly Ser Pro Ala Trp Asn Leu Pro Val Val Phe Gly Pro Ile Ala Val
100 105 110
Thr Tyr Asn Leu Asn Ser Val Ser Ser Leu Asn Leu Asp Gly Pro Thr
115 120 125
Leu Ala Lys Ile Phe Asn Gly Ser Ile Thr Gln Trp Asn Asn Pro Ala
130 135 140
Ile Gln Ala Leu Asn Arg Asp Phe Thr Leu Pro Gly Glu Arg Ile His
145 150 155 160
Val Val Phe Arg Ser Asp Glu Ser Gly Thr Thr Asp Asn Phe Gln Arg
165 170 175
Tyr Leu Gln Ala Ala Ser Asn Gly Ala Trp Gly Lys Gly Ala Gly Lys
180 185 190
Ser Phe Gln Gly Gly Val Gly Glu Gly Ala Arg Gly Asn Asp Gly Thr
195 200 205
Ser Ala Ala Ala Lys Asn Thr Pro Gly Ser Ile Thr Tyr Asn Glu Trp
210 215 220
Ser Phe Ala Gln Ala His Asp Leu Thr Met Ala Asn Ile Val Thr Ser
225 230 235 240
Ala Gly Gly Asp Pro Val Ala Ile Thr Ile Asp Ser Val Gly Gln Thr
245 250 255
Ile Ala Gly Ala Thr Ile Ser Gly Val Gly Asn Asp Leu Val Leu Asp
260 265 270
Thr Asp Ser Phe Tyr Arg Pro Lys Arg Pro Gly Ser Tyr Pro Ile Val
275 280 285
Leu Ala Thr Tyr Glu Ile Val Cys Ser Lys Tyr Pro Asp Ser Gln Val
290 295 300
Gly Thr Ala Val Lys Ala Phe Leu Gln Ser Thr Ile Gly Ala Gly Gln
305 310 315 320
Ser Gly Leu Gly Asp Asn Gly Tyr Ile Pro Ile Pro Asp Glu Phe Lys
325 330 335
Ser Arg Leu Ser Thr Ala Val Asn Ala Ile Ala
340 345
<210> 5
<211> 621
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of PPE39
<400> 5
Val Asn Phe Ser Val Leu Pro Pro Glu Ile Asn Ser Gly Arg Met Phe
1 5 10 15
Phe Gly Ala Gly Ser Gly Pro Met Leu Ala Ala Ala Ala Ala Trp Asp
20 25 30
Gly Leu Ala Ala Glu Leu Gly Leu Ala Ala Glu Ser Phe Gly Leu Val
35 40 45
Thr Ser Gly Leu Ala Gly Gly Ser Gly Gln Ala Trp Gln Gly Ala Ala
50 55 60
Ala Ala Ala Met Val Val Ala Ala Ala Pro Tyr Ala Gly Trp Leu Ala
65 70 75 80
Ala Ala Ala Ala Arg Ala Gly Gly Ala Ala Val Gln Ala Lys Ala Val
85 90 95
Ala Gly Ala Phe Glu Ala Ala Arg Ala Ala Met Val Asp Pro Val Val
100 105 110
Val Ala Ala Asn Arg Ser Ala Phe Val Gln Leu Val Leu Ser Asn Val
115 120 125
Phe Gly Gln Asn Ala Pro Ala Ile Ala Ala Ala Glu Ala Thr Tyr Glu
130 135 140
Gln Met Trp Ala Ala Asp Val Ala Ala Met Val Gly Tyr His Gly Gly
145 150 155 160
Ala Ser Ala Ala Ala Ala Ala Leu Ala Pro Trp Gln Gln Ala Val Pro
165 170 175
Gly Leu Ser Gly Leu Leu Asp Ser Ala Gln Ser Ser Ala Gln Ala Val
180 185 190
Thr Ala Gln Ala Val Gly Ser Thr Val Pro Gly Pro Leu Gln Gly Ile
195 200 205
Asn Phe Gly Phe Gly Asn Ile Gly Ser Leu Asn Leu Gly Ser Gly Asn
210 215 220
Thr Gly Asp Thr Asn Val Gly Ser Gly Asn Ile Gly Asn Thr Asn Leu
225 230 235 240
Gly Gly Gly Asn Ile Gly Ser Phe Asn Leu Gly Ser Gly Asn Gln Gly
245 250 255
Asp Ile Asn Leu Gly Ile Gly Asn Val Gly Asn Leu Asn Leu Gly Ser
260 265 270
Gly Asn Phe Gly Ser Gln Asn Leu Gly Ser Gly Asn Ile Gly Ser Thr
275 280 285
Asn Val Gly Ser Gly Asn Ile Gly Ser Thr Asn Val Gly Ser Gly Asn
290 295 300
Ile Gly Asp Thr Asn Phe Gly Asn Gly Asn Asn Gly Asn Phe Asn Phe
305 310 315 320
Gly Ser Gly Asn Thr Gly Ser Asn Asn Ile Gly Phe Gly Asn Thr Gly
325 330 335
Ser Gly Asn Phe Gly Phe Gly Asn Thr Gly Asn Asn Asn Ile Gly Ile
340 345 350
Gly Leu Thr Gly Asp Gly Gln Ile Gly Ile Gly Gly Leu Asn Ser Gly
355 360 365
Ser Gly Asn Ile Gly Phe Gly Asn Ser Gly Thr Gly Asn Val Gly Leu
370 375 380
Phe Asn Ser Gly Thr Gly Asn Val Gly Phe Gly Asn Ser Gly Thr Ala
385 390 395 400
Asn Thr Gly Phe Gly Asn Ala Gly Asn Val Asn Thr Gly Phe Trp Asn
405 410 415
Gly Gly Ser Thr Asn Thr Gly Leu Ala Asn Ala Gly Ala Gly Asn Thr
420 425 430
Gly Phe Phe Asp Ala Gly Asn Tyr Asn Phe Gly Ser Leu Asn Ala Gly
435 440 445
Asn Ile Asn Ser Ser Phe Gly Asn Ser Gly Asp Gly Asn Ser Gly Phe
450 455 460
Leu Asn Ala Gly Asp Val Asn Ser Gly Val Gly Asn Ala Gly Asp Val
465 470 475 480
Asn Thr Gly Leu Gly Asn Ser Gly Asn Ile Asn Thr Gly Gly Phe Asn
485 490 495
Pro Gly Thr Leu Asn Thr Gly Phe Phe Ser Ala Met Thr Gln Ala Gly
500 505 510
Pro Asn Ser Gly Phe Phe Asn Ala Gly Thr Gly Asn Ser Gly Phe Gly
515 520 525
His Asn Asp Pro Ala Gly Ser Gly Asn Ser Gly Ile Gln Asn Ser Gly
530 535 540
Phe Gly Asn Ser Gly Tyr Val Asn Thr Ser Thr Thr Ser Met Phe Gly
545 550 555 560
Gly Asn Ser Gly Val Leu Asn Thr Gly Tyr Gly Asn Ser Gly Phe Tyr
565 570 575
Asn Ala Ala Val Asn Asn Thr Gly Ile Phe Val Thr Gly Val Met Ser
580 585 590
Ser Gly Phe Phe Asn Phe Gly Thr Gly Asn Ser Gly Leu Leu Val Ser
595 600 605
Gly Asn Gly Leu Ser Gly Phe Phe Lys Asn Leu Phe Gly
610 615 620
<210> 6
<211> 740
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of GlcB
<400> 6
Thr Asp Arg Val Ser Val Gly Asn Leu Arg Ile Ala Arg Val Leu Tyr
1 5 10 15
Asp Phe Val Asn Asn Glu Ala Leu Pro Gly Thr Asp Ile Asp Pro Asp
20 25 30
Ser Phe Trp Ala Gly Val Asp Lys Val Val Ala Asp Leu Thr Pro Gln
35 40 45
Asn Gln Ala Leu Leu Asn Ala Arg Asp Glu Leu Gln Ala Gln Ile Asp
50 55 60
Lys Trp His Arg Arg Arg Val Ile Glu Pro Ile Asp Met Asp Ala Tyr
65 70 75 80
Arg Gln Phe Leu Thr Glu Ile Gly Tyr Leu Leu Pro Glu Pro Asp Asp
85 90 95
Phe Thr Ile Thr Thr Ser Gly Val Asp Ala Glu Ile Thr Thr Thr Ala
100 105 110
Gly Pro Gln Leu Val Val Pro Val Leu Asn Ala Arg Phe Ala Leu Asn
115 120 125
Ala Ala Asn Ala Arg Trp Gly Ser Leu Tyr Asp Ala Leu Tyr Gly Thr
130 135 140
Asp Val Ile Pro Glu Thr Asp Gly Ala Glu Lys Gly Pro Thr Tyr Asn
145 150 155 160
Lys Val Arg Gly Asp Lys Val Ile Ala Tyr Ala Arg Lys Phe Leu Asp
165 170 175
Asp Ser Val Pro Leu Ser Ser Gly Ser Phe Gly Asp Ala Thr Gly Phe
180 185 190
Thr Val Gln Asp Gly Gln Leu Val Val Ala Leu Pro Asp Lys Ser Thr
195 200 205
Gly Leu Ala Asn Pro Gly Gln Phe Ala Gly Tyr Thr Gly Ala Ala Glu
210 215 220
Ser Pro Thr Ser Val Leu Leu Ile Asn His Gly Leu His Ile Glu Ile
225 230 235 240
Leu Ile Asp Pro Glu Ser Gln Val Gly Thr Thr Asp Arg Ala Gly Val
245 250 255
Lys Asp Val Ile Leu Glu Ser Ala Ile Thr Thr Ile Met Asp Phe Glu
260 265 270
Asp Ser Val Ala Ala Val Asp Ala Ala Asp Lys Val Leu Gly Tyr Arg
275 280 285
Asn Trp Leu Gly Leu Asn Lys Gly Asp Leu Ala Ala Ala Val Asp Lys
290 295 300
Asp Gly Thr Ala Phe Leu Arg Val Leu Asn Arg Asp Arg Asn Tyr Thr
305 310 315 320
Ala Pro Gly Gly Gly Gln Phe Thr Leu Pro Gly Arg Ser Leu Met Phe
325 330 335
Val Arg Asn Val Gly His Leu Met Thr Asn Asp Ala Ile Val Asp Thr
340 345 350
Asp Gly Ser Glu Val Phe Glu Gly Ile Met Asp Ala Leu Phe Thr Gly
355 360 365
Leu Ile Ala Ile His Gly Leu Lys Ala Ser Asp Val Asn Gly Pro Leu
370 375 380
Ile Asn Ser Arg Thr Gly Ser Ile Tyr Ile Val Lys Pro Lys Met His
385 390 395 400
Gly Pro Ala Glu Val Ala Phe Thr Cys Glu Leu Phe Ser Arg Val Glu
405 410 415
Asp Val Leu Gly Leu Pro Gln Asn Thr Met Lys Ile Gly Ile Met Asp
420 425 430
Glu Glu Arg Arg Thr Thr Val Asn Leu Lys Ala Cys Ile Lys Ala Ala
435 440 445
Ala Asp Arg Val Val Phe Ile Asn Thr Gly Phe Leu Asp Arg Thr Gly
450 455 460
Asp Glu Ile His Thr Ser Met Glu Ala Gly Pro Met Val Arg Lys Gly
465 470 475 480
Thr Met Lys Ser Gln Pro Trp Ile Leu Ala Tyr Glu Asp His Asn Val
485 490 495
Asp Ala Gly Leu Ala Ala Gly Phe Ser Gly Arg Ala Gln Val Gly Lys
500 505 510
Gly Met Trp Thr Met Thr Glu Leu Met Ala Asp Met Val Glu Thr Lys
515 520 525
Ile Ala Gln Pro Arg Ala Gly Ala Ser Thr Ala Trp Val Pro Ser Pro
530 535 540
Thr Ala Ala Thr Leu His Ala Leu His Tyr His Gln Val Asp Val Ala
545 550 555 560
Ala Val Gln Gln Gly Leu Ala Gly Lys Arg Arg Ala Thr Ile Glu Gln
565 570 575
Leu Leu Thr Ile Pro Leu Ala Lys Glu Leu Ala Trp Ala Pro Asp Glu
580 585 590
Ile Arg Glu Glu Val Asp Asn Asn Cys Gln Ser Ile Leu Gly Tyr Val
595 600 605
Val Arg Trp Val Asp Gln Gly Val Gly Cys Ser Lys Val Pro Asp Ile
610 615 620
His Asp Val Ala Leu Met Glu Asp Arg Ala Thr Leu Arg Ile Ser Ser
625 630 635 640
Gln Leu Leu Ala Asn Trp Leu Arg His Gly Val Ile Thr Ser Ala Asp
645 650 655
Val Arg Ala Ser Leu Glu Arg Met Ala Pro Leu Val Asp Arg Gln Asn
660 665 670
Ala Gly Asp Val Ala Tyr Arg Pro Met Ala Pro Asn Phe Asp Asp Ser
675 680 685
Ile Ala Phe Leu Ala Ala Gln Glu Leu Ile Leu Ser Gly Ala Gln Gln
690 695 700
Pro Asn Gly Tyr Thr Glu Pro Ile Leu His Arg Arg Arg Arg Glu Phe
705 710 715 720
Lys Ala Arg Ala Ala Glu Lys Pro Ala Pro Ser Asp Arg Ala Gly Asp
725 730 735
Asp Ala Ala Arg
740
<210> 7
<211> 433
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of RipA
<400> 7
Asp Pro Gln Thr Asp Thr Ile Ala Ala Leu Ile Ala Asp Val Ala Lys
