CN113072647A

CN113072647A - Nucleic acid vaccine targeting human papilloma virus

Info

Publication number: CN113072647A
Application number: CN202110624820.8A
Authority: CN
Inventors: 齐海龙; 孙忠杰; 曲春枫; 赵宏; 陈立功; 王旭东; 陈坤; 谢皇帆; 刘德芳; 王晓芳; 姚艳玲
Original assignee: Nuowei Technology Beijing Co ltd
Current assignee: Nuowei Biotechnology (Wuxi) Co.,Ltd.; Newish Technology Beijing Co Ltd
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2021-07-06
Anticipated expiration: 2041-06-04
Also published as: CN113072647B

Abstract

The invention relates to the technical field of tumor immunotherapy and prevention, in particular to a nucleic acid vaccine targeting HPV E6 and E7. The present invention provides a fusion protein comprising: XCL1 chemokine and antigenic protein of HPV virus. The invention expresses the antigen protein of HPV virus and DC cell ligand XCL1 in a fusion way, and transfers the antigen protein of HPV virus without pathogenicity to DC cells with cross presentation by using XCL1 as a carrier, thereby improving the efficiency of phagocytosis, processing and presentation of the antigen protein of HPV virus by the DC cells and improving the effect of preventing and treating diseases related to HPV virus infection. Experiments prove that the protein expressed by the nucleic acid vaccine can effectively bind DC cells, induce E6/E7 specific T cell response and remarkably inhibit the growth of tumors with high expression of E6/E7 in various models.

Description

Nucleic acid vaccine targeting human papilloma virus

Technical Field

The invention relates to the technical field of tumor immunotherapy and prevention, in particular to a tumor nucleic acid vaccine targeting metastatic cancers of human papilloma viruses.

Background

Human Papilloma Virus (HPV) is a non-enveloped double-stranded DNA virus. This virus can infect epithelial and mucosal tissues in humans. Can cause squamous epithelial cell proliferation of human skin mucosa, and can induce cervical cancer or many genital related cancers. Among the 130 HPV viruses that have been found, 13 viruses are identified as inducing cancers of the reproductive system such as cervical cancer and rectal cancer, anal cancer, and penile cancer. The two most threatening HPV types are HPV16 and HPV18, which account for over 70% of the overall cervical cancer causative factors. Human papillomaviruses, a virus that is widely prevalent in developing and developed countries, has created a significant threat to human life.

After HPV viral nucleic acid substances are integrated into human genome, the expressed translated E6 and E7 proteins are the most important protein molecules for inducing generation and metastasis of matrix malignant tumors, and the two proteins are recognized and partially eliminated by an in vivo immune system because the two proteins belong to foreign protein molecules after being translated in a human body. Therefore, anti-tumor vaccines and drugs against E6 and E7 are currently the most studied in tumor therapy.

Through scientific research and clinical research in recent 20 years, therapeutic vaccine research against the HPV virus E6E7 protein has gradually progressed into several major groups. The Vaccine comprises but is not limited to Bacterial Vector Vaccine (Bacterial Vector Vaccine), Viral Vector Vaccine (Viral Vector Vaccine), polypeptide Protein Vaccine (Peptide/Protein Vaccine), nucleic acid Vaccine (Nucleotide Vaccine) and Whole Cell type Vaccine (Whole Cell Based Vaccine), and has obvious advantages in the field of HPV therapeutic Vaccine, and the Vaccine has the advantages of quick project recommendation, simple preparation, easy storage and the like. On the other hand, it can be seen from the results of some clinical experiments that immune tolerance reaction is mostly formed by pure vaccine immunization using materials such as E6 or E7 protein of HPV genetic materials, the targeting ability is poor, and the effect of the vaccine is greatly reduced. Therefore, there is a need for a HPV vaccine that relies on nucleic acids to efficiently express HPV vaccines that target the immune system in humans. The aim of doubling the treatment effect of the vaccine is achieved by activating high-efficiency cellular immune response by the human immune system.

Tumor vaccines induce effector T cell function in patients by immunopotentiating existing anti-tumor responses or priming naive T cells, and antigen-specific CD8+ Cytotoxic T Lymphocytes (CTLs) play an important role in anti-tumor processes (Shankaran v. et al. Nature 2001). Dendritic Cells (DCs) are the only professional antigen presenting cell that activates primary CD8+ T cells, uptake, processing and cross-presentation of extracellular tumor antigens via MHC-I, and are critical for the production of effective CTLs (Romero p. et al, Annu Rev Immunol 2017). Therefore, delivery of tumor antigens to DC cells by conjugation to DC cell surface molecules is a tumor treatment strategy that is effective in inducing CD8+ T cell immune responses.

The ability of DC cells of different subsets to induce antigen-specific immune responses varies, XCL1 being the only ligand for the G protein-coupled receptor family member XCR1, and XCR1 being a chemokine receptor, expressed only on a specific subset of DC cells (i.e., "cross-presenting" DC cells, also known as XCR1+ DC or cDC 1). XCR1+ DC is powerful in "cross-presentation" of antigens, i.e., exogenous antigen is not presented by MHC class II CD4+ T cells, but rather by MHC class I antigen into CD8+ T cells (Kurts C. et al J Exp Med 1997; den Haan JM. J Exp Med 2000; Pooley JL. et al J Immunol 2001), XCR1+ DC is the most effective antigen cross-presenting cell inducing CD8+ T cell activation. Thus, XCR1 is an ideal target for inducing the body to generate cellular immune toxicity against tumors (Evelyn hartung. et al. J Immunol 2015), and the delivery of target antigen to XCR1+ DC by fusion with XCL1 is a viable tumor immunotherapy strategy.

Although the improvement of tumor treatment effect by combining XCR1 with a targeting factor has been reported, the improvement of delivery effect is not uniform, and thus, further research on the improvement of tumor treatment effect is still ongoing in the art.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a tumor nucleic acid vaccine targeting E6 and E7, so as to improve the efficiency of phagocytosis, processing and presentation of E6/E7 protein by DC cells and improve the effect of inhibiting tumor growth.

The fusion protein provided by the invention comprises: signal peptides and HPV viral antigen proteins;

or comprises the following steps: signal peptide, XCL1 chemokine, and HPV viral antigen protein;

wherein the content of the first and second substances,

the signal peptide is XCL1 signal peptide of an amino acid sequence shown in SEQ ID NO. 7 or IgE signal peptide of an amino acid sequence shown in SEQ ID NO. 5.

The amino acid sequence of the XCL1 chemokine is shown as SEQ ID NO 6.

The HPV virus antigen protein is the E6 protein and/or the E7 protein of HPV virus, and key amino acids mediating oncogenic mechanism are mutated and/or deleted. The HPV virus antigen protein is selected from at least one of the following proteins:

1, the E6 protein of HPV16 virus with the amino acid sequence shown in SEQ ID NO;

2, the E7 protein of HPV16 virus with the amino acid sequence shown in SEQ ID NO;

3, and the E6 protein of HPV18 virus with the amino acid sequence shown in SEQ ID NO;

4, and the HPV18 virus E7 protein.

In the invention, the XCL1 chemokine is connected with the E6/E7 protein of HPV virus by a linker. The amino acid sequence of the linker is (G)₅S) n. In some embodiments, the (G)₅S) n is 1-10. In the embodiment of the invention, the linker sequence is GGGGGSGGGGG. The E6 protein and the E7 protein of the HPV16 and the HPV18 are connected through a short linker of AGA.

In the embodiment of the invention, the N end of the fusion protein is XCL1 chemokine (SEQ ID NO: 6), and the C end is HPV virus antigen protein. Wherein the signal peptide of XCL1 chemokine (SEQ ID NO: 7) is retained or replaced with the IgE signal peptide (SEQ ID NO: 5).

