STRUCTURALLY MODIFIED CHIMERIC POLYPEPTIDE OF HUMAN PAPILLOMAVIRUS, RECOMBINANT PROTEIN COMPRISING SAME POLYPEPTIDE, AND USE OF SAME PROTEIN

Abstract

The present invention relates to a chimeric recombinant protein having a therapeutic effect on cervical cancer by fusing genetic modified E6 and E7, which are carcinogenesis-inducing proteins of human papillomavirus high-risk group type 16, with a fusion protein for increasing immunogenicity, the HPV type 16 E6, E7 chimeric recombinant protein fused with the flagellin fusion protein of the present invention showed the lowest tumor cell volume, and the immune response of specific T cells according to the recombinant antigen was significantly confirmed, and when the prophylactic effect was measured, it was confirmed that the volume of tumor cells was low and the antibody titer was increased, therefore human papillomavirus recombinant antigen of the present invention shows tumor treatment and prophylaxis and can be applied as a therapeutic/prophylactic vaccine composition.

Claims

1. A chimeric polypeptide in which the structures of E6 and E7 are modified, comprising residues from the 1.sup.st to the 155.sup.th of the E6 protein derived from human papillomavirus type 16, the 1.sup.st to the 37.sup.th amino acids of the E7 protein, and the 33.sup.rd to the 98.sup.th amino acid of the E7 protein derived from human papillomavirus type 16 wherein the E6 protein has substitution mutations at the 54.sup.th and the 57.sup.th amino acid residues and the E7 protein has substitution mutations at the 2.sup.nd, the 24.sup.th, the 80.sup.th and the 81.sup.st amino acid residues.

2. The chimeric polypeptide according to claim 1, wherein the 54.sup.th amino acid of the E6 protein is substituted from phenylalanine to arginine, and the 57.sup.th amino acid is substituted from leucine to glycine.

3. The chimeric polypeptide according to claim 1, wherein the 2.sup.nd amino acid of the E7 protein is substituted from histidine to proline, the 24.sup.th amino acid is substituted from cystine to glycine, and the 80.sup.th amino acid is substituted from glutamate to arginine, and the 81.sup.st amino acid is substituted from aspartate to arginine.

4. The chimeric polypeptide according to claim 1, wherein the chimeric polypeptide comprising the modified structure of E6 and E7 preferably comprising the amino acid sequence set forth in SEQ ID NO: 1.

5. A recombinant protein comprising the chimeric polypeptide of claim 1 and a protein for enhancing immunity of the chimeric polypeptide.

6. The recombinant protein according to claim 5, wherein the protein for enhancing immunity is selected from the group consisting of ubiquitin, flagellin, and cholera toxin A1B.

7. The recombinant protein according to claim 6, wherein the flagellin protein comprises amino acid residues from the 1.sup.st to the 143.sup.rd and amino acid residues from the 409.sup.th to the 495.sup.th.

8. The recombinant protein according to claim 6, wherein the cholera toxin A1B comprises the 19.sup.th to the 212.sup.th amino acid residues of the A1 subunit and the 22.sup.nd to the 124.sup.th amino acid residues of the B subunit, and has substitution mutations at the 81.sup.st, the 124.sup.th and the 130.sup.th amino acid residues of the A1 subunit.

9. The recombinant protein according to claim 5, wherein the recombinant protein preferably comprises the amino acid sequence set forth in SEQ ID NO: 5.

10. A polynucleotide encoding the chimeric polypeptide of claim 1.

11-16. (canceled)

17. The chimeric polypeptide according to claim 2, wherein the chimeric polypeptide comprising the modified structure of E6 and E7 preferably comprising the amino acid sequence set forth in SEQ ID NO: 1.

18. The chimeric polypeptide according to claim 3, wherein the chimeric polypeptide comprising the modified structure of E6 and E7 preferably comprising the amino acid sequence set forth in SEQ ID NO: 1.

19. The recombinant protein according to claim 6, wherein the recombinant protein preferably comprises the amino acid sequence set forth in SEQ ID NO: 5.

20. The recombinant protein according to claim 7, wherein the recombinant protein preferably comprises the amino acid sequence set forth in SEQ ID NO: 5.

Description

DESCRIPTION OF DRAWINGS

[0086] FIG. 1 is a schematic diagram showing the genetic modification sites encoding HPV16 E6 and E7 proteins. A is a schematic diagram of the linear structure of E6 and E7, which are carcinogenic proteins of the HPV16 type, and B is a schematic diagram of the chimeric structure of the E6 and E7 proteins.

[0087] FIG. 2 is a schematic diagram showing the fusion positions of HPV16 E6, E7 proteins and three candidate fusion proteins (ubiquitin, flagellin, cholera toxin A1B).