1 5 10 15
Ala Asn Gln Arg Leu Gln Asp Leu Ser Asp Glu Val Gln Ala Glu Gln
20 25 30
Glu Ser Val Asn Lys Ala Met Val Asp Val Glu Thr Ala Arg Asp Asn
35 40 45
Ala Ala Ala Ala Glu Asp Asp Leu Glu Val Ser Gln Arg Ala Val Lys
50 55 60
Asp Ala Asn Ala Ala Ile Ala Ala Ala Gln His Arg Phe Asp Thr Phe
65 70 75 80
Ala Ala Ala Thr Tyr Met Asn Gly Pro Ser Val Ser Tyr Leu Ser Ala
85 90 95
Ser Ser Pro Asp Glu Ile Ile Ala Thr Val Thr Ala Ala Lys Thr Leu
100 105 110
Ser Ala Ser Ser Gln Ala Val Met Ala Asn Leu Gln Arg Ala Arg Thr
115 120 125
Glu Arg Val Asn Thr Glu Ser Ala Ala Arg Leu Ala Lys Gln Lys Ala
130 135 140
Asp Lys Ala Ala Ala Asp Ala Lys Ala Ser Gln Asp Ala Ala Val Ala
145 150 155 160
Ala Leu Thr Glu Thr Arg Arg Lys Phe Asp Glu Gln Arg Glu Glu Val
165 170 175
Gln Arg Leu Ala Ala Glu Arg Asp Ala Ala Gln Ala Arg Leu Gln Ala
180 185 190
Ala Arg Leu Val Ala Trp Ser Ser Glu Gly Gly Gln Gly Ala Pro Pro
195 200 205
Phe Arg Met Trp Asp Pro Gly Ser Gly Pro Ala Gly Gly Arg Ala Trp
210 215 220
Asp Gly Leu Trp Asp Pro Thr Leu Pro Met Ile Pro Ser Ala Asn Ile
225 230 235 240
Pro Gly Asp Pro Ile Ala Val Val Asn Gln Val Leu Gly Ile Ser Ala
245 250 255
Thr Ser Ala Gln Val Thr Ala Asn Met Gly Arg Lys Phe Leu Glu Gln
260 265 270
Leu Gly Ile Leu Gln Pro Thr Asp Thr Gly Ile Thr Asn Ala Pro Ala
275 280 285
Gly Ser Ala Gln Gly Arg Ile Pro Arg Val Tyr Gly Arg Gln Ala Ser
290 295 300
Glu Tyr Val Ile Arg Arg Gly Met Ser Gln Ile Gly Val Pro Tyr Ser
305 310 315 320
Trp Gly Gly Gly Asn Ala Ala Gly Pro Ser Lys Gly Ile Asp Ser Gly
325 330 335
Ala Gly Thr Val Gly Phe Asp Cys Ser Gly Leu Val Leu Tyr Ser Phe
340 345 350
Ala Gly Val Gly Ile Lys Leu Pro His Tyr Ser Gly Ser Gln Tyr Asn
355 360 365
Leu Gly Arg Lys Ile Pro Ser Ser Gln Met Arg Arg Gly Asp Val Ile
370 375 380
Phe Tyr Gly Pro Asn Gly Ser Gln His Val Thr Ile Tyr Leu Gly Asn
385 390 395 400
Gly Gln Met Leu Glu Ala Pro Asp Val Gly Leu Lys Val Arg Val Ala
405 410 415
Pro Val Arg Thr Ala Gly Met Thr Pro Tyr Val Val Arg Tyr Ile Glu
420 425 430
Tyr
<210> 8
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of GS linker
<400> 8
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
1 5 10
<210> 9
<211> 23
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of tPA
<400> 9
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser
20
<210> 10
<211> 156
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of Flt3L
<400> 10
Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala
1 5 10 15
Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val
20 25 30
Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp
35 40 45
Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala
50 55 60
Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His
65 70 75 80
Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95
Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu
100 105 110
Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu
115 120 125
Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140
Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro
145 150 155
<210> 11
<211> 1281
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of GX-170A
<400> 11
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln
20 25 30
His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser
35 40 45
Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln
50 55 60
Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg
65 70 75 80
Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu
85 90 95
Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe
100 105 110
Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg
115 120 125
Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile
130 135 140
Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp
145 150 155 160
Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr
165 170 175
Ala Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Phe
180 185 190
Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Ser
195 200 205
Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Ala Asn Ser
210 215 220
Pro Ala Leu Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Phe Ser
225 230 235 240
Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gln Ser Gly
245 250 255
Leu Ser Val Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp
260 265 270
Trp Tyr Gln Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys Trp
275 280 285
Glu Thr Phe Leu Thr Ser Glu Leu Pro Gly Trp Leu Gln Ala Asn Arg
290 295 300
His Val Lys Pro Thr Gly Ser Ala Val Val Gly Leu Ser Met Ala Ala
305 310 315 320
Ser Ser Ala Leu Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Val Tyr
325 330 335
Ala Gly Ala Met Ser Gly Leu Leu Asp Pro Ser Gln Ala Met Gly Pro
340 345 350
Thr Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ser
355 360 365
Asp Met Trp Gly Pro Lys Glu Asp Pro Ala Trp Gln Arg Asn Asp Pro
370 375 380
Leu Leu Asn Val Gly Lys Leu Ile Ala Asn Asn Thr Arg Val Trp Val
385 390 395 400
Tyr Cys Gly Asn Gly Lys Pro Ser Asp Leu Gly Gly Asn Asn Leu Pro
405 410 415
Ala Lys Phe Leu Glu Gly Phe Val Arg Thr Ser Asn Ile Lys Phe Gln
420 425 430
Asp Ala Tyr Asn Ala Gly Gly Gly His Asn Gly Val Phe Asp Phe Pro
435 440 445
Asp Ser Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala
450 455 460
Met Lys Pro Asp Leu Gln Arg Ala Leu Gly Ala Thr Pro Asn Thr Gly
465 470 475 480
Pro Ala Pro Gln Gly Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
485 490 495
Ser Gln Ile Met Tyr Asn Tyr Pro Ala Met Leu Gly His Ala Gly Asp
500 505 510
Met Ala Gly Tyr Ala Gly Thr Leu Gln Ser Leu Gly Ala Glu Ile Ala
515 520 525
Val Glu Gln Ala Ala Leu Gln Ser Ala Trp Gln Gly Asp Thr Gly Ile
530 535 540
Thr Tyr Gln Ala Trp Gln Ala Gln Trp Asn Gln Ala Met Glu Asp Leu
545 550 555 560
Val Arg Ala Tyr His Ala Met Ser Ser Thr His Glu Ala Asn Thr Met
565 570 575
Ala Met Met Ala Arg Asp Thr Ala Glu Ala Ala Lys Trp Gly Gly Gly
580 585 590
Ser Gly Ser Gly Ser Gly Ser Gly Ser Ala Pro Pro Ala Leu Ser Gln
595 600 605
Asp Arg Phe Ala Asp Phe Pro Ala Leu Pro Leu Asp Pro Ser Ala Met
610 615 620
Val Ala Gln Val Gly Pro Gln Val Val Asn Ile Asn Thr Lys Leu Gly
625 630 635 640
Tyr Asn Asn Ala Val Gly Ala Gly Thr Gly Ile Val Ile Asp Pro Asn
645 650 655
Gly Val Val Leu Thr Asn Asn Ala Val Ile Ala Gly Ala Thr Asp Ile
660 665 670
Asn Ala Phe Ser Val Gly Ser Gly Gln Thr Tyr Gly Val Asp Val Val
675 680 685
Gly Tyr Asp Arg Thr Gln Asn Val Ala Val Leu Gln Leu Arg Gly Ala
690 695 700
Gly Gly Leu Pro Ser Ala Ala Ile Gly Gly Gly Val Ala Val Gly Glu
705 710 715 720
Pro Val Val Ala Met Gly Asn Ser Gly Gly Gln Gly Gly Thr Pro Arg
725 730 735
Ala Val Pro Gly Arg Val Val Ala Leu Gly Gln Thr Val Gln Ala Ser
740 745 750
Asp Ser Leu Thr Gly Ala Glu Glu Thr Leu Asn Gly Leu Ile Gln Phe
755 760 765
Asp Ala Ala Ile Gln Pro Gly Asp Ala Gly Gly Pro Val Val Asn Gly
770 775 780
Leu Gly Gln Val Val Gly Met Asn Thr Ala Ala Ser Asp Asn Phe Gln
785 790 795 800
Leu Ser Gln Gly Gly Gln Gly Phe Ala Ile Pro Ile Gly Gln Ala Met
805 810 815
Ala Ile Ala Gly Gln Ile Arg Ser Gly Gly Gly Ser Pro Thr Val His
820 825 830
Ile Gly Pro Thr Ala Phe Leu Gly Leu Gly Val Val Asp Asn Asn Gly
835 840 845
Asn Gly Ala Arg Val Gln Arg Val Val Gly Ser Ala Pro Ala Ala Ser
850 855 860
Leu Gly Ile Ser Thr Gly Asp Val Ile Thr Ala Val Asp Gly Ala Pro
865 870 875 880
Ile Asn Ser Ala Thr Ala Met Ala Asp Ala Leu Asn Gly His His Pro
885 890 895
Gly Asp Val Ile Ser Val Thr Trp Gln Thr Lys Ser Gly Gly Thr Arg
900 905 910
Thr Gly Asn Val Thr Leu Ala Glu Gly Pro Pro Ala Gly Ser Gly Ser
915 920 925
Gly Ser Gly Ser Gly Ser Cys Gly Asn Asp Asp Asn Val Thr Gly Gly
930 935 940
Gly Ala Thr Thr Gly Gln Ala Ser Ala Lys Val Asp Cys Gly Gly Lys
945 950 955 960
Lys Thr Leu Lys Ala Ser Gly Ser Thr Ala Gln Ala Asn Ala Met Thr
965 970 975
Arg Phe Val Asn Val Phe Glu Gln Ala Cys Pro Gly Gln Thr Leu Asn
980 985 990
Tyr Thr Ala Asn Gly Ser Gly Ala Gly Ile Ser Glu Phe Asn Gly Asn
995 1000 1005
Gln Thr Asp Phe Gly Gly Ser Asp Val Pro Leu Ser Lys Asp Glu Pro
1010 1015 1020
Ser Gly Ala Ala Arg Cys Gly Ser Pro Ala Trp Asn Leu Pro Val Val
1025 1030 1035 1040