In some embodiments, the fusion protein is sequentially from N-terminus to C-terminus an IgE signal peptide-XCL 1 chemokine-E6 protein of HPV virus-E7 protein of HPV virus. In some embodiments, the fusion protein comprises, in order from N-terminus to C-terminus, IgE signal peptide (SEQ ID No. 5) -XCL1 chemokine (SEQ ID No.6) -E6 protein of HPV16 virus (SEQ ID No. 1) -E7 protein of HPV16 virus (SEQ ID No. 2) -E6 protein of HPV18 virus (SEQ ID No. 3) -E7 protein of HPV18 virus (SEQ ID No. 4). The amino acid sequence of the fusion protein is shown as SEQ ID NO.8, the nucleic acid sequence of the DNA for coding the protein is shown as SEQ ID NO. 11, and the nucleic acid sequence of the RNA for coding the protein is shown as SEQ ID NO. 14. The DNA encoding the protein may be present independently or in a vector as one of the components of a vaccine. The RNA encoding the protein may be present independently or in a vector as one of the components of a vaccine.

In some embodiments, the fusion protein is sequentially from N-terminus to C-terminus an IgE signal peptide-E6 protein of HPV virus-E7 protein of HPV virus. In some embodiments, the fusion protein sequentially comprises an IgE signal peptide, an E6 protein of HPV16 virus, an E7 protein of HPV16 virus, an E6 protein of HPV18 virus and an E7 protein of HPV18 virus from N end to C end. The amino acid sequence of the fusion protein is shown as SEQ ID NO.9, the nucleic acid sequence of the DNA for coding the protein is shown as SEQ ID NO. 12, and the nucleic acid sequence of the RNA for coding the protein is shown as SEQ ID NO. 15. The DNA encoding the protein may be present independently or in a vector as one of the components of a vaccine. The RNA encoding the protein may be present independently or in a vector as one of the components of a vaccine.

In some embodiments, the fusion protein is, in order from N-terminus to C-terminus, XCL1 signal peptide-XCL 1 chemokine-E6 protein of HPV virus-E7 protein of HPV virus. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, XCL1 signal peptide, XCL1 chemokine, E6 protein of HPV16 virus, E7 protein of HPV16 virus, E6 protein of HPV18 virus, and E7 protein of HPV18 virus. The amino acid sequence of the fusion protein is shown as SEQ ID NO.10, the nucleic acid sequence of the DNA for coding the protein is shown as SEQ ID NO. 13, and the nucleic acid sequence of the RNA for coding the protein is shown as SEQ ID NO. 16. The DNA encoding the protein may be present independently or in a vector as one of the components of a vaccine. The RNA encoding the protein may be present independently or in a vector as one of the components of a vaccine.

The invention also provides nucleic acids encoding the fusion proteins. In some embodiments, the coding sequence is as shown in any one of SEQ ID NO 11-13 or SEQ ID NO 14-16.

The invention also provides a transcription unit containing the coding fusion protein. The transcription unit comprises a promoter and a nucleic acid for coding the fusion protein.

In some embodiments, the promoter is a CMV or CMV/R promoter. The transcription unit has CMV or CMV/R and nucleic acid encoding the fusion protein in sequence from 5 'end to 3' end.

The invention also provides a recombinant vector comprising a backbone vector and a nucleic acid encoding the fusion protein.

In some embodiments, the scaffold vector is selected from a pVAX1 series vector, or a pVR series vector. In some embodiments, the scaffold vector is a pVR. Wherein the pVR vector is formed by replacing the mutation from the 36 th base to the 663 th base on the pVAX1 vector with a sequence SEQ ID No. 17. The sequence of the pVR plasmid vector is shown in SEQ ID No. 18. The pVAX1 series vector or the pVR series vector is used for expression of fusion protein, thereby being used for preparing protein vaccine or DNA vaccine. And expressing mRNA encoding the fusion protein in animal cells by using pVAX1 or pVR vector as mRNA expression vector to prepare mRNA vaccine.

The invention also provides a recombinant host transformed or transfected with the recombinant vector. The host cell of the recombinant host of the present invention is a bacterial or mammalian cell. The bacteria is escherichia coli; the mammalian cell HEK293T cell.

The preparation method of the fusion protein is to culture the recombinant host of the invention and obtain a culture containing the fusion protein.

The preparation method of the nucleic acid is to culture the recombinant host of the invention and obtain a culture containing the nucleic acid.

The fusion protein, the nucleic acid, the recombinant vector, the recombinant host and/or the fusion protein prepared by the preparation method are applied to preparing vaccines; the vaccine is used for preventing and treating diseases caused by HPV virus infection.

The HPV viral infection causes the disease to be metastatic cancer or a tumor with high expression of HPV E6/E7. The metastatic cancer is cancer of the reproductive system, and in the present invention, the cancer of the reproductive system is cervical cancer, head and neck cancer, lung cancer, oral cancer, tonsil cancer, rectal cancer, anal cancer, penile cancer, bladder cancer, prostate cancer and/or vulvar cancer.

A vaccine for the prevention and treatment of diseases caused by HPV viral infection, comprising: the fusion protein, the nucleic acid, the recombinant vector and/or the fusion protein prepared by the preparation method are provided by the invention.

The control of the invention comprises: raise antibody level in serum, prevent tumor formation, inhibit tumor growth and raise the immunological reaction capacity of body to tumor.

The vaccine also comprises a pharmaceutically acceptable carrier, excipient and/or adjuvant.

A method for preventing and treating diseases caused by HPV virus infection is to administer the vaccine provided by the invention. The administration mode comprises injection, oral administration or electrotransformation.

The present invention provides a fusion protein comprising: XCL1 chemokine and antigenic protein of HPV virus. The invention expresses the antigen protein of HPV virus and DC cell ligand XCL1 in a fusion way, and transfers the antigen protein of HPV virus without pathogenicity to DC cells with cross presentation by using XCL1 as a carrier, thereby improving the efficiency of phagocytosis, processing and presentation of the antigen protein of HPV virus by the DC cells and improving the effect of preventing and treating diseases related to HPV virus infection. Experiments prove that the protein expressed by the nucleic acid vaccine can effectively bind DC cells, induce E6/E7 specific T cell response and remarkably inhibit the growth of tumors with high expression of E6/E7 in various models.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1A, FIG. 1B and FIG. 1C show maps of vectors encoding fusion protein nucleotides, respectively; wherein A in FIG. 1 shows a map of the nucleic acid vaccine plasmid pVR-IgE-hXCL1-HPV16-E6E7-HPV18-E6E 7; FIG. 1B shows a map of the nucleic acid vaccine plasmid pVR-hXCL1-HPV16-E6E7-HPV18-E6E 7; FIG. 1C shows a plasmid map of pVR of the nucleotide plasmid vector;

FIG. 2 shows the detection of expression of unrelated proteins on different plasmid vectors for pVAX1 and pVR. Transfecting HEK292T cells by a plasmid vector carrying nucleic acid for encoding a protein-M for 48 hours, and detecting the expression condition of the fusion protein encoding nucleotide with a Flag label at the C end by using a western blot technology (Westernblot);

FIG. 3 shows the detection of expression of a nucleic acid encoding a fusion protein in HEK293T cells; transfecting HEK292T cells by a plasmid vector carrying nucleic acid for encoding the fusion protein for 48 hours, and detecting the expression condition of the fusion protein encoding nucleotide with Flag tag at the C end by using a western blot technology (Westernblot);

FIG. 4 shows the time axis of immunization of mice and vaccination in an example of the intervention effect of the fusion gene vaccine on the development of mouse transplanted tumor cells TC-1 allografts;

FIG. 5 shows the results of the intervention of fusion gene DNA, mRNA and protein vaccines in the development of mouse transplanted tumor cells TC-1 allografts; wherein A in FIG. 5 shows the tumor growth curve after the group of female mice was prevented from immunization for tumor inoculation; b in fig. 5 shows the tumor growth curve after the group of male mice was prevented from immunization for vaccination; c in fig. 5 shows the levels of E6E 7-specific antibodies in serum detected on day 28 after female mice became established with neoplasia;