[0088] FIG. 3 is a schematic diagram showing the structure of expression vectors of E6 and E7 proteins and flagellin fusion proteins.

[0089] FIG. 4 is a result of confirming the size of the HPV16-E6E7 linear or HPV16-NE7-E6-CE7 chimera and the fusion protein or non-fusion protein using Coomassie blue staining method. M is molecular weight marker, 1 is non-fused HPV 16_E6-E7, 2 is non-fused HPV 16_NE7-E6-CE7, 3 is HPV 16_E6-E7 fused with ubiquitin, 4 is HPV 16_NE7-E6-CE7 fused with ubiquitin, 5 is HPV 16_E6-E7 fused with flagellin, 6 is HPV 16_NE7-E6-CE7 fused with flagellin, 7 is HPV 16_E6-E7 fused with cholera toxinA1B, 8 is HPV 16_NE7-E6-CE7 fused with cholera toxinA1B recombinant antigen.

[0090] FIG. 5 is the result of measuring the size of the tumor, after subcutaneously injecting the tumor cell line TC-1 cells into C57BL/6 mice, and then injecting each recombinant antigen to measure the size of the cancer over time, it was confirmed that the tumor volume was the lowest in the HPV 16_NE7-E6-CE7 fused with flagellin group.

[0091] FIG. 6 is an ELISPOT result showing a specific T cell immune response according to the HPV recombinant antigen, confirming that the antigen-dependent immune response was induced in HPV 16_NE7-E6-CE7 fused with flagellin.

[0092] FIG. 7 shows the results of measuring the size of the tumor according to the preventive effect by subcutaneously injecting TC-1 cells, a tumor cell line, after injecting two recombinant antigens into C57BL/6 mice, and a total of three concentrations (50 μg, 75 μg, 100 μg) of the HPV 16_NE7-E6-CE7 fused with flagellin group showed a low tumor volume.

[0093] FIG. 8 is an ELISA result, and it was confirmed that antibody titers were increased in all groups including HPV 16_NE7-E6-CE7 fused with flagellin by measuring antibody titers according to two recombinant antigens.

MODE FOR INVENTION

[0094] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are described with the intention of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1: Genetic Modification Encoding HPV E6 and E7 Proteins

[0095] For genetic modification of the anticancer-inducing protein of HPV type 16, in the case of the E6 protein, substitution mutations at residues 54 and 57, and deletion mutations at residues 156-158 were induced to inhibit the destabilization of the anticancer protein p53 and the interaction with the oncogenic protein due to the PDZ binding domain. In the case of E7 protein, substitution mutations at residues 2, 24, 80, and 81 were induced to strongly inhibit the interaction with pRb, the main target, and to suppress the transforming activity of E7 itself.

[0096] It included a polypeptide in a linear (E6E7) form in which amino acids of E6 and E7, which are oncogenic proteins derived from human papillomavirus type 16, were substituted, and deleted, and in addition, the sequence of SEQ ID NO: 1 comprising the same mutation as the linear polypeptide comprised a chimeric polypeptide. Specifically, it is a fusion protein in which amino acids 1 to 37 of the E7 protein, amino acids 1 to 155 of the E6 protein, and amino acids 33 to 98 of the E7 protein are linked in this order. (See FIG. 1)

Example 2: Gene Sequence and Genetic Modification of Fusion Protein

[0097] Among the fusion proteins, amino acids from 1 to 76 of the ubiquitin polypeptide sequence were used, and substitution mutation at residue 76 was induced to avoid cleavage by ubiquitin hydrolase. The two types of the genetically modified HPV 16 E6 and E7 polypeptides of FIG. 1 were bound to the carboxy terminus of ubiquitin. The flagellin polypeptide comprised amino acid sequences 1 to 143, the amino-terminal domain of the conserved region, and amino acids 409 to 495, the carboxy-terminal domain, of two types of genetically modified HPV 16 E6, E7 polypeptide was comprised between the amino-terminal domain and the carboxy-terminal domain of flagellin. In addition, in the polypeptide of cholera toxin A1B, polypeptides of A1 subunit (19-212 amino acids) and B subunit (22-124 amino acids) were included, and to reduce toxicity, substitution mutation at residue A1 subunit 81, 124, and 130 was induced, and two types of genetically modified HPV 16 E6 and E7 polypeptides were bound to the amino terminus of the A1 subunit of cholera toxin. (See FIG. 2)

Example 3: Gene Synthesis to be Used as a Non-Fusion/Fusion Protein with Genetically Modified HPV16 E6 and E7

[0098] Genes of oncoproteins E6 and E7 were modified, and synthetic genes encoding these proteins were obtained, and synthetic genes for three types of fusion proteins for immune enhancement were obtained. Each protein was codon-optimized for easy expression in E. coli by selecting a strain as shown in Table 1.