Phe Gly Pro Ile Ala Val Thr Tyr Asn Leu Asn Ser Val Ser Ser Leu
1045 1050 1055
Asn Leu Asp Gly Pro Thr Leu Ala Lys Ile Phe Asn Gly Ser Ile Thr
1060 1065 1070
Gln Trp Asn Asn Pro Ala Ile Gln Ala Leu Asn Arg Asp Phe Thr Leu
1075 1080 1085
Pro Gly Glu Arg Ile His Val Val Phe Arg Ser Asp Glu Ser Gly Thr
1090 1095 1100
Thr Asp Asn Phe Gln Arg Tyr Leu Gln Ala Ala Ser Asn Gly Ala Trp
1105 1110 1115 1120
Gly Lys Gly Ala Gly Lys Ser Phe Gln Gly Gly Val Gly Glu Gly Ala
1125 1130 1135
Arg Gly Asn Asp Gly Thr Ser Ala Ala Ala Lys Asn Thr Pro Gly Ser
1140 1145 1150
Ile Thr Tyr Asn Glu Trp Ser Phe Ala Gln Ala His Asp Leu Thr Met
1155 1160 1165
Ala Asn Ile Val Thr Ser Ala Gly Gly Asp Pro Val Ala Ile Thr Ile
1170 1175 1180
Asp Ser Val Gly Gln Thr Ile Ala Gly Ala Thr Ile Ser Gly Val Gly
1185 1190 1195 1200
Asn Asp Leu Val Leu Asp Thr Asp Ser Phe Tyr Arg Pro Lys Arg Pro
1205 1210 1215
Gly Ser Tyr Pro Ile Val Leu Ala Thr Tyr Glu Ile Val Cys Ser Lys
1220 1225 1230
Tyr Pro Asp Ser Gln Val Gly Thr Ala Val Lys Ala Phe Leu Gln Ser
1235 1240 1245
Thr Ile Gly Ala Gly Gln Ser Gly Leu Gly Asp Asn Gly Tyr Ile Pro
1250 1255 1260
Ile Pro Asp Glu Phe Lys Ser Arg Leu Ser Thr Ala Val Asn Ala Ile
1265 1270 1275 1280
Ala
<210> 12
<211> 2005
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of GX-170B
<400> 12
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln
20 25 30
His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser
35 40 45
Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln
50 55 60
Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg
65 70 75 80
Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu
85 90 95
Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe
100 105 110
Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg
115 120 125
Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile
130 135 140
Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp
145 150 155 160
Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr
165 170 175
Ala Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val
180 185 190
Asn Phe Ser Val Leu Pro Pro Glu Ile Asn Ser Gly Arg Met Phe Phe
195 200 205
Gly Ala Gly Ser Gly Pro Met Leu Ala Ala Ala Ala Ala Trp Asp Gly
210 215 220
Leu Ala Ala Glu Leu Gly Leu Ala Ala Glu Ser Phe Gly Leu Val Thr
225 230 235 240
Ser Gly Leu Ala Gly Gly Ser Gly Gln Ala Trp Gln Gly Ala Ala Ala
245 250 255
Ala Ala Met Val Val Ala Ala Ala Pro Tyr Ala Gly Trp Leu Ala Ala
260 265 270
Ala Ala Ala Arg Ala Gly Gly Ala Ala Val Gln Ala Lys Ala Val Ala
275 280 285
Gly Ala Phe Glu Ala Ala Arg Ala Ala Met Val Asp Pro Val Val Val
290 295 300
Ala Ala Asn Arg Ser Ala Phe Val Gln Leu Val Leu Ser Asn Val Phe
305 310 315 320
Gly Gln Asn Ala Pro Ala Ile Ala Ala Ala Glu Ala Thr Tyr Glu Gln
325 330 335
Met Trp Ala Ala Asp Val Ala Ala Met Val Gly Tyr His Gly Gly Ala
340 345 350
Ser Ala Ala Ala Ala Ala Leu Ala Pro Trp Gln Gln Ala Val Pro Gly
355 360 365
Leu Ser Gly Leu Leu Asp Ser Ala Gln Ser Ser Ala Gln Ala Val Thr
370 375 380
Ala Gln Ala Val Gly Ser Thr Val Pro Gly Pro Leu Gln Gly Ile Asn
385 390 395 400
Phe Gly Phe Gly Asn Ile Gly Ser Leu Asn Leu Gly Ser Gly Asn Thr
405 410 415
Gly Asp Thr Asn Val Gly Ser Gly Asn Ile Gly Asn Thr Asn Leu Gly
420 425 430
Gly Gly Asn Ile Gly Ser Phe Asn Leu Gly Ser Gly Asn Gln Gly Asp
435 440 445
Ile Asn Leu Gly Ile Gly Asn Val Gly Asn Leu Asn Leu Gly Ser Gly
450 455 460
Asn Phe Gly Ser Gln Asn Leu Gly Ser Gly Asn Ile Gly Ser Thr Asn
465 470 475 480
Val Gly Ser Gly Asn Ile Gly Ser Thr Asn Val Gly Ser Gly Asn Ile
485 490 495
Gly Asp Thr Asn Phe Gly Asn Gly Asn Asn Gly Asn Phe Asn Phe Gly
500 505 510
Ser Gly Asn Thr Gly Ser Asn Asn Ile Gly Phe Gly Asn Thr Gly Ser
515 520 525
Gly Asn Phe Gly Phe Gly Asn Thr Gly Asn Asn Asn Ile Gly Ile Gly
530 535 540
Leu Thr Gly Asp Gly Gln Ile Gly Ile Gly Gly Leu Asn Ser Gly Ser
545 550 555 560
Gly Asn Ile Gly Phe Gly Asn Ser Gly Thr Gly Asn Val Gly Leu Phe
565 570 575
Asn Ser Gly Thr Gly Asn Val Gly Phe Gly Asn Ser Gly Thr Ala Asn
580 585 590
Thr Gly Phe Gly Asn Ala Gly Asn Val Asn Thr Gly Phe Trp Asn Gly
595 600 605
Gly Ser Thr Asn Thr Gly Leu Ala Asn Ala Gly Ala Gly Asn Thr Gly
610 615 620
Phe Phe Asp Ala Gly Asn Tyr Asn Phe Gly Ser Leu Asn Ala Gly Asn
625 630 635 640
Ile Asn Ser Ser Phe Gly Asn Ser Gly Asp Gly Asn Ser Gly Phe Leu
645 650 655
Asn Ala Gly Asp Val Asn Ser Gly Val Gly Asn Ala Gly Asp Val Asn
660 665 670
Thr Gly Leu Gly Asn Ser Gly Asn Ile Asn Thr Gly Gly Phe Asn Pro
675 680 685
Gly Thr Leu Asn Thr Gly Phe Phe Ser Ala Met Thr Gln Ala Gly Pro
690 695 700
Asn Ser Gly Phe Phe Asn Ala Gly Thr Gly Asn Ser Gly Phe Gly His
705 710 715 720
Asn Asp Pro Ala Gly Ser Gly Asn Ser Gly Ile Gln Asn Ser Gly Phe
725 730 735
Gly Asn Ser Gly Tyr Val Asn Thr Ser Thr Thr Ser Met Phe Gly Gly
740 745 750
Asn Ser Gly Val Leu Asn Thr Gly Tyr Gly Asn Ser Gly Phe Tyr Asn
755 760 765
Ala Ala Val Asn Asn Thr Gly Ile Phe Val Thr Gly Val Met Ser Ser
770 775 780
Gly Phe Phe Asn Phe Gly Thr Gly Asn Ser Gly Leu Leu Val Ser Gly
785 790 795 800
Asn Gly Leu Ser Gly Phe Phe Lys Asn Leu Phe Gly Gly Ser Gly Ser
805 810 815
Gly Ser Gly Ser Gly Ser Thr Asp Arg Val Ser Val Gly Asn Leu Arg
820 825 830
Ile Ala Arg Val Leu Tyr Asp Phe Val Asn Asn Glu Ala Leu Pro Gly
835 840 845
Thr Asp Ile Asp Pro Asp Ser Phe Trp Ala Gly Val Asp Lys Val Val
850 855 860
Ala Asp Leu Thr Pro Gln Asn Gln Ala Leu Leu Asn Ala Arg Asp Glu
865 870 875 880
Leu Gln Ala Gln Ile Asp Lys Trp His Arg Arg Arg Val Ile Glu Pro
885 890 895
Ile Asp Met Asp Ala Tyr Arg Gln Phe Leu Thr Glu Ile Gly Tyr Leu
900 905 910
Leu Pro Glu Pro Asp Asp Phe Thr Ile Thr Thr Ser Gly Val Asp Ala
915 920 925
Glu Ile Thr Thr Thr Ala Gly Pro Gln Leu Val Val Pro Val Leu Asn
930 935 940
Ala Arg Phe Ala Leu Asn Ala Ala Asn Ala Arg Trp Gly Ser Leu Tyr
945 950 955 960
Asp Ala Leu Tyr Gly Thr Asp Val Ile Pro Glu Thr Asp Gly Ala Glu
965 970 975
Lys Gly Pro Thr Tyr Asn Lys Val Arg Gly Asp Lys Val Ile Ala Tyr
980 985 990
Ala Arg Lys Phe Leu Asp Asp Ser Val Pro Leu Ser Ser Gly Ser Phe
995 1000 1005
Gly Asp Ala Thr Gly Phe Thr Val Gln Asp Gly Gln Leu Val Val Ala
1010 1015 1020
Leu Pro Asp Lys Ser Thr Gly Leu Ala Asn Pro Gly Gln Phe Ala Gly
1025 1030 1035 1040
Tyr Thr Gly Ala Ala Glu Ser Pro Thr Ser Val Leu Leu Ile Asn His
1045 1050 1055
Gly Leu His Ile Glu Ile Leu Ile Asp Pro Glu Ser Gln Val Gly Thr
1060 1065 1070
Thr Asp Arg Ala Gly Val Lys Asp Val Ile Leu Glu Ser Ala Ile Thr
1075 1080 1085
Thr Ile Met Asp Phe Glu Asp Ser Val Ala Ala Val Asp Ala Ala Asp
1090 1095 1100
Lys Val Leu Gly Tyr Arg Asn Trp Leu Gly Leu Asn Lys Gly Asp Leu
1105 1110 1115 1120
Ala Ala Ala Val Asp Lys Asp Gly Thr Ala Phe Leu Arg Val Leu Asn
1125 1130 1135
Arg Asp Arg Asn Tyr Thr Ala Pro Gly Gly Gly Gln Phe Thr Leu Pro
1140 1145 1150
Gly Arg Ser Leu Met Phe Val Arg Asn Val Gly His Leu Met Thr Asn
1155 1160 1165
Asp Ala Ile Val Asp Thr Asp Gly Ser Glu Val Phe Glu Gly Ile Met
1170 1175 1180
Asp Ala Leu Phe Thr Gly Leu Ile Ala Ile His Gly Leu Lys Ala Ser
1185 1190 1195 1200
Asp Val Asn Gly Pro Leu Ile Asn Ser Arg Thr Gly Ser Ile Tyr Ile
1205 1210 1215
Val Lys Pro Lys Met His Gly Pro Ala Glu Val Ala Phe Thr Cys Glu
1220 1225 1230
Leu Phe Ser Arg Val Glu Asp Val Leu Gly Leu Pro Gln Asn Thr Met
1235 1240 1245
Lys Ile Gly Ile Met Asp Glu Glu Arg Arg Thr Thr Val Asn Leu Lys
1250 1255 1260
Ala Cys Ile Lys Ala Ala Ala Asp Arg Val Val Phe Ile Asn Thr Gly
1265 1270 1275 1280
Phe Leu Asp Arg Thr Gly Asp Glu Ile His Thr Ser Met Glu Ala Gly
1285 1290 1295
Pro Met Val Arg Lys Gly Thr Met Lys Ser Gln Pro Trp Ile Leu Ala
1300 1305 1310
Tyr Glu Asp His Asn Val Asp Ala Gly Leu Ala Ala Gly Phe Ser Gly
1315 1320 1325
Arg Ala Gln Val Gly Lys Gly Met Trp Thr Met Thr Glu Leu Met Ala
1330 1335 1340
Asp Met Val Glu Thr Lys Ile Ala Gln Pro Arg Ala Gly Ala Ser Thr
1345 1350 1355 1360
Ala Trp Val Pro Ser Pro Thr Ala Ala Thr Leu His Ala Leu His Tyr
1365 1370 1375
His Gln Val Asp Val Ala Ala Val Gln Gln Gly Leu Ala Gly Lys Arg
1380 1385 1390
Arg Ala Thr Ile Glu Gln Leu Leu Thr Ile Pro Leu Ala Lys Glu Leu
1395 1400 1405
Ala Trp Ala Pro Asp Glu Ile Arg Glu Glu Val Asp Asn Asn Cys Gln
1410 1415 1420