FIG. 6 shows the results of intervention of low dose of the fusion gene vaccine in the development of mouse transplanted tumor cells TC-1 allografts; wherein A in FIG. 6 shows the tumor growth curve of mice after prophylactic immunization of tumors; b in fig. 6 shows the results of measuring E7-specific T cell detection at different times for all mouse groups;

FIG. 7 shows the results of the treatment of mouse transplanted tumor cell TC-1 allografts with the fusion gene vaccine; wherein a in figure 7 shows the timeline scenario for tumor inoculation and subsequent therapeutic immunization of mice; b in FIG. 7 shows the mouse tumor growth curve during the whole experiment;

FIG. 8 shows the results of the treatment of mouse transplanted tumor cells TC-1 with the fusion gene vaccine in a large-volume transplanted tumor; time-axis scenario in which mice a in figure 8 were tumorigenic and subsequently therapeutically immunized; b in FIG. 8 shows the mouse tumor growth curve during the whole experiment; c in fig. 8 represents the number of E7-specific T cells measured in mice at the ethical death phase of the control group.

Detailed Description

The invention provides tumor nucleic acid vaccines targeting E6 and E7, which can be realized by appropriately modifying process parameters by the skilled person with reference to the contents. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The fusion protein is formed by fusing XCL1 chemokine and HPV virus antigen protein. In the embodiment of the application, the human XCL1 fragment and the E6 and E7 proteins of human papilloma virus subtypes HPV16 and HPV18 are taken as experimental subjects to verify that the XCL1 has the improvement on the presentation effect of the antigen proteins of HPV viruses. In the present invention, the sequence of linkage between XCL1 chemokine and HPV virus antigen protein is not limited. Earlier studies show that the scheme that the N terminal is XCL1 chemokine is more favorable for protein expression, thereby improving the treatment effect on tumors.

In the present invention, the XCL1 chemokine comprises an IgE signal peptide. In some embodiments, the XCL1 chemokine has an amino acid sequence as set forth in SEQ ID NO 5. The HPV viral antigen protein adopts full-length protein, and key amino acid which mediates oncogenic mechanism is mutated and/or deleted. In the present invention, the mutation sites are as follows: e6 protein sequence mutations C70G and I135T of HPV16 (amino acid sequence after mutation is SEQ ID NO: 1); e7 protein sequence mutations C24G and E26G of HPV16 (amino acid sequence after mutation is SEQ ID NO: 2); e6 protein sequence mutation of HPV 18R 10S and P11G, mutation deletion of amino acids 45-47 and 111-115 (amino acid sequence after mutation is SEQ ID NO: 3); the E7 protein sequence of HPV18 mutations C27G and E29G (post-mutation amino acid sequence of SEQ ID NO: 4). The invention respectively carries out point mutation and deletion mutation on E6 and E7 of HPV, so that the proteins E6 and E7 lose the carcinogenic capacity.

In order to ensure that each functional fragment in the fusion protein is smoothly folded without being influenced by steric hindrance, a linker is added between the fragments, wherein the linker between XCL1 and HPV virus antigen protein is GGGGGSGGGGG. The E6 protein and the E7 protein of the HPV16 and the HPV18 are connected through a short linker of AGA.

The nucleic acid encoding the protein of the present invention may be DNA, RNA, cDNA or PNA. In embodiments of the invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. Nucleic acids may include nucleotide sequences having different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acids can be linear or circular in topology. The nucleic acid may be, for example, part of a vector, such as an expression or cloning vector, or a fragment. The nucleic acids may be obtained directly from natural sources, or may be prepared with the aid of recombinant, enzymatic or chemical techniques.

In the present invention, the nucleic acid sequence for expressing the fusion protein is optimized, and these optimizations include but are not limited to: codon usage bias, elimination of secondary structures that are detrimental to expression (such as hairpin structures), alteration of GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splice sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA instability regions, repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

The invention also provides a transcription unit of the fusion protein, wherein the transcription unit refers to a DNA sequence from the beginning of a promoter to the end of a terminator. Regulatory segments may also be included on either side of or between the promoter and terminator, which may include a promoter operably linked to a nucleic acid sequence, an enhancer, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites, such as enhancers for promoters, poly (A) signals, and the like. The transcription unit provided by the invention comprises a CMV or CMV/R promoter, a CMV enhancer and a nucleic acid segment for encoding the fusion protein.

The recombinant vector of the present invention, which refers to a recombinant nucleic acid vector, is a recombinant DNA molecule comprising the desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding gene in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotic cells include promoters, optionally including operator sequences, ribosome binding sites and possibly other sequences. Prokaryotic cells are known to utilize promoters, enhancers and termination and polyadenylation signals. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In the present specification, "plasmid" and "vector" may sometimes be used interchangeably, since plasmids are the most commonly used form of vector at present. However, the present invention is intended to include such other forms of expression vectors which serve equivalent functions, which are or will become known in the art, including, but not limited to: plasmids, phage particles, viral vectors and/or simply potential genomic inserts.

The host cell of the invention is a prokaryotic or eukaryotic host containing a nucleic acid vector and/or a target gene. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are competent to replicate the vector encoding the protein or to express the desired protein.

In the embodiment of the invention, the preparation method of the fusion protein adopts a mode of inducing recombinant host expression, and the culture can be thalli, cell bodies, culture solution obtained by culture or substances obtained by extraction and/or purification of the culture.

In the present invention, the prevention and/or treatment includes prevention and/or treatment of diseases. The disease is caused by HPV virus infection, and further, the disease is a tumor with high expression of E6/E7 of HPV. Further, the disease is metastatic cancer. In some embodiments, the metastatic cancer is a cancer of the reproductive system, and in the present invention, the cancer of the reproductive system is cervical cancer, head and neck cancer, lung cancer, oral cancer, tonsil cancer, rectal cancer, anal cancer, penile cancer, bladder cancer, prostate cancer, and/or vulvar cancer. In the embodiment of the invention, the effect of the fusion protein vaccine is verified by taking a mouse transplanted tumor cell TC-1 as an experimental object.

The prevention of the invention means that the medicine can play a role in reducing the risk of tumor occurrence when being administered before the tumor occurrence. The treatment of the invention refers to that the medicine provided by the invention can inhibit the growth of tumor, reduce the volume of tumor or delay the growth speed of tumor after the tumor is generated.

In the examples of the invention, the amino acid sequences of the fragments involved and the nucleic acid fragments encoded are shown in Table 1:

amino acid sequences of the fragments of Table 1 and the encoded nucleic acid fragments

SEQ ID No.1	Amino acid sequence of E6 protein of HPV16
		SEQ ID No.2	Amino acid sequence of E6 protein of HPV18
SEQ ID No.3	Amino acid sequence of E7 protein of HPV16
		SEQ ID No.4	Amino acid sequence of E7 protein of HPV18
SEQ ID No.5	Amino acid sequence of IgE signal peptide
		SEQ ID No.6	Amino acid sequence of hXCL1
SEQ ID No.7	Amino acid sequence of hXCL1 signal peptide
		SEQ ID No.8	Fusion protein IgE signal peptide-hXCL 1-HPV16-E6E7-HPV18-E6E7 amino acid sequence
SEQ ID No.9	Fusion protein IgE signal peptide-HPV 16-E6E7-HPV18-E6E7 amino acid sequence
		SEQ ID No.10	Amino acid sequence of fusion protein hXCL1 signal peptide-hXCL 1-HPV16-E6E7-HPV18-E6E7
SEQ ID No.11	DNA sequence encoding SEQ ID No.8
		SEQ ID No.12	DNA sequence encoding SEQ ID No.9
SEQ ID No.13	DNA sequence encoding SEQ ID No.10
		SEQ ID No.14	mRNA sequence coding for SEQ ID No.8
SEQ ID No.15	mRNA sequence coding for SEQ ID No.9
		SEQ ID No.16	mRNA sequence coding for SEQ ID No.10
SEQ ID No.17	The sequence obtained by mutating and replacing base numbers 36-663 on pVAX1 vector
		SEQ ID No.18	pVR vector sequence