TABLE-US-00001 TABLE 1 Accession no. HPV 16_E6-E7 (Linear E6; NP_041325.1, E7; form) NP_041326.1 HPV 16_NE7-E6-CE7 E6; NP_041325.1, E7; (Chimeric form) NP_041326.1 Ubiquitin, Ubi) NP_003324.1 Flagellin, FliC) NP_460912.1 Cholera toxin A1B, CTA; NP_231100.1, CTA1B) CTB; NP_231099.1

[0099] Table 1 shows the genetic information of HPV16 type E6, E7 and fusion protein candidates.

Example 4: HPV16-E6E7-Non-Fusion/Fusion Protein Recombinant Protein Cloning

[0100] An attempt was made to express a recombinant protein derived from Escherichia coli by fusing a synthetic gene of HPV 16 type linear or chimeric with a synthetic gene of ubiquitin, flagellin, and cholera toxin A1B. To obtain the corresponding gene, first, in first cloning, the three fusion protein genes and the HPV 16 type linear or chimeric synthetic gene are fused to the fusion protein using the primary cloning restriction enzyme shown in Table 2 and T4 DNA ligase. In addition, PCR was performed to add a stop codon using the primers in Table 3 to obtain a non-fused form of DNA. After obtaining the target DNA in the non-fusion form and in the fusion form, the target DNA was subcloned into the expression vector pET-28a using the restriction enzymes used for secondary cloning in Table 2 and T4 DNA ligase. As shown in Table 4, a total of 8 recombinant proteins were cloned. In addition, a total of 8 vectors shown in Table 4 were transformed into the BL21(DE3) strain using a medium comprising kanamycin, and then each strain was selected.

TABLE-US-00002 TABLE 2 Restriction enzymes Restriction enzymes used for primary used for secondary cloning cloning Non-fusion — NdeI, XhoI (NEB, USA) Ubiquitin BamHI, PstI (Roche, NdeI, XhoI (NEB, fusion Swiss) USA) flagellin BamHI, HindIII NdeI, XhoI (NEB, fusion (Roche, Swiss) USA) Cholera MfeI, HindIII (Takara, BamHI, XhoI (NEB, toxin A1B Japan) USA) fusion

[0101] Table 2 lists restriction enzymes used for cloning

TABLE-US-00003 TABLE 3 Forward CGACGCG CATATG ATGCATCAAAAACGCAC Primer 1 Forward GAACGCG CATATG ATGCCGGGCGACAC Primer 2 Reverse CTCGC CTCGAG TTA CGGCTTCTGAGAGCAG Primer

[0102] Table 3 shows primer sequences for stop codon insertion

TABLE-US-00004 TABLE 4 pET-28a_HPV 16_E6-E7 pET-28a_HPV 16_NE7-E6-CE7 (Linear form) (Chimeric form) pET-28a_Ubi_HPV 16_E6-E7 pET-28a_Ubi_HPV 16_ NE7-E6- CE7 pET-28a_FliC_HPV 16_E6-E7 pET-28a_FliC_HPV 16_ NE7-E6- CE7 pET-28a_CTA1B_HPV 16_E6- pET-28a_CTA1B_HPV 16_ NE7- E7 E6-CE7

[0103] Table 4 shows the types of recombinant protein cloning

Example 5: Expression of HPV16-E6E7-Non-Fusion/Fusion Protein-pET-BL21 Recombinant Protein

[0104] To confirm the expression of a total of 8 recombinant proteins in Table 4, the selected strain expressed the protein through induction of IPTG (Isopropyl β-D-1-thiogalactopyranoside). When expressing the selected bacteria, the expression temperature, absorbance (OD600), the concentration of the expression induction substance IPTG, shaking culture conditions, expression time after induction, etc. are applied as experimental conditions. Through the condition test, the optimal conditions for expression were established for 4 hours in a shaking incubator(shaker) at 37° C. and 200 rpm using the inducer IPTG 1.0 mM in absorbance (OD600=0.5-0.6).

[0105] To confirm the expression of the recombinant protein in bacterial cells, 5 ml of cell lysis solution BugBuster® (Novagen) per 1 g of the cells was used for 20 minutes stirring at room temperature, and then protein extraction was performed, and overexpression patterns were confirmed at each position through 10% SDS-PAGE. All expressed recombinant proteins were identified in the insoluble pellet, not the soluble supernatant.