Ser Ile Leu Gly Tyr Val Val Arg Trp Val Asp Gln Gly Val Gly Cys
1425 1430 1435 1440
Ser Lys Val Pro Asp Ile His Asp Val Ala Leu Met Glu Asp Arg Ala
1445 1450 1455
Thr Leu Arg Ile Ser Ser Gln Leu Leu Ala Asn Trp Leu Arg His Gly
1460 1465 1470
Val Ile Thr Ser Ala Asp Val Arg Ala Ser Leu Glu Arg Met Ala Pro
1475 1480 1485
Leu Val Asp Arg Gln Asn Ala Gly Asp Val Ala Tyr Arg Pro Met Ala
1490 1495 1500
Pro Asn Phe Asp Asp Ser Ile Ala Phe Leu Ala Ala Gln Glu Leu Ile
1505 1510 1515 1520
Leu Ser Gly Ala Gln Gln Pro Asn Gly Tyr Thr Glu Pro Ile Leu His
1525 1530 1535
Arg Arg Arg Arg Glu Phe Lys Ala Arg Ala Ala Glu Lys Pro Ala Pro
1540 1545 1550
Ser Asp Arg Ala Gly Asp Asp Ala Ala Arg Gly Ser Gly Ser Gly Ser
1555 1560 1565
Gly Ser Gly Ser Asp Pro Gln Thr Asp Thr Ile Ala Ala Leu Ile Ala
1570 1575 1580
Asp Val Ala Lys Ala Asn Gln Arg Leu Gln Asp Leu Ser Asp Glu Val
1585 1590 1595 1600
Gln Ala Glu Gln Glu Ser Val Asn Lys Ala Met Val Asp Val Glu Thr
1605 1610 1615
Ala Arg Asp Asn Ala Ala Ala Ala Glu Asp Asp Leu Glu Val Ser Gln
1620 1625 1630
Arg Ala Val Lys Asp Ala Asn Ala Ala Ile Ala Ala Ala Gln His Arg
1635 1640 1645
Phe Asp Thr Phe Ala Ala Ala Thr Tyr Met Asn Gly Pro Ser Val Ser
1650 1655 1660
Tyr Leu Ser Ala Ser Ser Pro Asp Glu Ile Ile Ala Thr Val Thr Ala
1665 1670 1675 1680
Ala Lys Thr Leu Ser Ala Ser Ser Gln Ala Val Met Ala Asn Leu Gln
1685 1690 1695
Arg Ala Arg Thr Glu Arg Val Asn Thr Glu Ser Ala Ala Arg Leu Ala
1700 1705 1710
Lys Gln Lys Ala Asp Lys Ala Ala Ala Asp Ala Lys Ala Ser Gln Asp
1715 1720 1725
Ala Ala Val Ala Ala Leu Thr Glu Thr Arg Arg Lys Phe Asp Glu Gln
1730 1735 1740
Arg Glu Glu Val Gln Arg Leu Ala Ala Glu Arg Asp Ala Ala Gln Ala
1745 1750 1755 1760
Arg Leu Gln Ala Ala Arg Leu Val Ala Trp Ser Ser Glu Gly Gly Gln
1765 1770 1775
Gly Ala Pro Pro Phe Arg Met Trp Asp Pro Gly Ser Gly Pro Ala Gly
1780 1785 1790
Gly Arg Ala Trp Asp Gly Leu Trp Asp Pro Thr Leu Pro Met Ile Pro
1795 1800 1805
Ser Ala Asn Ile Pro Gly Asp Pro Ile Ala Val Val Asn Gln Val Leu
1810 1815 1820
Gly Ile Ser Ala Thr Ser Ala Gln Val Thr Ala Asn Met Gly Arg Lys
1825 1830 1835 1840
Phe Leu Glu Gln Leu Gly Ile Leu Gln Pro Thr Asp Thr Gly Ile Thr
1845 1850 1855
Asn Ala Pro Ala Gly Ser Ala Gln Gly Arg Ile Pro Arg Val Tyr Gly
1860 1865 1870
Arg Gln Ala Ser Glu Tyr Val Ile Arg Arg Gly Met Ser Gln Ile Gly
1875 1880 1885
Val Pro Tyr Ser Trp Gly Gly Gly Asn Ala Ala Gly Pro Ser Lys Gly
1890 1895 1900
Ile Asp Ser Gly Ala Gly Thr Val Gly Phe Asp Cys Ser Gly Leu Val
1905 1910 1915 1920
Leu Tyr Ser Phe Ala Gly Val Gly Ile Lys Leu Pro His Tyr Ser Gly
1925 1930 1935
Ser Gln Tyr Asn Leu Gly Arg Lys Ile Pro Ser Ser Gln Met Arg Arg
1940 1945 1950
Gly Asp Val Ile Phe Tyr Gly Pro Asn Gly Ser Gln His Val Thr Ile
1955 1960 1965
Tyr Leu Gly Asn Gly Gln Met Leu Glu Ala Pro Asp Val Gly Leu Lys
1970 1975 1980
Val Arg Val Ala Pro Val Arg Thr Ala Gly Met Thr Pro Tyr Val Val
1985 1990 1995 2000
Arg Tyr Ile Glu Tyr
2005
<210> 13
<211> 885
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of Ag85A
<400> 13
tttagcaggc caggcctgcc cgtggagtat ctgcaggtgc cctccccttc tatgggccgc 60
gacatcaagg tgcagttcca gtccggagga gccaactctc cagccctgta tctgctggac 120
ggcctgagag cccaggacga tttcagcggc tgggacatca atacacccgc ctttgagtgg 180
tacgaccaga gcggactgtc cgtggtcatg ccagtgggag gacagtcctc tttttactcc 240
gactggtatc agcctgcatg cggcaaggca ggatgtcaga cctacaagtg ggagaccttc 300
ctgacaagcg agctgcctgg ctggctgcag gccaacaggc acgtgaagcc aacaggctcc 360
gccgtggtgg gactgtctat ggcagccagc tccgccctga ccctggcaat ctaccaccct 420
cagcagttcg tgtacgcagg agcaatgagc ggactgctgg acccctccca ggcaatggga 480
ccaaccctga tcggactggc aatgggcgac gcaggaggat ataaggcaag cgacatgtgg 540
ggaccaaagg aggaccccgc atggcagagg aacgacccac tgctgaatgt gggcaagctg 600
atcgccaaca ataccagagt gtgggtgtac tgtggaaacg gcaagccatc cgatctggga 660
ggaaacaatc tgcctgccaa gttcctggag ggctttgtgc ggacatctaa tatcaagttc 720
caggacgcat ataacgcagg aggaggacac aatggcgtgt tcgactttcc cgattccggc 780
acccactctt gggagtactg gggagcacag ctgaacgcaa tgaagcccga cctgcagaga 840
gccctgggag caacccccaa tacaggacca gcaccacagg gagca 885
<210> 14
<211> 285
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of TB10.4
<400> 14
agccagatca tgtacaacta tcctgccatg ctgggacacg caggcgacat ggcaggatac 60
gcaggcaccc tgcagtctct gggagcagag atcgcagtgg agcaggccgc cctgcagagc 120
gcctggcagg gcgataccgg catcacatat caggcatggc aggcacagtg gaaccaggca 180
atggaggacc tggtgcgggc ctaccacgcc atgtcctcta cccacgaggc caatacaatg 240
gccatgatgg ccagagatac cgcagaggca gcaaagtggg gagga 285
<210> 15
<211> 969
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of Mtb32b
<400> 15
gccccccctg ccctgtccca ggaccggttc gccgattttc ctgccctgcc actggacccc 60
tctgccatgg tggcacaagt gggacctcag gtggtgaaca tcaatacaaa gctgggctac 120
aacaatgcag tgggagcagg caccggaatc gtgatcgacc caaacggcgt ggtgctgaca 180
aacaatgccg tgatcgccgg cgccaccgac atcaatgcct tttctgtggg cagcggccag 240
acatacggcg tggacgtggt gggctatgat aggacccaga atgtggccgt gctgcagctg 300
aggggagcag gaggcctgcc ttccgccgcc atcggcggcg gcgtggccgt gggcgagcca 360
gtggtggcaa tgggcaacag cggaggacag ggaggcacac cacgggccgt gcccggcaga 420
gtggtggccc tgggacagac cgtgcaggca agcgattccc tgaccggagc cgaggagaca 480
ctgaacggcc tgatccagtt tgatgcagca atccagccag gcgacgcagg aggacctgtg 540
gtgaatggcc tgggacaggt ggtgggaatg aacaccgcag cctctgacaa tttccagctg 600
agccagggag gacagggatt tgcaatccca atcggacagg caatggcaat cgcaggacag 660
atcaggagcg gaggaggctc cccaaccgtg cacatcggac caacagcctt cctgggactg 720
ggagtggtgg acaacaatgg caacggcgcc cgggtgcaga gagtggtggg ctccgcccca 780
gcagcatctc tgggcatcag caccggcgac gtgatcacag ccgtggatgg cgcccctatc 840
aacagcgcca ccgccatggc cgatgccctg aatggccacc acccaggcga cgtgatcagc 900
gtgacctggc agacaaagtc cggaggcacc aggacaggaa acgtgacact ggcagaggga 960
ccaccagca 969
<210> 16
<211> 1044
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of PstS3
<400> 16
tgcggcaacg acgataatgt gaccggagga ggagcaacca caggacaggc ctccgccaag 60
gtggattgtg gcggcaagaa gaccctgaag gcatccggct ctacagcaca ggcaaacgcc 120
atgaccaggt tcgtgaacgt gttcgagcag gcatgcccag gacagaccct gaactataca 180
gcaaatggca gcggagcagg catctccgag ttcaacggca atcagacaga ctttggcggc 240
tctgatgtgc ctctgagcaa ggacgagcca tccggagcag caagatgtgg cagccctgcc 300
tggaatctgc cagtggtgtt tggccccatc gccgtgacct acaacctgaa tagcgtgagc 360
tccctgaacc tggatggccc cacactggcc aagattttca acggctccat cacccagtgg 420
aacaatcccg ccatccaggc cctgaaccgg gacttcaccc tgcctggcga gagaatccac 480
gtggtgtttc gctccgacga gtctggcacc acagataact tccagagata cctgcaggca 540
gcatctaatg gagcatgggg caagggagca ggcaagagct ttcagggagg agtgggagag 600
ggagccagag gcaacgatgg caccagcgcc gccgccaaga acaccccagg ctctatcaca 660
tacaatgagt ggagcttcgc ccaggcccac gacctgacca tggccaatat cgtgacaagc 720
gccggaggcg atcctgtggc aatcaccatc gactccgtgg gacagaccat cgcaggagcc 780
acaatctctg gcgtgggcaa cgatctggtg ctggacacag atagcttcta taggcctaag 840
cgcccaggct cctacccaat cgtgctggcc acctatgaga tcgtgtgctc taagtacccc 900
gacagccaag tgggcacagc cgtgaaggcc tttctgcagt ccaccatcgg agcaggacag 960
tctggactgg gcgataacgg ctacatccct atcccagacg agttcaagag cagactgtcc 1020
accgccgtga atgccatcgc ctaa 1044
<210> 17
<211> 1863
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of PPE39
<400> 17
gtgaatttca gcgtgctgcc ccctgagatc aactccggcc ggatgttctt tggagcaggc 60
tccggaccta tgctggctgc cgccgccgcc tgggacggac tggcagcaga gctgggcctg 120
gccgccgaga gctttggcct ggtgacaagc ggcctggccg gcggctctgg ccaggcctgg 180
cagggcgctg ccgccgccgc catggtggtg gctgccgccc catacgccgg ctggctggcc 240
gccgccgccg ccagagccgg aggagccgcc gtgcaggcca aggccgtggc cggcgccttc 300
gaggccgcca gggccgcaat ggtggaccca gtggtggtgg cagcaaaccg ctctgccttc 360
gtgcagctgg tgctgagcaa cgtgttcgga cagaacgcac cagcaatcgc tgccgccgag 420
gcaacctacg agcagatgtg ggcagcagat gtggcagcaa tggtgggcta tcacggagga 480
gcaagcgccg ccgccgccgc cctggcccca tggcagcagg ccgtgcccgg cctgagcgga 540
ctgctggact ccgcccagtc ctctgcccag gccgtgaccg cacaggcagt gggctccaca 600
gtgcccggcc ctctgcaggg catcaatttc ggctttggca acatcggctc tctgaatctg 660
ggcagcggca acacaggcga taccaatgtg ggcagcggca acatcggcaa taccaacctg 720
ggcggcggca atatcggctc cttcaacctg ggctctggca atcagggcga catcaacctg 780