The invention also provides a vaccine for preventing and treating diseases caused by HPV virus infection, which comprises the following components: the fusion protein, the nucleic acid, the recombinant vector, the recombinant host and/or the fusion protein prepared by the preparation method are disclosed. In some embodiments, the vaccine provided by the invention is a protein consisting of human papillomavirus subtype HPV16 and E6 and E7 proteins of HPV18, and an XCL1 molecule which is fused at the N end of the protein and expresses DC with specific binding antigen cross-presentation capacity, nucleic acid containing the fusion protein and a carrier containing the nucleic acid, and application of the fusion protein, the nucleic acid and the carrier in preventing and treating E6/E7 high-expression tumors and metastatic cancers. The invention improves the phagocytosis, processing and presentation efficiency of E6/E7 protein by DC cells and improves the effect of inhibiting tumor growth by fusing and expressing E6 and E7 proteins of human papilloma virus subtypes HPV16 and HPV18 and a DC cell ligand XCL 1. Aiming at the proteins E6 and E7, the invention carries out point mutation and deletion mutation on E6 and E7 of HPV16 and HPV18 respectively, so that the proteins E6 and E7 lose the carcinogenic capacity. The E6 and E7 proteins of HPV16 and HPV18 are connected through a short linker of AGA, and can be independently used as immunogens respectively. The invention connects E6 and E7 of HPV16 and E6 and E7 of HPV18 through AGA short linker, and can be used as immunogen in whole. The protein expressed by the nucleic acid vaccine can be effectively combined with DC cells, induces E6/E7 specific T cell response and obviously inhibits the growth of tumors with high expression of E6/E7 in various models.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:

example 1 design of fusion protein vaccine antigens and construction and preparation of mammalian cell expression plasmids

Construction of mammalian cell expression vector of fusion gene:

hXCL1-HPV16-E6E7-HPV18-E6E 7: according to the amino acid sequences (SEQ ID No.6) of E6 and E7 proteins of human papillomavirus subtypes HPV16 and HPV18 and human XCL1 protein, a fusion protein hXCL1-HPV16-E6E7-HPV18-E6E7 is constructed, and a short linker of AGA is added between the amino acid sequences of the E6 and E7 proteins to ensure that the two proteins as antigens cannot interfere with each other. In order to promote efficient secretion of the fusion protein from the nucleic acid expressing the fusion protein in vivo to the outside of the cell and chemotaxis of MHC-II + CD11c + CD8a + antigen cross-presenting DC cells, we replaced the secretory signal peptide (SEQ ID NO: 7) of the XCL1 protein with the IgE signal peptide (SEQ ID NO: 5). At the same time, the key amino acid mutation mediating the cancer mechanism in the E6/E7 protein makes the E6 and E7 proteins lose the carcinogenic capacity:

e6 protein sequence mutations of HPV16, C70G and I135T (SEQ ID NO. 1);

e7 protein sequence mutations of HPV16, C24G and E26G (SEQ ID NO. 2);

e6 protein sequence mutation of HPV 18R 10S and P11G, mutation deletion of amino acids 45-47 and amino acids 111-115 (SEQ ID NO. 3);

the E7 protein sequence of HPV18 mutations C27G and E29G (SEQ ID NO. 4).

The finally obtained fusion protein comprises the following components from the N end → the C end in sequence: IgE signal peptide, hXCL1 fragment with signal peptide removed, HPV 16E 6 protein, HPV 16E 7 protein, HPV 18E 6 protein and HPV 18E 7 protein, and an amino acid sequence thereof (SEQ ID No. 8). Nucleotide sequences corresponding to the amino acid sequences are subjected to codon optimization of mammal cell expression preference, and the encoding nucleic acid sequences are determined to be shown as SEQ ID NO. 9.

HPV16-E6E7-HPV18-E6E 7: an IgE secretion signal peptide (SEQ ID NO: 5) is added to the N-terminal of the E6E7 protein to ensure that the E6E7 protein expressed by the E6E7 expression vector alone can be secreted outside the cell similarly. Obtaining the fusion protein comprising, in order from N-terminus → C-terminus: IgE signal peptide, HPV 16E 6 protein, HPV 16E 7 protein, HPV 18E 6 protein and HPV 18E 7 protein, wherein the amino acid sequences of the proteins are shown as SEQ ID No.10, the nucleotide sequences corresponding to the amino acid sequences are subjected to codon optimization of mammal cell expression preference, and the encoding nucleic acid sequences are determined to be shown as SEQ ID No. 11.

Synthesizing the gene and connecting the gene to a pVR expression vector, and respectively recording two vectors obtained by construction as:

pVR-IgE-hXCL1-HPV16-E6E7-HPV18-E6E7

pVR-IgE-HPV16-E6E7-HPV18-E6E7。

example 2 comparison of protein expression Capacity of plasmid vector pVR and pVAX1

To facilitate the detection of gene expression effect, we linked a 3 XFlag tag consisting of DYKDHDGDYKDHDIDYKDDDDK 22 amino acids to the C-terminus of a protein-M gene to detect the expression of fusion protein using Flag tag antibody. protein-M was constructed into pVAX1 and pVR plasmids for mammalian cell expression assays, respectively.

24 hours prior to transfection, 1X 10 cells were inoculated in 6-well cell culture plates⁶HEK293T cells, the transfection assay was started when the cell density reached 70% -80%. Cell culture medium and serum-free Opti-MEM medium were pre-warmed in a 37 ℃ water bath at the time of transfection. 5 micrograms of empty Vector (Vector), pVAX1-protein-M expression Vector, pVR-protein-M expression Vector and 20. mu.L PEI transfection reagent were added to 200. mu.L of serum-free Opti-MEM in sequence for transfection, mixed well and allowed to stand at room temperature for 10 minutes. The cells to be transfected are replaced by fresh culture medium, and the cells are added into the transfection system gently and shaken up. The cells were returned to the cell incubator and incubated for 6 hours before changing the medium. Cells were harvested after 48 hours of transfer and Western Blot was used to examine the expression effect in protein-M fusion gene plasmid HEK293T cells.

Cells were harvested and 60 μ L of 0.5% NP40 lysis buffer containing PMSF or Cocktail protease inhibitor was added. Resuspend the cells well and spin lyse the cells at 4 ℃ for 30 min. The lysate was centrifuged at 12000 rpm for 10 min at 4 ℃ and the supernatant was collected in a fresh 1.5 mL EP tube and the pellet discarded. Adding a 5 xSDS-PAGE protein loading buffer according to the actual volume of the sample, uniformly mixing, placing the sample in an air bath at 100 ℃ for heating for 10 minutes, immediately carrying out Western blot, and detecting by using a Flag tag antibody (Sigma, F3165), wherein the result is shown in figure 2 that an empty Vector (Vector) has no protein expression, and the expression protein amount of pVR-protein-M is obviously higher than that of pVAX1-protein-M, which indicates that the expression capability of pVR as a plasmid Vector is better.

Example 3 detection of expression Effect of mammalian cell expression vectors encoding XCL1-E6E7 fusion proteins

24 hours prior to transfection, 1X 10 cells were inoculated in 6-well cell culture plates⁶HEK293T cells, the transfection assay was started when the cell density reached 70% -80%. Cell culture medium and serum-free Opti-MEM medium were pre-warmed in a 37 ℃ water bath at the time of transfection. When in transfection, 5 micrograms of empty Vector (Vector), single HPV16-E6E7-HPV18-E6E7 expression Vector, hXCL1-HPV16-E6E7-HPV18-E6E7 fusion gene plasmid and 20 microliters of PEI transfection reagent are added into 200 microliters of serum-free Opti-MEM in sequence, mixed uniformly and then kept stand for 10 minutes at room temperature. The cells to be transfected are replaced by fresh culture medium, and the cells are added into the transfection system gently and shaken up. The cells were returned to the cell incubator and incubated for 6 hours before changing the medium. Cells are harvested after 48 hours of transfer, and Western Blot is used for detecting the expression effect of hXCL1-HPV16-E6E7-HPV18-E6E7 fusion gene plasmid HEK293T cells.