Example 6: Isolation of HPV16-E6E7-Non-Fusion/Fusion Protein-pET-BL21 Recombinant Protein Inclusion Body

[0106] The recombinant protein expressed in the insoluble pellet was stirred with the cell lysis solution and the supernatant was removed by centrifugation, and the pellet was suspended with 50 mM Tris ((Tris-(hydroxymethyl) aminomethane) buffer in the same volume as the supernatant and was lysed with ultrasonication and centrifugation were repeated three times in total. The chaotropic denaturant 8 M urea (Urea), 2 mM reduced glutathione (GSH) and 1 mM oxidized glutathione disulfide (GSSG) were added to final insoluble pellet, and then the pellet was suspended and was ultrasonically lysed and stored at 4° C. for 16 hours to obtain a denatured protein through a protein denaturation step to release the tertiary structure of the protein.

Example 7: Purification of HPV16-E6E7-Non-Fusion/Fusion Protein-pET-BL21 Recombinant Protein

[0107] For the purification of the obtained denatured protein, open column purification was performed using a combination of 6×His-tag recombinant protein and nitrilotriacetic acid (Ni-NTA). The supernatant was obtained through centrifugation of the denatured protein that had undergone the solubilization step before purification, and impurities were removed using a syringe filter. This supernatant was stirred at room temperature for 2 hours to bind to the 6× His-tag of the N-term end of the target recombinant protein using ProBond™ nickel resin (NOVEX). Buffers used for protein purification all included 8 M urea, and buffers comprising 20 mM sodium phosphate and 0.5 M sodium chloride were used. For the buffers used for washing, pH 7.8, pH 6.5, pH 5.9, and pH 5.5 were used, and for the buffer for protein eluting, pH 4.0, pH 3.5, and pH 3.0 were used.

[0108] During purification, 5 ml of ProBond™ nickel resin was added to the column and washed with distilled water. Then, a pH 7.8 buffer was poured into the washed resin at 5 times the volume of the resin. After the buffer was all removed, the denatured protein was loaded into the column, mixed with the resin, and then transferred to a 15 ml tube and stirred at room temperature for 2 hours. After stirring was finished, the resin-bound denatured protein was transferred back to the column, and the buffer is flowed in the order of pH 7.8, pH 6.5, pH 5.9, and pH 5.5. And finally, to elute the protein, the eluted protein was obtained by flowing the buffer in the order of pH 4.0, pH 3.5, and pH 3.0. To confirm the obtained protein, the eluted protein was confirmed through 10% SDS/PAGE.

Example 8: Solubilization Method of HPV16-E6E7-Non-Fusion/Fusion Protein-pET-BL21 Recombinant Protein

[0109] To solubilize the denatured protein purified by the above method, 48 kinds of compositions as shown in Table 5 were tested. Purified protein denatured with 8 M urea was added to 1 ml of each composition in a 1/100 volume ratio and mixed, followed by reaction at 4° C. for 16 hours or more. After the reaction, a composition in which the formation of an insoluble precipitate was not confirmed even after centrifugation at 4° C. 16,000 g for 10 minutes or more was established as a solubilization method.

[0110] As shown in Table 5, it was confirmed that the composition range for solubilization of the recombinant protein was suitable for compositions Nos. 31-36 and 43-48 comprising Tris-HCl pH8.5 buffer and 0.5 M Arginine. Among them, the composition that can most suitably solubilize a total of 8 recombinant proteins was identified as composition 31 in Table 4, and this composition includes 50 mM Tris-HCl pH8.5, 2 mM GSH, 0.2 mM GSSG, 20 mM NaCl, 0.5 M Arginine. Eight kinds of eluted denatured proteins were added in a volume ratio of 1/20 through the solubilized composition identified in this way and mixed, followed by reaction at 4° C. for 16 hours or more (Cited Patent_KR20170103473A).

TABLE-US-00005 TABLE 5 Buffer 50 mM Arginine 0M Arginine 0.5M HEPES  1  2  3  4  5  6  7  8  9 10 11 12 GSH/ (pH GSSG 7.5) 2/0.2 mM 13 14 15 16 17 18 19 20 21 22 23 24 GSH/ GSSG 10/2 mM Tris- 25 26 27 28 29 30 31 32 33 34 35 36 GSH/ HCl GSSG (pH 2/0.2 8.5) mM 37 38 39 40 41 42 43 44 45 46 47 48 GSH/ GSSG 10/2 mM MgCl2/CaCl2 0 mM MgCl2/CaCl2 2 mM MgCl2/CaCl2 0 mM MgCl2/CaCl2 2 mM NaCl 20 60 180 20 60 180 20 60 180 20 60 180 NaCl mM mM mM mM mM mM mM mM mM mM mM mM

[0111] Table 5 shows the composition of 48 kinds of solubilization buffer screening

Example 9: Obtaining Protein Through Dialysis and Concentration of HPV16-E6E7-Non-Fusion/Fusion Protein-pET-BL21

[0112] Since the composition comprising the solubilized protein includes the diluted target protein and various chemical compositions, it is difficult to use it as an antigen material. Therefore, the remaining chemical composition was removed by exchanging with the final buffer using a dialysis membrane. The final buffer solution used was a 10 mM carbonate pH9.8 buffer, and the dialysis solution was replaced every 1 hour to remove the residual chemical composition. Thereafter, a concentrated recombinant protein was obtained through a concentration process using a centrifugal-type Amicon® tube including a 10 kDa protein separation membrane. To confirm the finally obtained protein, the protein was confirmed through 10% SDS/PAGE (see FIG. 4).