ggcatcggca atgtgggcaa tctgaacctg ggcagcggca actttggctc ccagaatctg 840
ggctctggca acatcggcag caccaatgtg ggctccggca acatcggctc tacaaatgtg 900
ggcagcggaa atatcggcga taccaatttc ggcaacggca acaatggcaa tttcaacttt 960
ggctccggca acacaggctc taacaatatc ggctttggca ataccggcag cggcaacttc 1020
ggctttggca atacaggcaa caataacatc ggcatcggcc tgaccggcga cggacagatc 1080
ggaatcggag gcctgaactc tggcagcggc aatatcggct tcggcaactc tggcacaggc 1140
aatgtgggcc tgttcaacag cggcaccggc aatgtgggct ttggcaactc cggcacagcc 1200
aataccggct tcggcaacgc cggcaatgtg aacacaggct tttggaatgg cggctccaca 1260
aacaccggac tggcaaatgc aggagcagga aacaccggct tctttgacgc cggcaattac 1320
aactttggct ctctgaacgc cggcaatatc aacagctcct tcggcaatag cggcgacggc 1380
aactccggct ttctgaatgc cggcgatgtg aactctggcg tgggcaatgc cggcgacgtg 1440
aacacaggcc tgggcaatag cggcaatatc aacaccggcg gcttcaaccc aggcacactg 1500
aataccggct tcttttccgc catgacacag gccggcccca actctggctt ctttaatgca 1560
ggcaccggca acagcggatt tggacacaac gacccagcag gcagcggcaa ttccggcatc 1620
cagaactctg gcttcggcaa tagcggctac gtgaacacat ctaccacaag catgtttggc 1680
ggcaattctg gcgtgctgaa caccggctac ggcaatagcg gcttctataa cgccgccgtg 1740
aataacacag gcatctttgt gaccggcgtg atgtctagcg gcttctttaa tttcggcaca 1800
ggcaactccg gcctgctggt gtctggcaat ggcctgagcg gcttctttaa gaacctgttt 1860
gga 1863
<210> 18
<211> 2220
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of GlcB
<400> 18
accgaccgcg tgtccgtggg caacctgaga atcgccaggg tgctgtatga tttcgtgaat 60
aacgaggccc tgcctggcac agacatcgac ccagacagct tttgggcagg agtggataag 120
gtggtggccg acctgacccc tcagaatcag gccctgctga acgccagaga cgagctgcag 180
gcccagatcg acaagtggca ccggcggaga gtgatcgagc caatcgacat ggacgcctac 240
aggcagttcc tgacagagat cggctatctg ctgccagagc ccgacgactt caccatcacc 300
acatccggcg tggatgccga gatcaccaca accgcaggac ctcagctggt ggtgccagtg 360
ctgaacgcaa ggttcgccct gaatgcagca aacgccagat ggggcagcct gtacgatgcc 420
ctgtatggca cagacgtgat cccagagacc gatggagcag agaagggacc tacatacaat 480
aaggtgaggg gcgacaaagt gatcgcctat gcccgcaagt tcctggacga ttctgtgccc 540
ctgtcctctg gcagcttcgg cgacgccaca ggctttaccg tgcaggatgg acagctggtg 600
gtggccctgc ctgataagtc caccggactg gcaaacccag gacagtttgc aggatacacc 660
ggagcagcag agtcccccac atctgtgctg ctgatcaatc acggcctgca catcgagatc 720
ctgatcgacc ctgagagcca agtgggcaca accgacagag caggagtgaa ggatgtgatc 780
ctggagtccg ccatcacaac catcatggat ttcgaggact ctgtggcagc agtggatgca 840
gcagacaagg tgctgggcta cagaaattgg ctgggcctga acaagggcga tctggctgcc 900
gccgtggata aggacggcac cgcctttctg agggtgctga atagagacag gaactatacc 960
gccccaggag gaggacagtt cacactgcca ggccggtctc tgatgtttgt gagaaatgtg 1020
ggccacctga tgaccaacga cgccatcgtg gatacagacg gcagcgaggt gttcgagggc 1080
atcatggatg ccctgtttac aggcctgatc gccatccacg gcctgaaggc ctccgacgtg 1140
aatggccccc tgatcaactc ccgcaccggc tctatctaca tcgtgaagcc aaagatgcac 1200
ggacctgcag aggtggcctt cacatgcgag ctgtttagcc gggtggagga cgtgctgggc 1260
ctgcctcaga ataccatgaa gatcggcatc atggatgagg agcgccggac aaccgtgaac 1320
ctgaaggcct gtatcaaggc tgccgccgac agggtggtgt tcatcaatac aggctttctg 1380
gatcgcaccg gcgacgagat ccacacatcc atggaggccg gcccaatggt gagaaagggc 1440
accatgaagt ctcagccctg gattctggcc tatgaggatc acaacgtgga cgcaggactg 1500
gcagcaggct tcagcggcag agcccaagtg ggcaagggca tgtggacaat gaccgagctg 1560
atggccgata tggtggagac aaagatcgca cagcctaggg ccggagccag caccgcatgg 1620
gtgccttccc caacagcagc caccctgcac gcactgcact accaccaggt ggacgtggca 1680
gcagtgcagc agggactggc aggcaagaga agggccacca tcgagcagct gctgacaatc 1740
cctctggcaa aggagctggc atgggcacca gacgagatca gggaggaggt ggataataac 1800
tgccagtcca tcctgggcta tgtggtgcgg tgggtggatc agggagtggg atgttctaag 1860
gtgccagaca tccacgacgt ggccctgatg gaggacaggg ccacactgcg gatcagctcc 1920
cagctgctgg ccaactggct gaggcacggc gtgatcacct ctgccgacgt gcgcgcaagc 1980
ctggagagga tggcaccact ggtggacagg cagaatgcag gcgatgtggc ctacagacct 2040
atggccccaa acttcgacga tagcatcgcc tttctggcag cacaggagct gatcctgtcc 2100
ggagcacagc agccaaacgg atataccgag cctatcctgc accgccggag aagggagttc 2160
aaggccagag ccgcagagaa gccagcacct tccgataggg caggcgacga tgcagcccgc 2220
2220
<210> 19
<211> 1302
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of RipA
<400> 19
gatccccaga cagacaccat cgccgccctg atcgcagacg tggcaaaggc caaccagagg 60
ctgcaggatc tgagcgacga ggtgcaggca gagcaggagt ccgtgaataa ggcaatggtg 120
gacgtggaga ccgcccggga taacgctgcc gccgccgagg acgatctgga ggtgagccag 180
agagccgtga aggatgcaaa cgcagcaatc gctgccgccc agcacaggtt cgacaccttc 240
gccgccgcca cctacatgaa tggcccctct gtgagctatc tgagcgcctc tagccctgac 300
gagatcatcg ccacagtgac cgcagcaaag accctgtccg cctcctctca ggccgtgatg 360
gcaaatctgc agagggcacg gacagagagg gtgaacaccg agtccgccgc ccgcctggca 420
aagcagaagg cagataaggc tgccgccgac gcaaaggcat ctcaggatgc agcagtggcc 480
gccctgacag agaccaggcg gaagttcgac gagcagagag aggaggtgca gaggctggca 540
gcagagaggg atgcagcaca ggcccggctg caggccgcca gactggtggc atggagctcc 600
gagggaggac agggcgcccc accctttcgg atgtgggacc ccggcagcgg accagcaggc 660
ggcagagcct gggatggact gtgggacccc accctgccta tgatcccatc cgccaatatc 720
cccggcgacc ctatcgccgt ggtgaaccag gtgctgggca tcagcgccac atccgcccag 780
gtgacagcca acatgggcag aaagttcctg gagcagctgg gcatcctgca gcccacagac 840
accggaatca ccaatgcacc tgcaggcagc gcccagggac gcatcccacg ggtgtacggc 900
aggcaggcat ccgagtatgt gatcagaagg ggcatgtccc agatcggcgt gccttactct 960
tggggaggag gaaacgcagc aggaccatcc aagggaatcg actctggagc aggcaccgtg 1020
ggattcgatt gttccggcct ggtgctgtat tcttttgccg gcgtgggcat caagctgcca 1080
cactactccg gctctcagta taatctgggc cgcaagatcc cctctagcca gatgcgccgg 1140
ggcgatgtga tcttctacgg ccctaatggc agccagcacg tgaccatcta tctgggaaac 1200
ggacagatgc tggaggcacc agacgtgggc ctgaaggtga gggtggcacc tgtgcgcaca 1260
gcaggcatga ccccatacgt ggtgcggtac atcgagtatt aa 1302
<210> 20
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of GS linker
<400> 20
ggcagcggct ccggctctgg cagcggctct 30
<210> 21
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of GS linker
<400> 21
ggctccggct ctggcagcgg ctccggctct 30
<210> 22
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of GS linker
<400> 22
ggctctggca gcggctccgg ctctggcagc 30
<210> 23
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of GS linker
<400> 23
ggcagcggct ccggctctgg cagcggctcc 30
<210> 24
<211> 69
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of tPA
<400> 24
atggatgcca tgaagagggg actgtgctgc gtgctgctgc tgtgcggagc cgtgttcgtg 60
tctccaagc 69
<210> 25
<211> 69
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of tPA
<400> 25
atggatgcca tgaagagggg actgtgctgc gtgctgctgc tgtgcggagc cgtgttcgtg 60
tccccatct 69
<210> 26
<211> 474
<212> DNA
<213> artificial sequence
<220>
<223> Flt3L nucleic acid sequence
<400> 26
acccaggatt gttctttcca gcacagcccc atcagctccg actttgccgt gaagatccgc 60
gagctgagcg actacctgct gcaggattat cccgtgacag tggcctccaa tctgcaggac 120
gaggagctgt gcggaggact gtggaggctg gtgctggcac agcggtggat ggagagactg 180
aagaccgtgg caggctccaa gatgcaggga ctgctggagc gcgtgaacac cgagatccac 240
ttcgtgacaa agtgcgcctt tcagccccct ccaagctgtc tgcggtttgt gcagacaaac 300
atctccagac tgctgcagga gacctctgag cagctggtgg ccctgaagcc ctggatcaca 360
aggcagaatt tctctcgctg cctggagctg cagtgtcagc ctgactctag caccctgcca 420
cctccatgga gccctcggcc actggaggca accgcaccaa cagcacctgg ctcc 474
<210> 27
<211> 474
<212> DNA
<213> artificial sequence
<220>
<223> Flt3L nucleic acid sequence
<400> 27
cacgccaccc aggattgttc tttccagcac agccctatca gctccgactt tgccgtgaag 60
atccgggagc tgtctgatta cctgctgcag gactatcccg tgacagtggc cagcaacctg 120
caggacgagg agctgtgcgg aggactgtgg aggctggtgc tggcacagag atggatggag 180
aggctgaaga ccgtggcagg ctccaagatg cagggactgc tggagcgggt gaatacagag 240
atccacttcg tgaccaagtg cgcctttcag ccccctccaa gctgtctgcg gttcgtgcag 300
accaacatca gcagactgct gcaggagaca tccgagcagc tggtggccct gaagccttgg 360
atcacaagac agaatttttc taggtgcctg gagctgcagt gtcagccaga ttctagcacc 420
ctgccacctc catggagccc tcgcccactg gaggccacag caccaaccgc acca 474
<210> 28
<211> 3846
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of GX-170A
<400> 28
atggatgcca tgaagagggg actgtgctgc gtgctgctgc tgtgcggagc cgtgttcgtg 60
tctccaagcc acgccaccca ggattgttct ttccagcaca gccccatcag ctccgacttt 120
gccgtgaaga tccgcgagct gagcgactac ctgctgcagg attatcccgt gacagtggcc 180