To facilitate the detection of the expression effect of the fusion gene, a 3 XFlag tag consisting of DYKDHDGDYKDHDIDYKDDDDK amino acids was attached to the C-terminus of the fusion protein to detect the expression of the fusion protein using a Flag tag antibody. Cells were harvested and 60 μ L of 0.5% NP40 lysis buffer containing PMSF or Cocktail protease inhibitor was added. Resuspend the cells well and spin lyse the cells at 4 ℃ for 30 min. The lysate was centrifuged at 12000 rpm for 10 min at 4 ℃ and the supernatant was collected in a fresh 1.5 mL EP tube and the pellet discarded. Adding a 5 xSDS-PAGE protein loading buffer according to the actual volume of a sample, uniformly mixing, placing the sample in an air bath at 100 ℃ for 10 minutes, immediately carrying out Western blot, and detecting by using Flag tag antibodies (Sigma, F3165), wherein the result is shown in figure 3 that an empty Vector (Vector) has no protein expression, an individual HPV16-E6E7-HPV18-E6E7 expression Vector and an hXCL1-HPV16-E6E7-HPV18-E6E7 fusion gene plasmid can be effectively expressed and have equivalent expression quantity.

Example 4 Effect of fusion of Gene DNA and mRNA and intervention of the protein-form vaccine in the development of murine transplanted tumor cell TC-1 allografts

Since the fusion gene can be normally expressed in mammalian cells. The individual HPV16-E6E7-HPV18-E6E7 and hXCL1-HPV16-E6E7-HPV18-E6E7 plasmids are extracted, an TERESA living gene introduction instrument is used for carrying out immune plasmid electrotransformation on a mouse, and the mouse is transformed outside the body to prepare mRNA vaccine expressing hXCL1-HPV16-E6E7-HPV18-E6E7 and an in-vivo transfection reagent in vivo transfection reagent in a vitamin-jetPEI (mRNA) encapsulated into lipid nanoparticles serving as mRNA form vaccine (denoted as mRNA).

And purifying the fusion protein as a protein vaccine, wherein the proteins of HPV16-E6E7-HPV18-E6E7 are designated as E6E7, and the proteins of hXCL1-HPV16-E6E7-HPV18-E6E7 are designated as XCL1-E6E 7.

After transplanting TC-1 cells in the same species, the inhibition of the fusion gene immunity on the growth of TC-1 transplanted tumor cells is observed.

(1) Preventive effect on development of TC-1 cell allograft tumor after immunization with fusion gene

After determination of TC-1 cell neoplasia, we performed plasmid electrotransformation and mRNA intramuscular injection as well as protein subcutaneous injection in mice according to the immunization strategy annotated by the time axis of fig. 4. C57B6 (purchased from Torilica) week-old female and male mice were divided into A, B groups, each group being five groups injected with PBS, the E6E7 and XCL1-E6E7 plasmids alone and XCL1-E6E7mRNA and XCL1-E6E7 protein, and five mice were depilated with depilatory cream on the right side of the mice near the inguinal lymph node. Then mRNA and protein are directly injected or plasmid is injected at the unhairing position by an electrotransfer instrument, each 50 mu g of the plasmid is injected once every two weeks, the injection is carried out twice, TC-1 tumor cells with good tumorigenic conditions are searched before inoculation is carried out for one week after the last injection, the tumor formation time is observed, the major diameter a and the minor diameter b of the tumor are measured every two days, the tumor volume is calculated according to a multiplied by b/2, and a tumor growth curve is drawn. The results are shown in A, B in fig. 5, and the immune injections XCL1-E6E7 and E6E7 both can prevent the transplanted tumor from forming tumor in the mouse body, and the vaccine effect is remarkable. And all mice were challenged with secondary tumor inoculation at D30, showing that the transplanted tumors still failed to form tumors. This shows that both vaccines have very significant tumor prevention effect.

(2) Exploration on whether fusion gene immunization induces E6E7 specific antibody reaction

All mice in the female group were bled from the eye at D28, and the serum was frozen after centrifugation to remove blood cells. Recombinant E6E7 protein was added to an Elisa plate of corning (9018) at 10. mu.g/well and incubated overnight at 4 ℃. The next day the plates were washed three times with PBST and patted dry. Add 1 XELISAPOT Diluent and block for 2h at room temperature. Mouse serum was diluted 10-fold and incubated with the plate overnight at 4 ℃. The plates were then washed three more times, diluted 1:5000 to goat anti-mouse lgG-BIOT was added and incubated for 1h at room temperature. Plates were washed three more times and incubated with streptavidin-HRP for 30 min at room temperature. After the final three washes, the reaction was quenched by peroxidase development with 100. mu.L of TMB 1-Component (Invitrogen, 00-4201-56) for 2-5min and addition of 50. mu.L of 2M hydrochloric acid. And reading the light absorption value of 450nm by using a microplate reader. The 450nm absorbance of the sera from each group of mice was statistically plotted as C in FIG. 5. From the results, it can be seen that the group XCL1-E6E7 has a distinct advantage in serum antibody detection, and although the two groups are not distinguished from each other in terms of graft tumor prevention, the antibody level in serum indicates that XCL1-E6E7 have a greater advantage in terms of inducing the immune system to produce antibodies.

Example 5 Effect of Low dose of the fusion Gene vaccine on the intervention of murine transplantation tumor cell TC-1 allogenic transplantation tumorigenesis

(1) Preventive effect on TC-1 cell allogenemia after low-dose fusion gene immunization

With reference to example 4 (1), the mice were subjected to preventive plasmid electroporation in part of the relevant procedures, and then were subjected to transplantation of the hybridomas in the same time axis. However, the immune plasmids were divided into two low dose groups, 5. mu.g and 25. mu.g, respectively. The growth of the transplanted tumor was observed and the tumor volume was calculated in the same manner, and a tumor growth curve was plotted as shown in A of FIG. 6. It can be seen that when the mass of the prophylactic immunophilin is reduced by half, the mice still can not form tumor after receiving the tumor. After the quality of the preventive immune plasmid is reduced to one tenth of the original quality, the group E6E7 and the group XCL1-E6E7 begin to have a difference in tumorigenesis. The results show that the XCL1-E6E7 group has slow tumor growth although the group starts to form tumors, and the tumor prevention effect is obviously better than that of the E6E7 group, which indicates that the plasmid vaccine has excellent tumor prevention effect.

(2) Exploring whether fusion gene immunity induces stronger cell-specific T cell response

Each group of mice was bled and added to heparin-added PBS solution at D7, D14, D21 and D28 in the above 5. mu.g and 25. mu.g prophylactic immunization models, respectively. The whole was centrifuged at 3000rpm for 5 min. The supernatant was discarded, and the remaining precipitate was broken up by shaking and then lysed for 1min at room temperature by adding 1mL of a lysis buffer. After this time, all samples were centrifuged at 1200rpm for 6 min. The supernatant was discarded and washed once with 700. mu.L of PBS solution. Centrifuge for 6min at 1200rpm for the second time. After discarding the supernatant, 300. mu.L of inactivated 10% FBS 1640 medium was added, and after resuspending the pellet, 5. mu. L E7 protein tetramer (E7-tetramer) was added and stained for 1 hour. Flow dyeing: cd 8-pe; E7-tetramer-Fitc. The streaming result is shown as B in fig. 6. The results show that all XCL1-E6E7 groups had a far greater number of E7-specific T cells than the E6E7 group in comparison of the same dose group. In comparison of the different dose groups, the specific T cells were produced at a significantly faster rate in the 25 μ g low dose group, but their cellular proportion also rapidly declined as the tumor had completely disappeared. The specific T cells in the low dose 5. mu.g group were produced slowly, but the XCL1-E6E7 group still produced a large number of specific T cells at 21 days after tumor inoculation. Indicating that it also activates the immune response of the body.

