KR101166516B1 - Composition for preventing or treating cervical cancer comprising structurally modified E6 and E7 derived from human papillomavirus and immune enhancement agent - Google Patents

Abstract

The present invention relates to a composition for preventing or treating cervical cancer comprising human papillomavirus variants and immune enhancers, and more specifically, E6 and antigens of types 16 and 18 of human papillomavirus (HPV); Fusion proteins comprising recombinant fusion polypeptides to modify the three-dimensional structure of E7, signal peptides for secreting them extracellularly, and immune enhancing peptides in individuals can induce HPV type 16 and 18 antigen-specific immune responses to be induced by HPV. The tumor that is caused can be treated.

Human papillomavirus, cervical cancer, signal peptide, immune enhancing peptide

Description

Composition for preventing or treating cervical cancer comprising structurally modified E6 and E7 derived from human papillomavirus and immune enhancement agent}

The present invention relates to a composition for preventing or treating cervical cancer comprising human papillomavirus variants and immune enhancers, and more specifically, E6 and antigens of types 16 and 18 of human papillomavirus (HPV); Fusion proteins comprising recombinant fusion polypeptides to modify the three-dimensional structure of E7, signal peptides for secreting them extracellularly, and immune enhancing peptides in individuals can induce HPV type 16 and 18 antigen-specific immune responses to be induced by HPV. The tumor that is caused can be treated.

Cervical cancer is known to be caused by infection of high-risk human papillomaviruses, such as types 16 and 18 of human papillomavirus (HPV) (zur Hausen, H et al. Biochem Biophys Acta) 1996, 1288; F55-F78, Mark H et al. J Natl Cancer Inst 1993, 85; 958-964). From HPV proteins E6 and E7 proteins play a key role in cervical cancer, and is found to be 99% expressed in tumor tissue of patients with cervical cancer is a major target substance for preparing a cervical cancer therapeutic and preventive vaccines (von Knebel Doeberitz et al Int. J. Cancer 1992, 51; 831-834). In the case of E6, it binds to p53, a tumor suppressor protein, promotes the breakdown of p53, which interferes with the progression of the cell cycle through the apoptosis pathway, and E7 binds to and inactivates the tumor suppressor retinoblastoma protein pRb. And promote degradation to allow the cell cycle to proceed to S phase (Cobrinik et al., Trends Biochem Sci 1992, 17: 312-5).

However, in clinical trials using nucleic acid sequence compositions expressing HPV16 E6 and E7 proteins for the treatment of cervical cancer, the therapeutic effect was negligible (Garcia F et al. Obstet Gynecol 2004, 103; 317-326). These results indicate that simply administering HPV antigen alone does not produce an antigen-specific immune response sufficient to treat or inhibit cervical cancer.

Thus, for the treatment of cervical cancer, strategies for enhancing the immunogenicity of E6 and E7 and removing the carcinogenic capacity of the protein itself are essential.

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel fusion protein and a polynucleotide encoding the same, which have enhanced immunogenicity while inhibiting the carcinogenic ability of HPV's E6 and E7 proteins in preventing or treating a disease caused by HPV. .

Another object of the present invention is to provide a recombinant vector expressing the fusion protein, a host cell comprising the vector, and a method of expressing a protein using the host cell.

Still another object of the present invention is to provide a composition for preventing or treating a disease caused by HPV using the fusion protein.

Another object of the present invention is to provide a method for preventing or treating a disease caused by HPV using the composition.

In order to achieve the above object,

Fusion polypeptides in which the three-dimensional structure of E6 and E7 having the amino acid sequence set forth in SEQ ID NO: 1 from human papillomavirus types 16 and 18 has been modified;

Signal peptide for secretion of the polypeptide; And

Provided are fusion proteins comprising immune enhancing peptides.

The invention also provides a polynucleotide encoding a fusion protein of the invention.

The invention also provides a recombinant vector comprising the polynucleotide according to the invention.

The invention also provides a host cell transformed with the vector of the invention.

The present invention also provides a method of expressing the fusion protein of the present invention by culturing the transgenic host cell of the present invention.