tccaatctgc aggacgagga gctgtgcgga ggactgtgga ggctggtgct ggcacagcgg 240
tggatggaga gactgaagac cgtggcaggc tccaagatgc agggactgct ggagcgcgtg 300
aacaccgaga tccacttcgt gacaaagtgc gcctttcagc cccctccaag ctgtctgcgg 360
tttgtgcaga caaacatctc cagactgctg caggagacct ctgagcagct ggtggccctg 420
aagccctgga tcacaaggca gaatttctct cgctgcctgg agctgcagtg tcagcctgac 480
tctagcaccc tgccacctcc atggagccct cggccactgg aggcaaccgc accaacagca 540
cctggctccg gctctggcag cggctccggc tcttttagca ggccaggcct gcccgtggag 600
tatctgcagg tgccctcccc ttctatgggc cgcgacatca aggtgcagtt ccagtccgga 660
ggagccaact ctccagccct gtatctgctg gacggcctga gagcccagga cgatttcagc 720
ggctgggaca tcaatacacc cgcctttgag tggtacgacc agagcggact gtccgtggtc 780
atgccagtgg gaggacagtc ctctttttac tccgactggt atcagcctgc atgcggcaag 840
gcaggatgtc agacctacaa gtgggagacc ttcctgacaa gcgagctgcc tggctggctg 900
caggccaaca ggcacgtgaa gccaacaggc tccgccgtgg tgggactgtc tatggcagcc 960
agctccgccc tgaccctggc aatctaccac cctcagcagt tcgtgtacgc aggagcaatg 1020
agcggactgc tggacccctc ccaggcaatg ggaccaaccc tgatcggact ggcaatgggc 1080
gacgcaggag gatataaggc aagcgacatg tggggaccaa aggaggaccc cgcatggcag 1140
aggaacgacc cactgctgaa tgtgggcaag ctgatcgcca acaataccag agtgtgggtg 1200
tactgtggaa acggcaagcc atccgatctg ggaggaaaca atctgcctgc caagttcctg 1260
gagggctttg tgcggacatc taatatcaag ttccaggacg catataacgc aggaggagga 1320
cacaatggcg tgttcgactt tcccgattcc ggcacccact cttgggagta ctggggagca 1380
cagctgaacg caatgaagcc cgacctgcag agagccctgg gagcaacccc caatacagga 1440
ccagcaccac agggagcagg cagcggctcc ggctctggca gcggctctag ccagatcatg 1500
tacaactatc ctgccatgct gggacacgca ggcgacatgg caggatacgc aggcaccctg 1560
cagtctctgg gagcagagat cgcagtggag caggccgccc tgcagagcgc ctggcagggc 1620
gataccggca tcacatatca ggcatggcag gcacagtgga accaggcaat ggaggacctg 1680
gtgcgggcct accacgccat gtcctctacc cacgaggcca atacaatggc catgatggcc 1740
agagataccg cagaggcagc aaagtgggga ggaggctccg gctctggcag cggctccggc 1800
tctgcccccc ctgccctgtc ccaggaccgg ttcgccgatt ttcctgccct gccactggac 1860
ccctctgcca tggtggcaca agtgggacct caggtggtga acatcaatac aaagctgggc 1920
tacaacaatg cagtgggagc aggcaccgga atcgtgatcg acccaaacgg cgtggtgctg 1980
acaaacaatg ccgtgatcgc cggcgccacc gacatcaatg ccttttctgt gggcagcggc 2040
cagacatacg gcgtggacgt ggtgggctat gataggaccc agaatgtggc cgtgctgcag 2100
ctgaggggag caggaggcct gccttccgcc gccatcggcg gcggcgtggc cgtgggcgag 2160
ccagtggtgg caatgggcaa cagcggagga cagggaggca caccacgggc cgtgcccggc 2220
agagtggtgg ccctgggaca gaccgtgcag gcaagcgatt ccctgaccgg agccgaggag 2280
acactgaacg gcctgatcca gtttgatgca gcaatccagc caggcgacgc aggaggacct 2340
gtggtgaatg gcctgggaca ggtggtggga atgaacaccg cagcctctga caatttccag 2400
ctgagccagg gaggacaggg atttgcaatc ccaatcggac aggcaatggc aatcgcagga 2460
cagatcagga gcggaggagg ctccccaacc gtgcacatcg gaccaacagc cttcctggga 2520
ctgggagtgg tggacaacaa tggcaacggc gcccgggtgc agagagtggt gggctccgcc 2580
ccagcagcat ctctgggcat cagcaccggc gacgtgatca cagccgtgga tggcgcccct 2640
atcaacagcg ccaccgccat ggccgatgcc ctgaatggcc accacccagg cgacgtgatc 2700
agcgtgacct ggcagacaaa gtccggaggc accaggacag gaaacgtgac actggcagag 2760
ggaccaccag caggctctgg cagcggctcc ggctctggca gctgcggcaa cgacgataat 2820
gtgaccggag gaggagcaac cacaggacag gcctccgcca aggtggattg tggcggcaag 2880
aagaccctga aggcatccgg ctctacagca caggcaaacg ccatgaccag gttcgtgaac 2940
gtgttcgagc aggcatgccc aggacagacc ctgaactata cagcaaatgg cagcggagca 3000
ggcatctccg agttcaacgg caatcagaca gactttggcg gctctgatgt gcctctgagc 3060
aaggacgagc catccggagc agcaagatgt ggcagccctg cctggaatct gccagtggtg 3120
tttggcccca tcgccgtgac ctacaacctg aatagcgtga gctccctgaa cctggatggc 3180
cccacactgg ccaagatttt caacggctcc atcacccagt ggaacaatcc cgccatccag 3240
gccctgaacc gggacttcac cctgcctggc gagagaatcc acgtggtgtt tcgctccgac 3300
gagtctggca ccacagataa cttccagaga tacctgcagg cagcatctaa tggagcatgg 3360
ggcaagggag caggcaagag ctttcaggga ggagtgggag agggagccag aggcaacgat 3420
ggcaccagcg ccgccgccaa gaacacccca ggctctatca catacaatga gtggagcttc 3480
gcccaggccc acgacctgac catggccaat atcgtgacaa gcgccggagg cgatcctgtg 3540
gcaatcacca tcgactccgt gggacagacc atcgcaggag ccacaatctc tggcgtgggc 3600
aacgatctgg tgctggacac agatagcttc tataggccta agcgcccagg ctcctaccca 3660
atcgtgctgg ccacctatga gatcgtgtgc tctaagtacc ccgacagcca agtgggcaca 3720
gccgtgaagg cctttctgca gtccaccatc ggagcaggac agtctggact gggcgataac 3780
ggctacatcc ctatcccaga cgagttcaag agcagactgt ccaccgccgt gaatgccatc 3840
gcctaa 3846
<210> 29
<211> 6018
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of GX-170B
<400> 29
atggatgcca tgaagagggg actgtgctgc gtgctgctgc tgtgcggagc cgtgttcgtg 60
tccccatctc acgccaccca ggattgttct ttccagcaca gccctatcag ctccgacttt 120
gccgtgaaga tccgggagct gtctgattac ctgctgcagg actatcccgt gacagtggcc 180
agcaacctgc aggacgagga gctgtgcgga ggactgtgga ggctggtgct ggcacagaga 240
tggatggaga ggctgaagac cgtggcaggc tccaagatgc agggactgct ggagcgggtg 300
aatacagaga tccacttcgt gaccaagtgc gcctttcagc cccctccaag ctgtctgcgg 360
ttcgtgcaga ccaacatcag cagactgctg caggagacat ccgagcagct ggtggccctg 420
aagccttgga tcacaagaca gaatttttct aggtgcctgg agctgcagtg tcagccagat 480
tctagcaccc tgccacctcc atggagccct cgcccactgg aggccacagc accaaccgca 540
ccaggcagcg gctccggctc tggcagcggc tccgtgaatt tcagcgtgct gccccctgag 600
atcaactccg gccggatgtt ctttggagca ggctccggac ctatgctggc tgccgccgcc 660
gcctgggacg gactggcagc agagctgggc ctggccgccg agagctttgg cctggtgaca 720
agcggcctgg ccggcggctc tggccaggcc tggcagggcg ctgccgccgc cgccatggtg 780
gtggctgccg ccccatacgc cggctggctg gccgccgccg ccgccagagc cggaggagcc 840
gccgtgcagg ccaaggccgt ggccggcgcc ttcgaggccg ccagggccgc aatggtggac 900
ccagtggtgg tggcagcaaa ccgctctgcc ttcgtgcagc tggtgctgag caacgtgttc 960
ggacagaacg caccagcaat cgctgccgcc gaggcaacct acgagcagat gtgggcagca 1020
gatgtggcag caatggtggg ctatcacgga ggagcaagcg ccgccgccgc cgccctggcc 1080
ccatggcagc aggccgtgcc cggcctgagc ggactgctgg actccgccca gtcctctgcc 1140
caggccgtga ccgcacaggc agtgggctcc acagtgcccg gccctctgca gggcatcaat 1200
ttcggctttg gcaacatcgg ctctctgaat ctgggcagcg gcaacacagg cgataccaat 1260
gtgggcagcg gcaacatcgg caataccaac ctgggcggcg gcaatatcgg ctccttcaac 1320
ctgggctctg gcaatcaggg cgacatcaac ctgggcatcg gcaatgtggg caatctgaac 1380
ctgggcagcg gcaactttgg ctcccagaat ctgggctctg gcaacatcgg cagcaccaat 1440
gtgggctccg gcaacatcgg ctctacaaat gtgggcagcg gaaatatcgg cgataccaat 1500
ttcggcaacg gcaacaatgg caatttcaac tttggctccg gcaacacagg ctctaacaat 1560
atcggctttg gcaataccgg cagcggcaac ttcggctttg gcaatacagg caacaataac 1620
atcggcatcg gcctgaccgg cgacggacag atcggaatcg gaggcctgaa ctctggcagc 1680
ggcaatatcg gcttcggcaa ctctggcaca ggcaatgtgg gcctgttcaa cagcggcacc 1740
ggcaatgtgg gctttggcaa ctccggcaca gccaataccg gcttcggcaa cgccggcaat 1800
gtgaacacag gcttttggaa tggcggctcc acaaacaccg gactggcaaa tgcaggagca 1860
ggaaacaccg gcttctttga cgccggcaat tacaactttg gctctctgaa cgccggcaat 1920
atcaacagct ccttcggcaa tagcggcgac ggcaactccg gctttctgaa tgccggcgat 1980
gtgaactctg gcgtgggcaa tgccggcgac gtgaacacag gcctgggcaa tagcggcaat 2040
atcaacaccg gcggcttcaa cccaggcaca ctgaataccg gcttcttttc cgccatgaca 2100
caggccggcc ccaactctgg cttctttaat gcaggcaccg gcaacagcgg atttggacac 2160
aacgacccag caggcagcgg caattccggc atccagaact ctggcttcgg caatagcggc 2220
tacgtgaaca catctaccac aagcatgttt ggcggcaatt ctggcgtgct gaacaccggc 2280
tacggcaata gcggcttcta taacgccgcc gtgaataaca caggcatctt tgtgaccggc 2340
gtgatgtcta gcggcttctt taatttcggc acaggcaact ccggcctgct ggtgtctggc 2400
aatggcctga gcggcttctt taagaacctg tttggaggct ccggctctgg cagcggctcc 2460
ggctctaccg accgcgtgtc cgtgggcaac ctgagaatcg ccagggtgct gtatgatttc 2520
gtgaataacg aggccctgcc tggcacagac atcgacccag acagcttttg ggcaggagtg 2580
gataaggtgg tggccgacct gacccctcag aatcaggccc tgctgaacgc cagagacgag 2640
ctgcaggccc agatcgacaa gtggcaccgg cggagagtga tcgagccaat cgacatggac 2700
gcctacaggc agttcctgac agagatcggc tatctgctgc cagagcccga cgacttcacc 2760
atcaccacat ccggcgtgga tgccgagatc accacaaccg caggacctca gctggtggtg 2820
ccagtgctga acgcaaggtt cgccctgaat gcagcaaacg ccagatgggg cagcctgtac 2880
gatgccctgt atggcacaga cgtgatccca gagaccgatg gagcagagaa gggacctaca 2940
tacaataagg tgaggggcga caaagtgatc gcctatgccc gcaagttcct ggacgattct 3000