Example 6: therapeutic effect of fusion gene vaccine on mouse transplantation tumor cell TC-1 allografting tumor

In view of the excellent effect of XCL1-E6E7 in prophylactic immunization experiments. We also conducted experimental exploration on the therapeutic effect of TC-1 allografts immunized by the fusion gene. Mice were vaccinated and plasmid-treated using the timeline as in a in figure 7. The amount of each immunization was 25. mu.g. Mice were immunized twice, D4 and D11, respectively, starting on the third day after inoculation. Tumor quantification was then performed according to the same method, and tumor volumes were counted. The tumor growth curve was plotted as shown in B of fig. 7. The results show that the XCL1-E6E7 group had earlier tumor growth inhibition than the E6E7 group, and the XCL1-E6E7 group had completely disappeared the transplanted tumor on the day 17 of tumor inoculation, achieving complete tumor treatment.

Example 7: therapeutic effect of fusion gene vaccine on mouse transplanted tumor cell TC-1 large-volume transplanted tumor

(1) Exploring the therapeutic effect of the fusion gene vaccine on the mouse transplanted tumor cell TC-1 large-volume transplanted tumor

In view of the superior efficacy of the above vaccines in tumor therapy, we challenge tumor therapy for TC-1 large-volume transplantable tumors. Under the same experimental conditions, when the average tumor volume reaches 50mm³The administration is started. Mice were therefore vaccinated and plasmid-treated using the timeline as in a in figure 8. The amount of immunization was 25. mu.g. Tumor quantification was performed according to the same method, and tumor volume was counted. Tumor growth curves were plotted as shown in B of fig. 8. The results showed that inhibition of tumor growth occurred earlier in XCL1-E6E7 than in E6E7 group, and that inhibition of tumor growth started at D19 and almost complete tumor clearance at D25 in XCL1-E6E7 group. In contrast, the E6E7 group also exhibited inhibition of tumor growth, but failed to completely clear the tumor. The XCL1-E6E7 has absolute effect advantage in the process of treating tumors and even treating large-volume tumors.

All ethical deaths (tumor volume 2000 mm) were performed under the same experimental conditions in the control group³) Mice at time were sacrificed. The same experimental method as in example 5 (2) was used to treat the blood of the mice. And finally, carrying out flow dyeing: cd 8-pe; E7-tetramer-Fitc. The results are shown in fig. 8C. The number of specific T cells of mice in the XCL1-E6E7 group was significantly higher than those of the E6E7 group and the control group. The XCL1-E6E7 vaccine is proved to have absolutely strong capability of activating the immune response of the organism.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

<110> Nouchi Tech (Beijing) Ltd

<120> nucleic acid vaccine targeting human papillomavirus

<130> MP21007635

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His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Ser Gly Arg

1 5 10 15

Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile

20 25 30

Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val

35 40 45

Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn

50 55 60

Pro Tyr Ala Val Gly Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser

65 70 75 80

Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln

85 90 95

Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys

100 105 110

Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys

115 120 125

Gln Arg Phe His Asn Thr Arg Gly Arg Trp Thr Gly Arg Cys Met Ser

130 135 140

Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu

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<213> Artificial Sequence (Artificial Sequence)

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His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro

1 5 10 15

Glu Thr Thr Asp Leu Tyr Gly Tyr Gly Gln Leu Asn Asp Ser Ser Glu

20 25 30

Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg

35 40 45

Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu

50 55 60

Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp

65 70 75 80

Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys

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Pro

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<213> Artificial Sequence (Artificial Sequence)

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Ala Arg Phe Glu Asp Pro Thr Arg Ser Gly Tyr Lys Leu Pro Asp Leu

1 5 10 15

Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys Val

20 25 30

Tyr Cys Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu Lys Asp Leu

35 40 45

Phe Val Val Tyr Arg Asp Ser Ile Pro His Ala Ala Cys His Lys Cys

50 55 60

Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg His Tyr Ser Asp Ser

65 70 75 80

Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr Gly Leu Tyr Asn

85 90 95

Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys Pro Leu Leu Arg His Leu

100 105 110

Asn Glu Lys Arg Arg Phe His Asn Ile Ala Gly His Tyr Arg Gly Gln

115 120 125

Cys His Ser Cys Cys Asn Arg Ala Arg Gln Glu Arg Leu Gln Arg Arg

130 135 140

Arg Glu Thr Gln Val

145

<210> 4

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<213> Artificial Sequence (Artificial Sequence)

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His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu Pro

1 5 10 15

Gln Asn Glu Ile Pro Val Asp Leu Leu Gly His Gly Gln Leu Ser Asp

20 25 30

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

35 40 45

Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met Cys

50 55 60

Cys Lys Cys Glu Ala Arg Ile Glu Leu Val Val Glu Ser Ser Ala Asp

65 70 75 80

Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe Val

85 90 95

Cys Pro Trp Cys Ala Ser Gln Gln

100

<210> 5

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser

<210> 6

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Val Gly Ser Glu Val Ser Asp Lys Arg Thr Cys Val Ser Leu Thr Thr

1 5 10 15

Gln Arg Leu Pro Val Ser Arg Ile Lys Thr Tyr Thr Ile Thr Glu Gly

20 25 30

Ser Leu Arg Ala Val Ile Phe Ile Thr Lys Arg Gly Leu Lys Val Cys

35 40 45

Ala Asp Pro Gln Ala Thr Trp Val Arg Asp Val Val Arg Ser Met Asp

50 55 60

Arg Lys Ser Asn Thr Arg Asn Asn Met Ile Gln Thr Lys Pro Thr Gly

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Thr Gln Gln Ser Thr Asn Thr Ala Val Thr Leu Thr Gly

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<210> 7

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<213> Artificial Sequence (Artificial Sequence)

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Met Arg Leu Leu Ile Leu Ala Leu Leu Gly Ile Cys Ser Leu Thr Ala

1 5 10 15

Tyr Ile Val Glu Gly

20

<210> 8

<211> 638

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser Val Gly Ser Glu Val Ser Asp Lys Arg Thr Cys Val Ser Leu