The present invention also provides a fusion protein of the present invention, a host cell transformed with a recombinant vector expressing the protein and one or more selected from the group consisting of lysates thereof as an active ingredient, caused by human papillomavirus in an individual Provided is a composition for preventing or treating a disease.

The invention also provides a method of preventing or treating a disease caused by human papillomavirus in a subject, comprising administering to the subject an effective amount of a composition of the invention.

The fusion protein prepared to include a recombinant fusion protein to modify the three-dimensional structure of the E6 and E7 proteins derived from HPV types 16 and 18 of the present invention, a signal peptide for secreting it extracellularly, and an immune enhancing peptide in a subject are highly HPV. Type 16 and 18 antigen specific immune responses can be induced to treat tumors caused by HPV.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention

Fusion polypeptides in which the three-dimensional structure of E6 and E7 having the amino acid sequence set forth in SEQ ID NO: 1 from human papillomavirus types 16 and 18 has been modified;

Signal peptide for secretion of the polypeptide; And

A fusion protein comprising an immune enhancing peptide.

The fusion protein of the present invention may comprise a recombinant fusion polypeptide such that the three-dimensional structure of E6 and E7 from human papillomavirus types 16 and 18 is modified. More specifically, the fusion polypeptide is amino acid 1 to 85 of the E6 protein derived from human papillomavirus type 16, amino acids 1 to 65 of the E7 protein, amino acids 70 to 158 of the E6 protein, Amino acids 50 to 98, amino acids 1 to 85 of E6 protein derived from human papillomavirus type 18, amino acids 1 to 65 of E7 protein, amino acids 70 to 158 of E6 protein, amino acid 50 of E7 protein Is a fusion polypeptide to which 105 is bound.

Most specifically, the fusion polypeptide may have an amino acid sequence set forth in SEQ ID NO: 1.

In addition, the signal peptide is a peptide containing about 20 to 30 amino acids, and refers to a peptide that secretes into the cell extracellularly expressed proteins, especially proteins including E6 and E7 fusion polypeptides. The nucleic acid sequence encoding it is also referred to as a "secretory signal sequence". E6 and E7 fusion polypeptides of the present invention is a protein (nucleus protein) expressed in the nucleus of a cell infected with the virus, so its immunity is weak. The signal peptide expressed by the secretory signal sequence induces the extracellular secretion of E6 and E7 antigens with modified three-dimensional structure, thereby antigen-specific humoral immune response and cellular immune response. It can lead to an increase in cellular immune response.

The signal peptide may be a signal peptide used in higher eukaryotic cells, including mammals, and the like, for example, tPA, HSV gDs, or a secretion signal sequence of growth hormone may be used. More preferably, tissue plasminogen activation (TPA) can be used. Most preferably, they may have the amino acid sequence set forth in SEQ ID NO: 2.

In addition, immune enhancing peptides refer to peptides that increase an immune response by activating cells (eg, dendritic cells, etc.) involved in the immune response.

CD40 ligand, Flt3 ligand (fms-like tyrosine kinase 3 ligand), Flagellin (Flagellin), or OX40 may be used as the immune enhancing peptide. More preferably, Flt3 ligand can be used. The Flt3 ligand is a factor that induces proliferation and maturation of dendritic cells (DCs), which increases the immune response by the antigen and has an excellent tumor reduction effect in the state of fusion with the tumor antigen. Can be represented. Most preferably, the Flt3 ligand may have an amino acid sequence set forth in SEQ ID NO: 3.

The invention also relates to a polynucleotide encoding a fusion protein of the invention.

The polynucleotide encodes a fusion protein of the present invention, and the fusion polypeptides of E6 and E7 may be encoded from the nucleotide sequence set forth in SEQ ID NO: 4, but are not particularly limited thereto. In addition, the signal peptide may be encoded from the nucleotide sequence set forth in SEQ ID NO: 5, but is not particularly limited thereto. The immune enhancing peptide may be encoded from the nucleotide sequence set forth in SEQ ID NO: 6, but is not particularly limited thereto.