gtgcccctgt cctctggcag cttcggcgac gccacaggct ttaccgtgca ggatggacag 3060
ctggtggtgg ccctgcctga taagtccacc ggactggcaa acccaggaca gtttgcagga 3120
tacaccggag cagcagagtc ccccacatct gtgctgctga tcaatcacgg cctgcacatc 3180
gagatcctga tcgaccctga gagccaagtg ggcacaaccg acagagcagg agtgaaggat 3240
gtgatcctgg agtccgccat cacaaccatc atggatttcg aggactctgt ggcagcagtg 3300
gatgcagcag acaaggtgct gggctacaga aattggctgg gcctgaacaa gggcgatctg 3360
gctgccgccg tggataagga cggcaccgcc tttctgaggg tgctgaatag agacaggaac 3420
tataccgccc caggaggagg acagttcaca ctgccaggcc ggtctctgat gtttgtgaga 3480
aatgtgggcc acctgatgac caacgacgcc atcgtggata cagacggcag cgaggtgttc 3540
gagggcatca tggatgccct gtttacaggc ctgatcgcca tccacggcct gaaggcctcc 3600
gacgtgaatg gccccctgat caactcccgc accggctcta tctacatcgt gaagccaaag 3660
atgcacggac ctgcagaggt ggccttcaca tgcgagctgt ttagccgggt ggaggacgtg 3720
ctgggcctgc ctcagaatac catgaagatc ggcatcatgg atgaggagcg ccggacaacc 3780
gtgaacctga aggcctgtat caaggctgcc gccgacaggg tggtgttcat caatacaggc 3840
tttctggatc gcaccggcga cgagatccac acatccatgg aggccggccc aatggtgaga 3900
aagggcacca tgaagtctca gccctggatt ctggcctatg aggatcacaa cgtggacgca 3960
ggactggcag caggcttcag cggcagagcc caagtgggca agggcatgtg gacaatgacc 4020
gagctgatgg ccgatatggt ggagacaaag atcgcacagc ctagggccgg agccagcacc 4080
gcatgggtgc cttccccaac agcagccacc ctgcacgcac tgcactacca ccaggtggac 4140
gtggcagcag tgcagcaggg actggcaggc aagagaaggg ccaccatcga gcagctgctg 4200
acaatccctc tggcaaagga gctggcatgg gcaccagacg agatcaggga ggaggtggat 4260
aataactgcc agtccatcct gggctatgtg gtgcggtggg tggatcaggg agtgggatgt 4320
tctaaggtgc cagacatcca cgacgtggcc ctgatggagg acagggccac actgcggatc 4380
agctcccagc tgctggccaa ctggctgagg cacggcgtga tcacctctgc cgacgtgcgc 4440
gcaagcctgg agaggatggc accactggtg gacaggcaga atgcaggcga tgtggcctac 4500
agacctatgg ccccaaactt cgacgatagc atcgcctttc tggcagcaca ggagctgatc 4560
ctgtccggag cacagcagcc aaacggatat accgagccta tcctgcaccg ccggagaagg 4620
gagttcaagg ccagagccgc agagaagcca gcaccttccg atagggcagg cgacgatgca 4680
gcccgcggca gcggctccgg ctctggcagc ggctccgatc cccagacaga caccatcgcc 4740
gccctgatcg cagacgtggc aaaggccaac cagaggctgc aggatctgag cgacgaggtg 4800
caggcagagc aggagtccgt gaataaggca atggtggacg tggagaccgc ccgggataac 4860
gctgccgccg ccgaggacga tctggaggtg agccagagag ccgtgaagga tgcaaacgca 4920
gcaatcgctg ccgcccagca caggttcgac accttcgccg ccgccaccta catgaatggc 4980
ccctctgtga gctatctgag cgcctctagc cctgacgaga tcatcgccac agtgaccgca 5040
gcaaagaccc tgtccgcctc ctctcaggcc gtgatggcaa atctgcagag ggcacggaca 5100
gagagggtga acaccgagtc cgccgcccgc ctggcaaagc agaaggcaga taaggctgcc 5160
gccgacgcaa aggcatctca ggatgcagca gtggccgccc tgacagagac caggcggaag 5220
ttcgacgagc agagagagga ggtgcagagg ctggcagcag agagggatgc agcacaggcc 5280
cggctgcagg ccgccagact ggtggcatgg agctccgagg gaggacaggg cgccccaccc 5340
tttcggatgt gggaccccgg cagcggacca gcaggcggca gagcctggga tggactgtgg 5400
gaccccaccc tgcctatgat cccatccgcc aatatccccg gcgaccctat cgccgtggtg 5460
aaccaggtgc tgggcatcag cgccacatcc gcccaggtga cagccaacat gggcagaaag 5520
ttcctggagc agctgggcat cctgcagccc acagacaccg gaatcaccaa tgcacctgca 5580
ggcagcgccc agggacgcat cccacgggtg tacggcaggc aggcatccga gtatgtgatc 5640
agaaggggca tgtcccagat cggcgtgcct tactcttggg gaggaggaaa cgcagcagga 5700
ccatccaagg gaatcgactc tggagcaggc accgtgggat tcgattgttc cggcctggtg 5760
ctgtattctt ttgccggcgt gggcatcaag ctgccacact actccggctc tcagtataat 5820
ctgggccgca agatcccctc tagccagatg cgccggggcg atgtgatctt ctacggccct 5880
aatggcagcc agcacgtgac catctatctg ggaaacggac agatgctgga ggcaccagac 5940
gtgggcctga aggtgagggt ggcacctgtg cgcacagcag gcatgacccc atacgtggtg 6000
cggtacatcg agtattaa 6018
<210> 30
<211> 253
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of IL-12p35
<400> 30
Met Trp Pro Pro Gly Ser Ala Ser Gln Pro Pro Pro Ser Pro Ala Ala
1 5 10 15
Ala Thr Gly Leu His Pro Ala Ala Arg Pro Val Ser Leu Gln Cys Arg
20 25 30
Leu Ser Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val
35 40 45
Leu Leu Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro
50 55 60
Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
65 70 75 80
Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr
85 90 95
Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys
100 105 110
Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu
115 120 125
Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys
130 135 140
Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser
145 150 155 160
Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn
165 170 175
Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn
180 185 190
Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
195 200 205
Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys
210 215 220
Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala
225 230 235 240
Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
245 250
<210> 31
<211> 328
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of IL-12p40
<400> 31
Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu
1 5 10 15
Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val
20 25 30
Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu
35 40 45
Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln
50 55 60
Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys
65 70 75 80
Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val
85 90 95
Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp
100 105 110
Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe
115 120 125
Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp
130 135 140
Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg
145 150 155 160
Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser
165 170 175
Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu
180 185 190
Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile
195 200 205
Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr
210 215 220
Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn
225 230 235 240
Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp
245 250 255
Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr
260 265 270
Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg
275 280 285
Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala
290 295 300
Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser
305 310 315 320
Glu Trp Ala Ser Val Pro Cys Ser
325
<210> 32
<211> 155
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of IL-21
<400> 32
Met Glu Arg Ile Val Ile Cys Leu Met Val Ile Phe Leu Gly Thr Leu
1 5 10 15
Val His Lys Ser Ser Ser Gln Gly Gln Asp Arg His Met Ile Arg Met
20 25 30
Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp
35 40 45
Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys
50 55 60
Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala
65 70 75 80
Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu
85 90 95
Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg
100 105 110
Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu
115 120 125
Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His
130 135 140
Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser
145 150 155
<210> 33
<211> 92
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of MIP-1alpha
<400> 33
Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala
1 5 10 15
Leu Cys Asn Gln Phe Ser Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala
20 25 30
Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile Ala
35 40 45
Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Gly Val Ile Phe
50 55 60
Leu Thr Lys Arg Ser Arg Gln Val Cys Ala Asp Pro Ser Glu Glu Trp
65 70 75 80
Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala
85 90
<210> 34
<211> 712
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of IL-12p35
<400> 34
atgtggcccc ctgggtcagc ctcccagcca ccgccctcac ctgccgcggc cacaggtctg 60
catccagcgg ctcgccctgt gtccctgcag tgccggctca gcatgtgtcc agcgcgcagc 120
ctcctccttg tggctaccct ggtcctcctg aatgttccca tgccttcacc actcccaaaa 180
cctgctgagg gccgtcagca acatgctcca gaaggccaga caaactctgg aattttaccc 240
ttgcacttct gaagagattg atcatgaaga tatcacaaaa gataaaacga gcacagtgga 300
ggcctgttta ccattggaat taaccaagaa tgagagttgc ctaaattcca gagagacctc 360