Example 10 Immunological Validation of HPV16-E6E7-Non-Fusion/Fusion Protein-pET-BL21 Recombinant Antigen

[0113] Western blot was performed for immunological verification of the final obtained 8 recombinant antigens. 5×loading dye was added to each recombinant protein, bathed at 100° C. for 15 minutes, and electrophoresed using 10% SDS/PAGE. After that, proteins were transferred to Immobilon-P PVDF membrane (Merck, Germany) using Mini Trans-Blot (Bio-Rad) at 100V for 1 hour and 30 minutes, and PVDF membrane was blocked by stirring in 5% skim milk solution for 1 hour. After blocking, repeated washing with stirring with TBST solution, diluted 1:800 mouse-derived anti-HPV16 E7 monoclonal antibody in 5% skim milk solution used for blocking, and stirring at 4 degrees for 16 hours to bind the antibody to the protein did it After repeated washing with agitation with TBST solution, the HRP-conjugated goat-derived anti-mouse immunoglobulin G secondary antibody was diluted 1:5000 in 5% skim milk solution and stirred at room temperature for 1 hour to react. This was repeatedly washed by stirring with a TBST solution, followed by color development using a DAB (3,3-diaminobenzidine) substrate.

[0114] A total of 8 recombinant protein antigens reacted antibody-specifically, with antigens 1 and 2 about 31.5 kDa, antigens 3 and 4 about 41.5 kDa, antigens 5 and 6 about 59 kDa, and antigens 7 and 8 about 67 kDa in size. of the protein was detected. Under the same experimental conditions, the band density was generally darker in the chimeric structure than in the HPV 16 type E6, E7 linear structure. As for the difference in the fusion protein, it was the most intense in the recombinant protein antigen fused with flagellin.

Example 11: Confirmation of Tumor Treatment Effect of HPV16-E6E7-Non-Fusion/Fusion Protein-pET-BL21 Recombinant Antigen

[0115] To confirm the therapeutic effect of the recombinant protein antigen on cervical cancer, TC-1 tumor cells and 1×10.sup.5 cells were subcutaneously injected into the flank of 6-week-old C57BL/6 female mice, and 6 mice were used in each group. Seven days after injection of TC-1 cells, each recombinant protein was injected subcutaneously in the vicinity of the tumor cells in an amount of 50 μg. On the 21.sup.st day after tumor cell injection, 50 μg of recombinant protein was subcutaneously injected into the vicinity of tumor cells a second time. Tumor size was estimated in mice by measuring the longest (length) and shortest dimension (width) using digital calipers every 4-5 days. Tumor volume was calculated by the following equation:

Tumor volume=(length×width.sup.2)/2.

[0116] As a result of measuring the tumor volume through the recombinant protein as shown in FIG. 5, the HPV16 type E6 and E7 proteins showed a lower tumor volume in the chimeric structural group than in the linear structural group, and the lowest tumor volume was confirmed in the chimeric structural group including the flagellin fusion protein, compared to the control group. (See FIG. 5)

Example 12: ELISPOT; Specific T Cell Immune Response by HPV16-E6E7-Non-Fusion/Fusion Protein-pET-BL21 Recombinant Antigen

[0117] On the 35th day from the day of TC-1 cell injection, spleens were harvested from 3 mice in each group. After crushing the spleen using a cell strainer (Falcon) product, the spleen cells were recovered, and the spleen cells were washed with PBS buffer. Thereafter, red blood cells were removed using red blood cell lysis buffer, and the number of cells was counted for use in the ELISPOT experiment. For this experiment, mouse IFN-gamma ELISpot kit of R&D system was used. Then, on a plate coated with mouse IFN-gamma monoclonal antibody, 2×10.sup.5 cells, 4×10.sup.4 cells of splenocytes recovered from each mouse and 10 μg of the recombinant protein antigen injected into each mouse were put together in each well, and in a 37° C., 5% CO.sub.2 incubator for 16 hours incubation. After washing 4 times using 1X Wash buffer, 100 μl of IFN-gamma detection antibody to which biotin is bound was added, followed by incubation by stirring at room temperature for 2 hours. After incubation, washing was performed 4 times using 1X Wash buffer, 100 μl of streptavidin-AKP (alkaline phosphate) was added and incubated at room temperature for 2 hours. After washing 4 times using 1X Wash buffer, 100 ul of BCIP/NBT substrate was added and incubated for 45 minutes at room temperature. When color development by the reaction appeared, washed with sterile water and counted the number of colored spots.