In addition, the polynucleotides of the present invention can be prepared by chemical synthesis or genetic engineering techniques. Chemical synthesis methods are well known to those skilled in the art, and any method may be used. It can also be purchased by commercial nucleic acid synthesis and provider. When prepared by genetic engineering techniques, for example, nucleic acid fragments encoding the known fusion polypeptides, signal peptides and immune enhancing peptides of E6 and E7 can be obtained, respectively, and produced by linking these fragments in a frame. . Methods for obtaining such nucleic acid fragments are well known in the art, and those skilled in the art can easily connect using appropriate restriction enzymes. In a specific embodiment of the present invention, a method for producing by chemical synthesis is disclosed.

The invention also relates to a recombinant vector comprising the polynucleotide of the invention.

In the present invention, "vector" refers to a gene construct comprising foreign DNA inserted into the genome encoding a polypeptide. A vector related to the present invention is a vector in which a secretory signal sequence, a nucleic acid sequence encoding an E6 and E7 fusion polypeptide having a modified three-dimensional structure of human papillomavirus, and a nucleic acid sequence encoding an immune enhancing peptide are inserted into the genome, These vectors include, for example, plasmid vectors, cosmid vectors, bacteriophage vectors, yeast vectors, or viral vectors such as adenovirus vectors, retrovirus vectors, adeno-associated virus vectors.

The secretion signal sequence is a nucleic acid sequence encoding a peptide that secretes tumor antigen expressed in the cell to the outside of cells to be recognized by the immune cells, for example, tPA, HSV gDs, or a growth hormone secretion signal sequence, etc. There is this. Preferably, secretory signal sequences used in higher eukaryotic cells, including mammals, may be used, and more preferably, tPA (tissue plasminogen activation) may be used. Most preferably, it may have a nucleotide sequence set forth in SEQ ID NO: 5. In addition, the secretion signal sequence of the present invention can be used by replacing the codon with high expression frequency in the host cell.

In addition, the nucleic acid sequence encoding the immune enhancing peptide refers to a nucleic acid sequence encoding a peptide that increases the immune response by activating a cell (eg, dendritic cells, etc.) involved in the immune response. CD40 ligand, Flt3 ligand, Flagellin (Flagellin), or OX40 may be used as the immune enhancing peptide. More preferably, Flt3 ligand can be used as an immune enhancing peptide. In addition, the nucleic acid sequence encoding the immune enhancing peptide of the present invention can be used by replacing with a codon having a high expression frequency in the host cell.

In addition, the polynucleotides included in the recombinant vector of the present invention may be substituted with codons with high expression frequency in the host cell. As used herein, "substituted with high-expression codons in host cells" or "codon optimization" refers to codons that command amino acids when DNA is transcribed and translated into proteins in host cells. Depending on the host, codons with high affinity exist, and substitution with these high affinity codons increases the expression efficiency of the amino acid or protein encoded by the nucleic acid.

Here, "host cell" includes prokaryotic or eukaryotic cells, and eukaryotic cells include higher eukaryotic cells, including mammals, as well as lower eukaryotic cells, including fungi, yeast, and the like.

The recombinant vector of the present invention may include a nucleic acid encoding the fusion protein in a form suitable for expression of the nucleic acid encoding the fusion protein of the present invention in a host cell. That is, the recombinant vector of the present invention contains one or more regulatory sequences selected based on the host cell to be used for expression, and these are operably linked with the nucleic acid sequence to be expressed.

“Operably linked” means that the nucleotide sequence of interest (eg, in an in vitro transcription / translation system or in a host cell) is linked to the regulatory sequence in such a way that its expression can be achieved. do.

The term "regulatory sequence" is meant to include promoters, enhancers and other regulatory elements (eg, polyadenylation signals). Regulatory sequences include directing the constitutive expression of a desired nucleic acid in many host cells and directing the desired nucleic acid to be expressed only in a particular host cell (eg, tissue specific regulatory sequences). do. It will be appreciated by those skilled 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 level of protein expression desired. The expression vector of the present invention can be introduced into a host cell to express the fusion protein.