tttcataact aatgggagtt gcctggcctc cagaaagacc tcttttatga tggccctgtg 420
ccttagtagt atttatgaag acttgaagat gtaccaggtg gagttcaaga ccatgaatgc 480
aaagcttctg atggatccta agaggcagat ctttctggat caaaacatgc tggcagttat 540
tgatgagctg atgcaggccc tgaatttcaa cagtgagact gtgccacaaa aatcctccct 600
tgaagaaccg gatttttata aaactaaaat caagctctgc atacttcttc atgctttcag 660
gattcgggca gtgactattg atagagtgat gagctatctg aatgcttcct aa 712
<210> 35
<211> 937
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of IL-12p40
<400> 35
atgtgccacc agcagctggt catcagctgg ttctccctgg tctttctggc ttctcctctg 60
gtggcaattt gggagctgaa gaaagacgtg tacgtggtcg aactggactg gtatccagat 120
gcccccggag agatggtggt cctgacctgc gacacaccag aggaagatgg catcacttgg 180
accctggacc agagctccga ggtcctggga agcggcaaga cactgactat tcaggtgaaa 240
gaattcgggg atgctggaca gtacacatgt cataagggcg gggaggtgct gtcccactct 300
ctgctgctgc tgcataagaa agaagatggc atctggtcta ctgacattct gaaggatcag 360
aaagagccca agaacaaaac cttcctgaga tgcgaagcca agaattatag cgggaggttt 420
acctgttggt ggctgaccac aatctctact agtgacctgc ggagcagcta cactgagcgc 480
agagcgggtc agaggagaca acaaggagta cgaatattcc gtggagtgcc aggaagattc 540
tgcatgtccc gcagccgagg aatccctgcc tatcgaagtg atggtggacg ccgtgcacaa 600
gctgaaatac gaaaactaca catcctcttt ctttatccgg gacatcatta agccagatcc 660
ccctaaaaac ctgcagctga agcccctgaa aaattcacga caggtggagg tcagctggga 720
ataccctgat acatggagca ctccacattc ttatttcagt ctgacttttt gcgtgcaggt 780
ccagggcaag agtaaacgag agaagaaaga ccgggtcttc accgataaga catccgctac 840
tgtgatctgt cggaaaaacg ccagtatttc agtgagggct caggaccgct actatagttc 900
aagctggtca gagtgggcaa gcgtgccctg ttcctag 937
<210> 36
<211> 468
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of IL-21
<400> 36
atggaacgga ttgtcatttg cctgatggtc atttttctgg gaaccctggt ccacaagtca 60
agcagtcagg gccaggatag gcacatgatt aggatgcgcc agctgatcga cattgtggat 120
cagctgaaga actacgtgaa tgacctggtc cctgagtttc tgcctgcacc agaggatgtc 180
gaaacaaact gcgaatggag cgccttctcc tgttttcaga aggcccagct gaaatccgct 240
aacaccggca acaatgagcg aatcatcaac gtgagcatca agaagctgaa gcggaaaccc 300
cctagcacta atgctgggcg gagacagaaa catagactga cctgcccctc ttgtgacagt 360
tatgaaaaga aaccacccaa ggagttcctg gaacgcttta aaagtctgct gcagaaaatg 420
attcaccagc acctgtcctc cagaactcac gggtccgaag attcctaa 468
<210> 37
<211> 279
<212> DNA
<213> artificial sequence
<220>
<223> nucleic acid sequence of MIP-1alpha
<400> 37
atgcaggtgt caaccgccgc cctggctgtc ctgctgtgca ctatggctct gtgcaatcag 60
ttttccgcaa gtctggccgc tgatactccc accgcctgct gtttctctta cacaagtagg 120
cagatccctc agaacttcat tgctgactat tttgagacta gctcccagtg cagcaagccc 180
ggcgtgatct ttctgaccaa gcggagccgg caggtctgtg ccgatccctc cgaagaatgg 240
gtgcagaagt atgtctccga cctggaactg tcagcataa 279
Claims (20)
1. A fusion protein comprising a fusion polypeptide derived from mycobacterium tuberculosis (Mycoba cterium tuberculosis).
2. The fusion protein of claim 1, wherein the fusion polypeptide is selected from the group consisting of Ag85A, TB10.4, mtb32b and PstS3.
3. The fusion protein of claim 2, wherein Ag85A consists of the amino acid sequence of seq id No. 1; the TB10.4 consists of the amino acid sequence of SEQ ID NO. 2; the Mtb32b consists of the amino acid sequence of SEQ ID NO. 3; the PstS3 consists of the amino acid sequence of SEQ ID NO. 4.
4. The fusion protein of claim 1, wherein the fusion polypeptide further comprises a polypeptide selected from the group consisting of PPE39, glcB and RipA.
5. The fusion protein of claim 4, wherein the PPE39 consists of the amino acid sequence of SEQ ID NO. 5; the above GlcB consists of the amino acid sequence of seq id No. 6; the RipA consists of the amino acid sequence of SEQ ID No. 7.
6. The fusion protein of claim 2 or 4, wherein the fusion polypeptide is linked via a GS linker.
7. The fusion protein of claim 6, wherein the GS linker consists of the amino acid sequence of seq id No. 8.
8. The fusion protein of claim 1, further comprising a signal peptide.
9. The fusion protein of claim 8, wherein the signal peptide is selected from the group consisting of a tissue-type plasminogen activator signal peptide, a herpes simplex virus glycoprotein Ds signal peptide, and a growth hormone signal peptide.
10. The fusion protein of claim 1, wherein the fusion protein further comprises an immunopotentiating peptide.
11. The fusion protein of claim 10, wherein the immunopotentiating peptide is selected from the group consisting of CD40 ligand, FMS-like tyrosine kinase 3 ligand, flagellin, and OX 40.
12. A polynucleotide for encoding the fusion protein according to any one of claims 1 to 11.
13. A recombinant vector comprising the polynucleotide of claim 12.
14. The recombinant vector according to claim 13, wherein the recombinant vector is a plasmid vector or a viral vector.
15. A pharmaceutical composition for preventing or treating tuberculosis, characterized by comprising the fusion protein according to any one of claims 1 to 11.
16. The composition of claim 15, wherein the composition further comprises an immunoadjuvant comprising interleukin-12, interleukin-21, and macrophage inflammatory protein-1 alpha.
17. The composition of claim 16, wherein said immunoadjuvant comprises the amino acid sequence of seq id No. 30 to 33.
18. A pharmaceutical composition for preventing or treating tuberculosis, comprising the polynucleotide of claim 12.
19. The composition of claim 18, wherein the composition further comprises a polynucleotide encoding an immunoadjuvant comprising interleukin-12, interleukin-21, and macrophage inflammatory protein-1 a.
20. A pharmaceutical composition for preventing or treating tuberculosis, comprising the recombinant vector of claim 13.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2020-0168502 | 2020-12-04 | ||
KR1020200168502A KR20220079081A (en) | 2020-12-04 | 2020-12-04 | Pharmaceutical composition for preventing or treating tuberculosis |
PCT/KR2021/018238 WO2022119384A1 (en) | 2020-12-04 | 2021-12-03 | Pharmaceutical composition for preventing or treating tuberculosis |
Publications (1)
Publication Number | Publication Date |
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CN116802197A true CN116802197A (en) | 2023-09-22 |
Family
ID=81854270
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202180092760.8A Pending CN116802197A (en) | 2020-12-04 | 2021-12-03 | Pharmaceutical composition for preventing or treating tuberculosis |
Country Status (3)
Country | Link |
---|---|
KR (1) | KR20220079081A (en) |
CN (1) | CN116802197A (en) |
WO (1) | WO2022119384A1 (en) |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7026465B2 (en) * | 2002-02-15 | 2006-04-11 | Corixa Corporation | Fusion proteins of Mycobacterium tuberculosis |
WO2005061534A2 (en) * | 2003-12-23 | 2005-07-07 | Statens Serum Institut | Improved tuberculosis vaccines |
ES2647070T3 (en) * | 2005-06-23 | 2017-12-19 | Statens Serum Institut | Tuberculosis vaccines comprising antigens expressed during the latent infection phase |
KR101378240B1 (en) | 2008-02-22 | 2014-03-28 | 포항공과대학교 산학협력단 | A pharmaceutical composition for preventing tuberculosis |
KR101166516B1 (en) * | 2009-07-02 | 2012-07-20 | 주식회사 바이오드 | Composition for preventing or treating cervical cancer comprising structurally modified E6 and E7 derived from human papillomavirus and immune enhancement agent |
KR20170011200A (en) * | 2015-07-22 | 2017-02-02 | 사단법인대한결핵협회 | Fusion protein of tuberculosis specific antigens, efficient method of producing thereof and uses thereof |
-
2020
- 2020-12-04 KR KR1020200168502A patent/KR20220079081A/en not_active Application Discontinuation
-
2021
- 2021-12-03 CN CN202180092760.8A patent/CN116802197A/en active Pending
- 2021-12-03 WO PCT/KR2021/018238 patent/WO2022119384A1/en active Application Filing
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Publication number | Publication date |
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WO2022119384A1 (en) | 2022-06-09 |
KR20220079081A (en) | 2022-06-13 |
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