[0118] As a result of measuring the specific T cell immune response to each recombinant protein antigen by ELISPOT, the group in which flagellin was fused to the HPV16 type E6, E7 protein chimeric form showed high results compared to the control group, confirming that a specific immune response to the antigen was induced (See FIG. 6).

Example 13: Confirmation of Tumor Prevention Effect of HPV16-E6E7-Non-Fusion/Fusion Protein-pET-BL21 Recombinant Antigen

[0119] To confirm the cervical cancer prevention effect of the recombinant protein antigen, 6-week-old C57BL/6 female mice were used in each group. From the results of confirming the therapeutic effect of FIGS. 5 and 6, the two most excellent recombinant protein antigens were selected to confirm the preventive effect. First, 50 μg of control (PBS) and one non-fusion recombinant protein were injected, and in the case of one fusion recombinant protein, 50, 75, and 100 μg of antigen were injected subcutaneously twice (14 days and 28 days before tumor cell injection). On the 14th day of injection of control and recombinant protein, 1×10.sup.5 cells were injected subcutaneously in the flank of TC-1 tumor cells, and the longest (length) and shortest dimensions (width) of the tumor cells were measured using a digital caliper every 4-5 days. was measured to estimate the tumor size of the mouse. Tumor volume was calculated by the following equation: Tumor volume=(length×width.sup.2)/2.

[0120] As a result of measuring the tumor volume through the recombinant protein as shown in FIG. 7, compared to the non-fusion chimeric recombinant protein, the tumor volume was lower in the chimeric structural group including all concentrations of the flagellin fusion protein, and the lowest tumor volume was confirmed in the group injected with 100 μg of the flagellin fusion protein. (See FIG. 7)

Example 14: ELISA; Antibody Titer Confirmation by the Preventive Effect of HPV16-E6E7-Non-Fusion/Fusion Protein-pET-BL21 Recombinant Antigen

[0121] Control and recombinant antigens were subcutaneously injected twice, and on days 1, 2, 3, and 4 from the first injection day, 6 mice in each group were anesthetized and blood was collected. The serum was obtained by centrifugation from the collected blood. In this experiment, each recombinant antigen was coated on an immune plate at 4° C. for 16 hours. Washing was performed three or more times using 1X Wash buffer, and blocking was performed at 37° C. for 1 hour using a blocking buffer to prevent non-specific binding of the antibody. After blocking, each separated serum was diluted 1:100 and reacted at 37° C. for 1 hour, washed 3 times or more using 1X Wash buffer, and then reacted with Goat anti-mouse IgG-HPR antibody at 37° C. for 1 hour. After washing 3 times using 1X Wash buffer, 50 μl of TMB substrate was added and reacted at room temperature for 20 minutes. After stopping the reaction with 2 M sulfuric acid, absorbance was measured at a wavelength of 450 nm.

[0122] As a result of measuring the antibody titer against each recombinant protein antigen by ELISA, there was no increase in titer of the non-fusion recombinant protein compared to the control group, but in all the groups injected with the flagellin fusion protein, it was confirmed that the antibody titer significantly increased at the 3rd and 4th weeks. (See FIG. 8)