In addition, the vectors of the present invention can be prepared, for example, by standard recombinant DNA techniques, which include, for example, blunt- and adhesive-terminal ligation, restriction enzyme treatment to provide appropriate ends, and inappropriate. Phosphoric acid group removal by alkaline phosphatase treatment and enzymatic linkage by T4 DNA ligase are included to prevent binding. DNA encoding a signal peptide obtained by chemical synthesis or genetic recombination technology, DNA encoding a fusion polypeptide of human papillomavirus E6 and E7, and DNA encoding an immune enhancing peptide are recombined into a vector containing appropriate regulatory sequences Thereby the vector of the present invention can be produced. The vector containing the control sequence can be purchased or produced commercially, in the present invention, pGX27, a vector for preparing a DNA vaccine, was prepared and used.

In one embodiment, the recombinant vector of the present invention can be used as a pharmaceutically active ingredient administered in an individual as a vector for gene delivery in gene therapy or as a vector for gene delivery in gene therapy. have.

The invention also relates to a host cell transformed with the vector of the invention.

The type of the host cell is as described above.

Transformation may use known methods for introducing nucleic acids into organisms, cells, tissues or organs, and may be carried out by selecting appropriate available techniques according to the host cell to the extent that can be understood by those skilled in the art. These methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agrobacterial mediated transformation, PEG, dextran sulfate , Lipofectamine, and the like.

The present invention also relates to a method of expressing the fusion protein of the present invention by culturing the transformed host cell of the present invention.

The fusion protein of the present invention can be easily expressed and mass-produced by culturing the transformed host cell in a suitable medium or by introducing the transformed host cell into any animal and then culturing in vivo.

The present invention also provides a fusion protein of the present invention, a host cell transformed with a recombinant vector expressing the protein and one or more selected from the group consisting of lysates thereof as an active ingredient, caused by human papillomavirus in an individual It relates to a composition for preventing or treating a disease.

In the present invention, "individual" is human, monkey. Mammals such as mice, pigs, cattle and rabbits, but are not limited to these examples.

In addition, diseases caused by the virus may include cervical cancer, gojirom, warts and the like.

In addition, the composition of the present invention may further comprise a pharmaceutically acceptable carrier. The carrier includes lactose, glucose, sucrose, sorbitol, mannitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzo Ate, propylhydroxybenzoate, talc, magnesium stearade and mineral oil. The composition may also further include lubricants, wetting agents, flavoring agents, emulsifiers, and preservatives.

The compositions of the present invention are administered directly to the subject by any means, such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. It may be, but is not limited to these methods. The composition of the present invention can be administered to cells cultured in vitro and indirectly administered to the subject by administering the cultured cells in the body. The compositions of the present invention can be administered systemically or locally.

The composition of the present invention can be formulated into parenteral formulations such as granules, powders, solutions, tablets, capsules, or dry or syrups or injections, but is not limited to such formulations. Preferably the composition of the present invention may be in the form of a liquid or injection.

An effective amount of the fusion protein of the invention, or recombinant expression vector, as an active ingredient may be about 0.05 to 500 mg / kg body weight, preferably 0.5 to 50 mg / kg body weight, and may be considered as a single dose and a distributed dose. . However, the amount of active ingredient to be administered is not limited to the above range because it should be determined in consideration of various factors such as the condition to be treated, the age and weight of the patient, the severity of the symptoms.

The invention also relates to a method of preventing or treating a disease caused by human papillomavirus in a subject, comprising administering to the subject an effective amount of a composition of the invention.

The pharmaceutical composition of the present invention, its efficacy, method of administration, dosage and the like are as described above.

In the methods of the invention, the subject includes, but is not limited to, mammals such as humans, monkeys, mice, pigs, cattle and rabbits.

In addition, the disease caused by the virus may include cervical cancer, gojirom, warts and the like.

Hereinafter, the present invention will be described in more detail through examples according to the present invention and comparative examples not according to the present invention, but the scope of the present invention is not limited to the examples given below.

Example 1 Composition of GX-188E DNA

Abbreviations used in the embodiments of the present invention are defined as follows. Optimized nucleic acid sequence, "tPa" or "t" means secretory signal sequence of tissue plasminogen activator, "F" or "Flt3L" means fms-like tyrosine kinase 3 ligand do.