20 25 30

Thr Thr Gln Arg Leu Pro Val Ser Arg Ile Lys Thr Tyr Thr Ile Thr

35 40 45

Glu Gly Ser Leu Arg Ala Val Ile Phe Ile Thr Lys Arg Gly Leu Lys

50 55 60

Val Cys Ala Asp Pro Gln Ala Thr Trp Val Arg Asp Val Val Arg Ser

65 70 75 80

Met Asp Arg Lys Ser Asn Thr Arg Asn Asn Met Ile Gln Thr Lys Pro

85 90 95

Thr Gly Thr Gln Gln Ser Thr Asn Thr Ala Val Thr Leu Thr Gly Gly

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly His Gln Lys Arg Thr Ala

115 120 125

Met Phe Gln Asp Pro Gln Glu Ser Gly Arg Lys Leu Pro Gln Leu Cys

130 135 140

Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr

145 150 155 160

Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg

165 170 175

Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Gly Asp

180 185 190

Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys

195 200 205

Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu

210 215 220

Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu Cys Pro

225 230 235 240

Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Thr

245 250 255

Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg

260 265 270

Thr Arg Arg Glu Thr Gln Leu Ala Gly Ala His Gly Asp Thr Pro Thr

275 280 285

Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr

290 295 300

Gly Tyr Gly Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp

305 310 315 320

Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val

325 330 335

Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser

340 345 350

Thr His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu

355 360 365

Gly Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Ala Gly Ala Ala Arg

370 375 380

Phe Glu Asp Pro Thr Arg Ser Gly Tyr Lys Leu Pro Asp Leu Cys Thr

385 390 395 400

Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys Val Tyr Cys

405 410 415

Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu Lys Asp Leu Phe Val

420 425 430

Val Tyr Arg Asp Ser Ile Pro His Ala Ala Cys His Lys Cys Ile Asp

435 440 445

Phe Tyr Ser Arg Ile Arg Glu Leu Arg His Tyr Ser Asp Ser Val Tyr

450 455 460

Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr Gly Leu Tyr Asn Leu Leu

465 470 475 480

Ile Arg Cys Leu Arg Cys Gln Lys Pro Leu Leu Arg His Leu Asn Glu

485 490 495

Lys Arg Arg Phe His Asn Ile Ala Gly His Tyr Arg Gly Gln Cys His

500 505 510

Ser Cys Cys Asn Arg Ala Arg Gln Glu Arg Leu Gln Arg Arg Arg Glu

515 520 525

Thr Gln Val Ala Gly Ala His Gly Pro Lys Ala Thr Leu Gln Asp Ile

530 535 540

Val Leu His Leu Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Gly

545 550 555 560

His Gly Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly

565 570 575

Val Asn His Gln His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His

580 585 590

Thr Met Leu Cys Met Cys Cys Lys Cys Glu Ala Arg Ile Glu Leu Val

595 600 605

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

610 615 620

Asn Thr Leu Ser Phe Val Cys Pro Trp Cys Ala Ser Gln Gln

625 630 635

<210> 9

<211> 534

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Ser

20 25 30

Gly Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His

35 40 45

Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg

50 55 60

Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp

65 70 75 80

Gly Asn Pro Tyr Ala Val Gly Asp Lys Cys Leu Lys Phe Tyr Ser Lys

85 90 95

Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu

100 105 110

Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile

115 120 125

Asn Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp

130 135 140

Lys Lys Gln Arg Phe His Asn Thr Arg Gly Arg Trp Thr Gly Arg Cys

145 150 155 160

Met Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu Ala

165 170 175

Gly Ala His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu

180 185 190

Gln Pro Glu Thr Thr Asp Leu Tyr Gly Tyr Gly Gln Leu Asn Asp Ser

195 200 205

Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro

210 215 220

Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser

225 230 235 240

Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu

245 250 255

Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser

260 265 270

Gln Lys Pro Ala Gly Ala Ala Arg Phe Glu Asp Pro Thr Arg Ser Gly

275 280 285

Tyr Lys Leu Pro Asp Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp

290 295 300

Ile Glu Ile Thr Cys Val Tyr Cys Lys Thr Val Leu Glu Leu Thr Glu

305 310 315 320

Val Phe Glu Lys Asp Leu Phe Val Val Tyr Arg Asp Ser Ile Pro His

325 330 335

Ala Ala Cys His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu

340 345 350

Arg His Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr

355 360 365

Asn Thr Gly Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys

370 375 380

Pro Leu Leu Arg His Leu Asn Glu Lys Arg Arg Phe His Asn Ile Ala

385 390 395 400

Gly His Tyr Arg Gly Gln Cys His Ser Cys Cys Asn Arg Ala Arg Gln

405 410 415

Glu Arg Leu Gln Arg Arg Arg Glu Thr Gln Val Ala Gly Ala His Gly

420 425 430

Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu Pro Gln Asn

435 440 445

Glu Ile Pro Val Asp Leu Leu Gly His Gly Gln Leu Ser Asp Ser Glu

450 455 460

Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His Leu Pro Ala

465 470 475 480

Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met Cys Cys Lys

485 490 495

Cys Glu Ala Arg Ile Glu Leu Val Val Glu Ser Ser Ala Asp Asp Leu

500 505 510

Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe Val Cys Pro

515 520 525

Trp Cys Ala Ser Gln Gln

530

<210> 10

<211> 641

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Arg Leu Leu Ile Leu Ala Leu Leu Gly Ile Cys Ser Leu Thr Ala

1 5 10 15

Tyr Ile Val Glu Gly Val Gly Ser Glu Val Ser Asp Lys Arg Thr Cys

20 25 30

Val Ser Leu Thr Thr Gln Arg Leu Pro Val Ser Arg Ile Lys Thr Tyr

35 40 45

Thr Ile Thr Glu Gly Ser Leu Arg Ala Val Ile Phe Ile Thr Lys Arg

50 55 60

Gly Leu Lys Val Cys Ala Asp Pro Gln Ala Thr Trp Val Arg Asp Val

65 70 75 80

Val Arg Ser Met Asp Arg Lys Ser Asn Thr Arg Asn Asn Met Ile Gln

85 90 95

Thr Lys Pro Thr Gly Thr Gln Gln Ser Thr Asn Thr Ala Val Thr Leu

100 105 110

Thr Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly His Gln Lys

115 120 125

Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Ser Gly Arg Lys Leu Pro

130 135 140

Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu

145 150 155 160

Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe

165 170 175

Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala

180 185 190

Val Gly Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg

195 200 205

His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn

210 215 220

Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro

225 230 235 240

Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe

245 250 255

His Asn Thr Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg

260 265 270

Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu Ala Gly Ala His Gly Asp

275 280 285

Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr

290 295 300

Asp Leu Tyr Gly Tyr Gly Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp

305 310 315 320

Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr

325 330 335

Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys

340 345 350

Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met

355 360 365

Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Ala Gly

370 375 380

Ala Ala Arg Phe Glu Asp Pro Thr Arg Ser Gly Tyr Lys Leu Pro Asp

385 390 395 400

Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys

405 410 415

Val Tyr Cys Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu Lys Asp

420 425 430

Leu Phe Val Val Tyr Arg Asp Ser Ile Pro His Ala Ala Cys His Lys

435 440 445

Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg His Tyr Ser Asp

450 455 460

Ser Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr Gly Leu Tyr

465 470 475 480

Asn Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys Pro Leu Leu Arg His

485 490 495

Leu Asn Glu Lys Arg Arg Phe His Asn Ile Ala Gly His Tyr Arg Gly

500 505 510

Gln Cys His Ser Cys Cys Asn Arg Ala Arg Gln Glu Arg Leu Gln Arg

515 520 525

Arg Arg Glu Thr Gln Val Ala Gly Ala His Gly Pro Lys Ala Thr Leu

530 535 540

Gln Asp Ile Val Leu His Leu Glu Pro Gln Asn Glu Ile Pro Val Asp

545 550 555 560

Leu Leu Gly His Gly Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp Glu

565 570 575

Ile Asp Gly Val Asn His Gln His Leu Pro Ala Arg Arg Ala Glu Pro

580 585 590

Gln Arg His Thr Met Leu Cys Met Cys Cys Lys Cys Glu Ala Arg Ile

595 600 605

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

610 615 620

Leu Phe Leu Asn Thr Leu Ser Phe Val Cys Pro Trp Cys Ala Ser Gln

625 630 635 640

Gln

<210> 11

<211> 1914

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atggactgga cctggattct gttcctggtg gctgctgcca ctagagtgca cagcgtgggc 60