TABLE-US-00006 [sequence information] Human papillomavirus type 16, E6, E7 modified amino acid sequence (E6 underlined) SEQ ID NO: 1 MPGDTPTLHEYMLDLQPETTDLYGYEQLNDSS EEEDEMHQKRTAMFQDPQERPRKLPQLCTELQ TTIHDIILECVYCKQQLLRREVYDFARRDGCI VYRDGNPYAVCDKCLKFYSKISEYRHYCYSLY GTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQ RHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRE EEEDEIDGPAGQAEPDRAHYNIVTFCCKCDST LRLCVQSTHVDIRTLRRLLMGTLGIVCPICSQ KP Human papillomavirus type 16, E6, E7 modified nucleic acid sequence (E6 underlined) SEQ ID NO: 2 ATG CCG GGC GAC ACG CCG ACC TTA CAT GAG TAC ATG CTT GAT TTA CAG CCG GAA ACG ACC GAT TTA TAC GGT TAC GAA CAG TTG AAC GAC TCT AGC GAG GAA GAA GAC GAG ATG CAT CAA AAA CGC ACC GCC ATG TTC CAA GAT CCG CAA GAA CGT CCG CGC AAA CTG CCG CAG CTT TGC ACC GAG CTG CAA ACT ACC ATT CAT GAC ATT ATC CTT GAG TGC GTG TAC TGT AAA CAG CAA TTA TTG CGT CGC GAA GTA TAC GAC TTC GCG CGC CGT GAC GGT TGT ATT GTG TAC CGT GAC GGT AAC CCG TAT GCA GTC TGC GAC AAA TGC CTG AAG TTT TAC AGC AAG ATA AGC GAG TAC CGT CAT TAT TGT TAT TCT TTG TAT GGC ACC ACC CTT GAG CAG CAG TAC AAT AAG CCG CTT TGT GAT TTG TTG ATC CGT TGC ATT AAT TGC CAG AAA CCG TTG TGC CCG GAA GAA AAG CAG CGC CAT TTA GAC AAG AAG CAG CGT TTC CAT AAT ATT CGC GGG CGC TGG ACC GGT CGT TGT ATG AGC TGT TGC CGT TCT AGC CGC ACT CGT CGT GAA GAG GAA GAA GAC GAG ATT GAC GGT CCG GCA GGG CAG GCC GAG CCG GAT CGC GCT CAT TAT AAT ATC GTG ACT TTT TGT TGT AAG TGT GAT AGC ACC CTT CGC CTT TGT GTG CAA AGC ACC CAT GTT GAC ATT CGT ACT TTG CGC CGC TTA TTA ATG GGC ACG CTT GGT ATT GTG TGC CCG ATC TGC TCT CAG AAG CCG Salmonella typhimurium str. LT2, Flagellin (FliC) modified amino acid sequence (Linker underlined, Arrow HPV 16 E6, E7 location) SEQ ID NO: 3 MAQVINTNSLSLLTQNNLNKSQSALGTAIERL SSGLRINSAKDDAAGQAIANRFTANIKGLTQA SRNANDGISIAQTTEGALNEINNNLQRVRELA VQSANSTNSQSDLDSIQAEITQRLNEIDRVSG QTQFNGVKVLAQDNTGGGGSGGGGSGSGXLQK IDAALAQVDTLRSDLGAVQNRFNSAITNLGNT VNNLTSARSRIEDSDYATEVSNMSRAQILQQA GTSVLAQANQVPQNVLSLLR Salmonella typhimurium str. LT2, Flagellin (FliC) modified nucleic acid sequence (Linker underlined, Arrow HPV 16 E6, E7 location) SEQ ID NO: 4 ATG GCG CAG GTT ATT AAC ACC AAT AGC TTA TCT TTG CTT ACC CAG AAT AAC TTA AAC AAA TCT CAA AGC GCT CTT GGG ACG GCA ATC GAG CGT TTA TCT AGC GGG CTG CGC ATT AAT TCT GCT AAA GAC GAC GCG GCA GGT CAA GCA ATC GCC AAC CGT TTC ACG GCA AAC ATT AAG GGC CTT ACC CAA GCC AGC CGT AAT GCT AAC GAC GGG ATC TCT ATC GCG CAG ACC ACC GAA GGC GCT CTG AAC GAG ATC AAC AAC AAC TTG CAG CGC GTT CGT GAG TTA GCG GTG CAA TCT GCC AAC AGC ACG AAT AGC CAA TCT GAC TTA GAC AGC ATC CAG GCC GAG ATC ACC CAG CGT CTT AAT GAA ATT GAT CGC GTA TCT GGC CAG ACG CAA TTT AAC GGG GTC AAG GTT CTT GCG CAA GAT AAT ACC GGT GGC GGT GGT TCT GGT GGG GGC GGC AGC GGA TCC GGC AAG CTT GGT GGG GGC GGC AGC GGC GGT GGC GGT TCT TTG CAG AAA ATT GAC GCG GCA CTT GCG CAG GTC GAC ACC CTT CGC AGT GAC CTG GGG GCA GTT CAG AAC CGC TTT AAT TCT GCC ATT ACG AAC CTG GGG AAC ACT GTG AAC AAC CTT ACG TCT GCG CGC TCT CGC