A codon optimized tPa secretion signal sequence having a nucleic acid sequence of SEQ ID NO: 5 and a codon optimized Flt3L having a nucleic acid sequence of SEQ ID NO: 6 were chemically synthesized in linked form. Kpn I (5 ′), Nhe I (3 ′) sites were added at the ends to facilitate insertion into the vector. After treating the DNA vaccine vector for producing the pGX10 (Republic of Korea Patent Publication No. 2003-0047667 No.) by Kpn I and Nhe I restriction enzyme was prepared pGX10 / tF by connecting a tPa-Flt3L synthesized with ligase.

Codon optimized nucleic acid sequences encoding amino acids 1 to 85 of HPV16 E6, Codon optimized nucleic acid sequences encoding amino acids 1 to 65 of HPV16 E7, HPV16 E6 amino acids encoding amino acids 70 to 158 Codon optimized nucleic acid sequence, HPV16 E7 Codon optimized nucleic acid sequence encoding amino acids 50 to 98, Codon optimized nucleic acid sequence encoding amino acids 1 to 85 of HPV18 E6, starting with amino acid 1 of HPV18 E7 Codon optimized nucleic acid sequence encoding amino acid 65, HPV18 E6 Codon optimized nucleic acid sequence encoding amino acids 70 to 158, HPV18 E7 Codon optimized nucleic acid sequence encoding amino acid amino acids 50 to 105 Chemical synthesis (hereinafter, 16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C: SEQ ID NO: 4). NheI (5 '), XbaI (3') sites were added at the ends to facilitate insertion into the vector. pGX10 / tF was treated with Nhe I and Xba I enzymes, and then synthesized 16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C was linked with a ligase to prepare pGX10 / tF16E6N16E7N16E6C16E7C18E6N18E7N7E18C6E. la pGX10 / processes the tF16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C with Kpn I and Xba I enzyme (enzyme) After removing the tF16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C and handle pGX10 with Kpn I and Xba I enzymes and Lee through to azepin pGX27 / tF16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C (hereinafter, "GX-188E" also ) Was prepared.

Example 2 Confirmation of GX-188E Cervical Cancer Treatment Effect

To determine the effect of GX-188E on the treatment of cervical cancer, mice were injected subcutaneously with TC-1 tumor cells 5 × 10 ⁵ cells in C57BL / 6 and injected with 50 ㎍ and 100 ㎍ of muscle at 3 and 8 days. Electroporation was performed. Observe the volume change of tumor cells from the day of injection of tumor cells to the 27th day and remove the spleen of the mice on the 36th day of antimicrobial 5 μg / mL anti-mouse IFN-g antibody (BD Pharmigen, Sandiego, CA) 50 1 × 10 ⁶ cells were plated in μl coated IL-2 and HPV16 E6 CD8 T cell epitopes (E6 _48-57 ; EVYDFAFRDL, Peptron, Korea) or HPV18 E7 CD8 T cell epitope (E7 _49-57 RAHYNIVTF, Peptron, Korea) or HPV18 E6 peptide pool or HPV18 E7 peptide pool were added together and incubated in 37 ° C. 5% CO ₂ incubator (Froma, Minnesota, USA) for 24 hours. After washing the plate with PBST, 50 μl of 2 μg / mL of biotin-containing IFN-g detection antibody (BD Pharmigen, Sandiego, CA) was added and incubated at room temperature for about 3 hours. After washing with PBST, streptavidin-AKP (alkaline phosphate) was diluted 1: 2000, 50 μl each and incubated at room temperature for 1 hour. After washing with PBST, 50 μl of 66 μl NBT (Promega, Madison, WT) and 33 μl of BCIP (Promega, Madison, WT) were added per 10 mL of alkaline phosphate buffer, followed by reaction. The color of the reaction was well left in the incubator for about 30 minutes at 37 ℃ incubator with distilled water (DW) and the number of dots produced was read with a reader.

T cell immunity specific for HPV16 E6, E7 and HPV18 E6, E7 using HPV16 E6 CD8 T cell epitope, HPV16 E7 CD8 T cell epitope, HPV18 E6 peptide pool, HPV18 E7 peptide pool As a result of measuring the response by ELISPOT, GX-188E induced high antigen-specific immune responses, and it was confirmed that HPV16 and HPV18 simultaneously induce specific immune responses against E6 and E7 (Fig. 1, Fig. 2, Fig. 1). 3, and FIG. 4).

In mice treated with TC-1 tumor cells with GX-188E, a significant decrease in tumor volume was observed compared to mice injected with the control group pGX27 (see FIG. 5).

1 is a graph showing HPV16 E6 specific CD8 ⁺ T cell responses seen through the treatment of GX-188E after subcutaneous injection of tumor cell line TC-1 into rat C57BL / 6 in an anticancer treatment model.

Figure 2 is a graph showing the HPV16 E7 specific CD8 ⁺ T cell response shown through the treatment of GX-188E of the present invention after subcutaneous injection of TC-1, a tumor cell line, to rat C57BL / 6 in an anticancer treatment model.

Figure 3 is a graph showing the HPV18 E6 specific CD8 ⁺ T cell response seen through the treatment of GX-188E of the present invention after subcutaneous injection of TC-1, a tumor cell line, to rat C57BL / 6 in an anticancer treatment model.

Figure 4 is a graph showing the HPV18 E7 specific CD8 ⁺ T cell response shown through the treatment of GX-188E of the present invention after subcutaneous injection of tumor cell line TC-1 to rat C57BL / 6 in an anticancer treatment model.