ATC GAG GAC TCT GAT TAT GCA ACT GAA GTG AGC AAT ATG AGC CGC GCC CAA ATC TTG CAG CAG GCA GGC ACC AGC GTT CTT GCC CAG GCA AAC CAA GTT CCG CAA AAT GTC CTT TCT CTT CTT CGT FliC. HPV16 E6, E7 modified amino acid sequence (E6 underlined) SEQ ID NO: 5 MAQVINTNSLSLLTQNNLNKSQSALGTAIERL SSGLRINSAKDDAAGQAIANRFTANIKGLTQA SRNANDGISIAQTTEGALNEINNNLQRVRELA VQSANSTNSQSDLDSIQAEITQRLNEIDRVSG QTQFNGVKVLAQDNTGGGGSGGGGSGSGGLQM PGDTPTLHEYMLDLQPETTDLYGYEQLNDSSE EEDEMHQKRTAMFQDPQERPRKLPQLCTELQT TIHDIILECVYCKQQLLRREVYDFARRDGCIV YRDGNPYAVCDKCLKFYSKISEYRHYCYSLYG TTLEQQYNKPLCDLLIRCINCQKPLCPEEKQR HLDKKQRFHNIRGRWTGRCMSCCRSSRTRREE EEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTL RLCVQSTHVDIRTLRRLLMGTLGIVCPICSQK PQLGGKLGGGGSGGGGSLQKIDAALAQVDTLR SDLGAVQNRFNSAITNLGNTVNNLTSARSRIE DSDYATEVSNMSRAQILQQAGTSVLAQANQVP QNVLSLLR FliC. HPV16 E6, E7 modified nucleic acid sequence (E6 underlined) SEQ ID NO: 6 ATG GCG CAG GTT ATT AAC ACC AAT AGC TTA TCT TTG CTT ACC CAG AAT AAC TTA AAC AAA TCT CAA AGC GCT CTT GGG ACG GCA ATC GAG CGT TTA TCT AGC GGG CTG CGC ATT AAT TCT GCT AAA GAC GAC GCG GCA GGT CAA GCA ATC GCC AAC CGT TTC ACG GCA AAC ATT AAG GGC CTT ACC CAA GCC AGC CGT AAT GCT AAC GAC GGG ATC TCT ATC GCG CAG ACC ACC GAA GGC GCT CTG AAC GAG ATC AAC AAC AAC TTG CAG CGC GTT CGT GAG TTA GCG GTG CAA TCT GCC AAC AGC ACG AAT AGC CAA TCT GAC TTA GAC AGC ATC CAG GCC GAG ATC ACC CAG CGT CTT AAT GAA ATT GAT CGC GTA TCT GGC CAG ACG CAA TTT AAC GGG GTC AAG GTT CTT GCG CAA GAT AAT ACC GGT GGC GGT GGT TCT GGT GGG GGC GGC AGC GGA TCC GGC GGT CTG CAG ATG CCG GGC GAC ACG CCG ACC TTA CAT GAG TAC ATG CTT GAT TTA CAG CCG GAA ACG ACC GAT TTA TAC GGT TAC GAA CAG TTG AAC GAC TCT AGC GAG GAA GAA GAC GAG ATG CAT CAA AAA CGC ACC GCC ATG TTC CAA GAT CCG CAA GAA CGT CCG CGC AAA CTG CCG CAG CTT TGC ACC GAG CTG CAA ACT ACC ATT CAT GAC ATT ATC CTT GAG TGC GTG TAC TGT AAA CAG CAA TTA TTG CGT CGC GAA GTA TAC GAC TTC GCG CGC CGT GAC GGT TGT ATT GTG TAC CGT GAC GGT AAC CCG TAT GCA GTC TGC GAC AAA TGC CTG AAG TTT TAC AGC AAG ATA AGC GAG TAC CGT CAT TAT TGT TAT TCT TTG TAT GGC ACC ACC CTT GAG CAG CAG TAC AAT AAG CCG CTT TGT GAT TTG TTG ATC CGT TGC ATT AAT TGC CAG AAA CCG TTG TGC CCG GAA GAA AAG CAG CGC CAT TTA GAC AAG AAG CAG CGT TTC CAT AAT ATT CGC GGG CGC TGG ACC GGT CGT TGT ATG AGC TGT TGC CGT TCT AGC CGC ACT CGT CGT GAA GAG GAA GAA GAC GAG ATT GAC GGT CCG GCA GGG CAG GCC GAG CCG GAT CGC GCT CAT TAT AAT ATC GTG ACT TTT TGT TGT AAG TGT GAT AGC ACC CTT CGC CTT TGT GTG CAA AGC ACC CAT GTT GAC ATT CGT ACT TTG CGC CGC TTA TTA ATG GGC ACG CTT GGT ATT GTG TGC CCG ATC TGC TCT CAG AAG CCG CAA TTG GGT GGC AAG CTT GGT GGG GGC GGC AGC GGC GGT GGC GGT TCT TTG CAG AAA ATT GAC GCG GCA CTT GCG CAG GTC GAC ACC CTT CGC AGT GAC CTG GGG GCA GTT CAG AAC CGC TTT AAT TCT GCC ATT ACG AAC CTG GGG AAC ACT GTG AAC AAC CTT ACG TCT GCG CGC TCT CGC ATC GAG GAC TCT GAT TAT GCA ACT GAA GTG AGC AAT ATG AGC CGC GCC CAA ATC TTG CAG CAG GCA GGC ACC AGC GTT CTT GCC CAG GCA AAC CAA GTT CCG CAA AAT GTC CTT TCT CTT CTT CGT