5 is a graph showing the anticancer effect of the GX-188E treatment of the present invention after subcutaneous injection of TC-1, a tumor cell line, to rat C57BL / 6 in an anticancer treatment model.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Composition for preventing or treating cervical cancer comprising          structurally modified E6 and E7 derived from human papillomavirus          and immune enhancement agent <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 579 <212> PRT <213> Human papillomavirus <220> <221> PEPTIDE (222) (1) .. (579) <223> structurally modified E6 and E7 derived from HPV16 and HPV18 <400> 1 Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro   1 5 10 15 Arg Lys Leu Pro His Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp              20 25 30 Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu          35 40 45 Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly      50 55 60 Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile  65 70 75 80 Ser Glu Tyr Arg Tyr Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser                  85 90 95 Glu Tyr Arg Tyr Tyr Cys Tyr Ser Val Tyr Gly Thr Thr Leu Glu Gln             100 105 110 Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys         115 120 125 Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys     130 135 140 Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met Ser 145 150 155 160 Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu Met His Gly                 165 170 175 Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr             180 185 190 Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu         195 200 205 Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His     210 215 220 Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu His Tyr 225 230 235 240 Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys                 245 250 255 Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met             260 265 270 Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Met Ala         275 280 285 Arg Phe Glu Asp Pro Thr Arg Arg Pro Tyr Lys Leu Pro Asp Leu Cys     290 295 300 Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys Val Tyr 305 310 315 320 Cys Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu Phe Ala Phe Lys                 325 330 335 Asp Leu Phe Val Val Tyr Arg Asp Ser Ile Pro His Ala Ala Cys His             340 345 350 Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg Tyr Tyr Ser         355 360 365 Asp Ser Val Phe Tyr Ser Arg Ile Arg Glu Leu Arg Tyr Tyr Ser Asp     370 375 380 Ser Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr Gly Leu Tyr 385 390 395 400 Asn Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys Pro Leu Asn Pro Ala                 405 410 415 Glu Lys Leu Arg His Leu Asn Glu Lys Arg Arg Phe His Lys Ile Ala             420 425 430 Gly His Tyr Arg Gly Gln Cys His Ser Cys Cys Asn Arg Ala Arg Gln         435 440 445 Glu Arg Leu Gln Arg Arg Arg Glu Thr Gln Val Met Tyr Gly Pro Lys     450 455 460 Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu Pro Gln Asn Glu Ile 465 470 475 480 Pro Val Asp Leu Leu Cys His Glu Gln Leu Ser Asp Ser Glu Glu Glu                 485 490 495 Asn Asp Glu Ile Asp Gly Val Asn His Gln His Leu Pro Ala Arg Arg             500 505 510 Ala Glu Pro Gln Arg His Thr Met Leu Cys Met Cys Ala Arg Arg Ala         515 520 525 Glu Pro Gln Arg His Thr Met Leu Cys Met Cys Cys Lys Cys Glu Ala     530 535 540 Arg Ile Glu Leu Val Val Glu Ser Ser Ala Asp Asp Leu Arg Ala Phe 545 550 555 560 Gln Gln Leu Phe Leu Ser Thr Leu Ser Phe Val Cys Pro Trp Cys Ala                 565 570 575 Ser Gln Gln             <210> 2 <211> 24 <212> PRT <213> Eukaryote <220> <221> PEPTIDE (222) (1) .. (24) <223> tissue plasminogen activator secretory signal sequence <400> 2 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 Ala              20 <210> 3 <211> 158 <212> PRT <213> Eukaryote <220> <221> PEPTIDE (222) (1) .. (158) <223> Amino acid sequence of fms-like tyrosine kinase 3 ligand <400> 3 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 Ala Ser 145 150 155 <210> 4 <211> 1746 <212> DNA <213> Human papillomavirus <220> <221> gene (222) (1) .. (1746) <223> Codon-optimized nucleotide sequence of structurally modified E6          and E7 derived from HPV16 and HPV18 <400> 4 atgcaccaga agagaaccgc catgttccag gaccctcagg agagacctag gaagctgcct 60 cacctgtgta cagagctcca gacaaccatc cacgacatca tcctggagtg cgtgtactgt 120 aagcagcagc tgctgagaag agaggtgtac gacttcgcct tcagagacct gtgcatcgtg 180 tacagagacg gcaaccctta cgccgtgtgc gataagtgtc tgaagttcta ttccaaaatc 240 tccgaatata ggtacatgca cggcgacacc cctaccctgc acgagtacat gctggacctc 300 cagcctgaga ccacagacct gtactgctac gagcagctga acgacagctc tgaggaagag 360 gacgagattg acggacctgc tggccaggcc gagcctgaca gagcccacta caatatcgtg 420 acattctgtt gcaaatgcga ctccacactg gacaagtgcc tgaagttcta cagcaagatc 480 tctgagtaca gatactactg ctactctgtg tacggcacca cactggagca gcagtacaac 540 aagcctctgt gcgacctcct gatccgctgc atcaactgcc agaagcctct gtgccctgag 600 gagaagcaga gacacctgga caagaagcag cggttccaca acatcagagg cagatggacc 660 ggcaggtgca tgtcctgctg tagatcctcc agaaccagac gggagaccca gctgcactac 720 aacatcgtga ccttctgctg caagtgcgac tctaccctga gactgtgcgt gcagtctacc 780 cacgtggaca tcagaaccct ggaggacctg ctgatgggca ccctgggcat cgtgtgccct 840 atctgctctc agaagcctat ggccaggttc gaggacccta ccagaagacc ctacaagctg 900 cctgacctgt gcaccgagct gaacacctct ctgcaagaca tcgagatcac ctgcgtgtac 960 tgcaagaccg tgctggagct gaccgaggtg ttcgagttcg ccttcaagga cctgttcgtg 1020 gtgtacagag acagcatccc tcacgctgcc tgccacaagt gcatcgactt ctattccagg 1080 atcagggagc tgcgctatta ctccgactct gtgatgtacg gccccaaggc caccctccag 1140 gacatcgtgc tgcacctgga gcctcagaac gagatccccg tggacctgct gtgccacgag 1200 cagctgtctg actctgaaga ggagaacgac gagatcgacg gcgtgaacca ccagcacctg 1260 cctgccagga gagctgaacc ccagcggcat accatgctgt gtatgtgctt ctactctagg 1320 atcagagagc tgaggtacta ctctgactct gtgtacggcg acaccctgga gaagctgacc 1380 aacaccggcc tgtacaacct gctgatccgg tgcctgaggt gccagaagcc tctgaaccct 1440 gccgagaagc tgagacacct gaacgagaag agaagattcc acaagatcgc tggccactac 1500 agaggccagt gccactcttg ctgcaacaga gccagacagg agagactcca gcggagaagg 1560 gagacccagg tggccagaag agccgagcct cagagacaca ccatgctgtg catgtgctgc 1620 aagtgcgagg ccagaatcga gctggtggtg gagagctctg ccgacgacct gagagccttc 1680 cagcagctgt tcctgtctac cctgagcttc gtgtgccctt ggtgcgcctc tcagcagtaa 1740 tctaga 1746 <210> 5 <211> 69 <212> DNA <213> Eukaryote <220> <221> gene (222) (1) .. (69) <223> Codon-optimized tissue plasminogen activator secretory signal          sequence nucleotide sequence <400> 5 atggatgcta tgaaacgggg cctgtgctgc gtgctgctcc tgtgcggcgc tgtgtttgtg 60 agccctagc 69 <210> 6 <211> 489 <212> DNA <213> Eukaryote <220> <221> gene (222) (1) .. (489) <223> Codon-optimized fms-like tyrosine kinase 3 ligand nucleotide          sequence <400> 6 atcacccagg actgctcctt ccaacacagc cccatctcct ccgacttcgc tgtcaaaatc 60 cgtgagctgt ctgactacct gcttcaagat tacccagtca ccgtggcctc caacctgcag 120 gacgaggagc tctgcggggg cctctggcgg ctggtcctgg cacagcgctg gatggagcgg 180 ctcaagactg tcgctgggtc caagatgcaa ggcttgctgg agcgcgtgaa cacggagata 240 cactttgtca ccaaatgtgc ctttcagccc ccccccagct gtcttcgctt cgtccagacc 300 aacatctccc gcctcctgca ggagacctcc gagcagctgg tggcgctgaa gccctggatc 360 actcgccaga acttctcccg gtgcctggag ctgcagtgtc agcccgactc ctcaaccctg 420 ccacccccat ggagtccccg gcccctggag gccacagccc cgacagcccc gggcggcggc 480 agcggcgat 489  

Claims (17)

Fusion polypeptides in which the three-dimensional structure of E6 and E7 having the amino acid sequence set forth in SEQ ID NO: 1 from human papillomavirus types 16 and 18 has been modified; Signal peptide for secretion of the polypeptide; And Fusion protein comprising an immune enhancing peptide. The method of claim 1, The signal peptide is at least one fusion protein selected from the group consisting of tPa, HSV gDs and growth hormone secretion signal peptide. The method of claim 1, The signal peptide is a fusion protein having the amino acid sequence set forth in SEQ ID NO: 2. The method of claim 1, A fusion protein wherein the immune enhancing peptide is at least one selected from the group consisting of CD40 ligand, Flt3 ligand (fms-like tyrosine kinase 3 ligand), Flagellin and OX40. The method of claim 1, The immune enhancing peptide is a fusion protein having the amino acid sequence set forth in SEQ ID NO: 3. A polynucleotide encoding the fusion protein of claim 1. The method of claim 6, A polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 4 encoding the fusion polypeptides of human papillomaviruses E6 and E7. The method of claim 6, A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5 encoding a signal peptide. The method of claim 6, A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6 encoding an immune enhancing peptide. Recombinant vector comprising the polynucleotide of claim 6. A host cell transformed with the recombinant vector of claim 10. A method of expressing the fusion protein of claim 1 by culturing the host cell of claim 11. Prevention of a disease caused by human papillomavirus in a subject comprising at least one selected from the group consisting of a fusion protein of claim 1, a host cell transformed with a recombinant vector expressing the protein and a lysate thereof. Therapeutic composition. 14. The method of claim 13, Wherein the disease is cervical cancer. 14. The method of claim 13, A composition further comprising a pharmaceutically acceptable carrier. A method for preventing or treating a disease caused by human papillomavirus comprising administering to a non-human animal an effective amount of the composition of claim 13. 17. The method of claim 16, The disease is cervical cancer.

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