agcgaagtgt ccgacaagag gacttgcgtg tccctgacaa cccagagact gccagtgtcc 120

cggatcaaga cctacaccat caccgagggc agcctgagag ccgtgatctt catcaccaag 180

cggggcctga aagtctgtgc cgatcctcag gctacttggg tcagagacgt cgtgaggagc 240

atggacagga agagcaacac ccggaacaac atgatccaga ccaagcccac cggaacacag 300

cagagcacca acacagccgt gaccctgaca ggaggaggag gaggaggaag cggaggagga 360

ggaggacacc agaagagaac agccatgttc caggaccctc aggagagcgg aaggaagctg 420

ccacagctct gtaccgagct gcagaccacc atccacgaca tcatcctcga gtgcgtctac 480

tgcaagcagc agctgctgcg gagagaggtg tacgacttcg ccttcaggga cctctgcatc 540

gtgtaccggg acggcaatcc ctacgccgtg ggcgacaagt gcctgaagtt ctacagcaag 600

atcagcgagt accggcacta ttgctacagc ctgtacggaa ccaccctgga gcagcagtac 660

aacaagcccc tctgcgacct gctcatccgc tgcatcaact gccagaagcc cctctgtccc 720

gaggaaaagc agaggcacct ggacaagaag cagcggttcc acaacaccag gggcaggtgg 780

accggccgct gcatgagttg ttgtaggagc agcaggacca gaagagagac ccagctggca 840

ggagcccacg gagatacacc tacactgcac gagtacatgc tggacctgca gccagagacc 900

accgatctgt acggctacgg acagctgaac gacagcagcg aggaggaaga cgagattgat 960

ggcccagccg gacaggcaga accagatcgg gcccactaca acatcgtcac cttctgctgc 1020

aagtgcgaca gcaccctgag actctgcgtg cagtctaccc acgtggacat caggaccctg 1080

gaggatctgc tgatgggcac actgggcatc gtctgcccca tttgtagcca gaagccagcc 1140

ggagccgcta gatttgagga tcctaccaga agcggctaca agctgccaga cctgtgtacc 1200

gagctgaaca ccagcctgca ggacatcgag atcacttgcg tctactgcaa gaccgtgctg 1260

gagctgaccg aggtgttcga gaaggacctg ttcgtggtgt accgggacag catccctcac 1320

gccgcttgcc acaagtgtat cgatttctac agccggatcc gggagctgag acactacagc 1380

gacagcgtgt acggcgacac actggagaag ctgaccaaca ccggcctgta caacctgctg 1440

atccgctgcc tccgctgtca gaaacctctg ctgaggcacc tgaacgagaa gaggcggttc 1500

cacaacatcg ccggacacta caggggccag tgccacagtt gttgcaacag ggctaggcag 1560

gagaggctgc agagaaggag agagacccag gtggcaggag ctcacggacc taaagctacc 1620

ctgcaggaca tcgtgctgca tctggagcct cagaacgaga tccccgtgga tctgctggga 1680

cacggacagc tgtcagacag cgaagaggag aacgacgaga tcgacggcgt gaaccaccag 1740

catctgccag ccagaagagc cgaacctcag cggcacacca tgctctgcat gtgttgcaag 1800

tgcgaggcca gaatcgagct ggtggtggaa agcagcgcag acgatctgag agccttccag 1860

cagctgttcc tgaacaccct gagcttcgtc tgcccttggt gcgcttctca gcag 1914

<210> 12

<211> 1602

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atggactgga cctggattct gttcctggtg gctgctgcca ctagagtgca cagccaccag 60

aagagaacag ccatgttcca ggaccctcag gagagcggaa ggaagctgcc acagctctgt 120

accgagctgc agaccaccat ccacgacatc atcctcgagt gcgtctactg caagcagcag 180

ctgctgcgga gagaggtgta cgacttcgcc ttcagggacc tctgcatcgt gtaccgggac 240

ggcaatccct acgccgtggg cgacaagtgc ctgaagttct acagcaagat cagcgagtac 300

cggcactatt gctacagcct gtacggaacc accctggagc agcagtacaa caagcccctc 360

tgcgacctgc tcatccgctg catcaactgc cagaagcccc tctgtcccga ggaaaagcag 420

aggcacctgg acaagaagca gcggttccac aacaccaggg gcaggtggac cggccgctgc 480

atgagttgtt gtaggagcag caggaccaga agagagaccc agctggcagg agcccacgga 540

gatacaccta cactgcacga gtacatgctg gacctgcagc cagagaccac cgatctgtac 600

ggctacggac agctgaacga cagcagcgag gaggaagacg agattgatgg cccagccgga 660

caggcagaac cagatcgggc ccactacaac atcgtcacct tctgctgcaa gtgcgacagc 720

accctgagac tctgcgtgca gtctacccac gtggacatca ggaccctgga ggatctgctg 780

atgggcacac tgggcatcgt ctgccccatt tgtagccaga agccagccgg agccgctaga 840

tttgaggatc ctaccagaag cggctacaag ctgccagacc tgtgtaccga gctgaacacc 900

agcctgcagg acatcgagat cacttgcgtc tactgcaaga ccgtgctgga gctgaccgag 960

gtgttcgaga aggacctgtt cgtggtgtac cgggacagca tccctcacgc cgcttgccac 1020

aagtgtatcg atttctacag ccggatccgg gagctgagac actacagcga cagcgtgtac 1080

ggcgacacac tggagaagct gaccaacacc ggcctgtaca acctgctgat ccgctgcctc 1140

cgctgtcaga aacctctgct gaggcacctg aacgagaaga ggcggttcca caacatcgcc 1200

ggacactaca ggggccagtg ccacagttgt tgcaacaggg ctaggcagga gaggctgcag 1260

agaaggagag agacccaggt ggcaggagct cacggaccta aagctaccct gcaggacatc 1320

gtgctgcatc tggagcctca gaacgagatc cccgtggatc tgctgggaca cggacagctg 1380

tcagacagcg aagaggagaa cgacgagatc gacggcgtga accaccagca tctgccagcc 1440

agaagagccg aacctcagcg gcacaccatg ctctgcatgt gttgcaagtg cgaggccaga 1500

atcgagctgg tggtggaaag cagcgcagac gatctgagag ccttccagca gctgttcctg 1560

aacaccctga gcttcgtctg cccttggtgc gcttctcagc ag 1602

<210> 13

<211> 1923

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgcgcctgc tgatcctggc cctgctgggc atctgcagcc tgaccgccta catcgtggag 60

ggcgtgggca gcgaagtgtc cgacaagagg acttgcgtgt ccctgacaac ccagagactg 120

ccagtgtccc ggatcaagac ctacaccatc accgagggca gcctgagagc cgtgatcttc 180

atcaccaagc ggggcctgaa agtctgtgcc gatcctcagg ctacttgggt cagagacgtc 240

gtgaggagca tggacaggaa gagcaacacc cggaacaaca tgatccagac caagcccacc 300

ggaacacagc agagcaccaa cacagccgtg accctgacag gaggaggagg aggaggaagc 360

ggaggaggag gaggacacca gaagagaaca gccatgttcc aggaccctca ggagagcgga 420

aggaagctgc cacagctctg taccgagctg cagaccacca tccacgacat catcctcgag 480

tgcgtctact gcaagcagca gctgctgcgg agagaggtgt acgacttcgc cttcagggac 540

ctctgcatcg tgtaccggga cggcaatccc tacgccgtgg gcgacaagtg cctgaagttc 600

tacagcaaga tcagcgagta ccggcactat tgctacagcc tgtacggaac caccctggag 660

cagcagtaca acaagcccct ctgcgacctg ctcatccgct gcatcaactg ccagaagccc 720

ctctgtcccg aggaaaagca gaggcacctg gacaagaagc agcggttcca caacaccagg 780

ggcaggtgga ccggccgctg catgagttgt tgtaggagca gcaggaccag aagagagacc 840

cagctggcag gagcccacgg agatacacct acactgcacg agtacatgct ggacctgcag 900

ccagagacca ccgatctgta cggctacgga cagctgaacg acagcagcga ggaggaagac 960

gagattgatg gcccagccgg acaggcagaa ccagatcggg cccactacaa catcgtcacc 1020

ttctgctgca agtgcgacag caccctgaga ctctgcgtgc agtctaccca cgtggacatc 1080

aggaccctgg aggatctgct gatgggcaca ctgggcatcg tctgccccat ttgtagccag 1140

aagccagccg gagccgctag atttgaggat cctaccagaa gcggctacaa gctgccagac 1200

ctgtgtaccg agctgaacac cagcctgcag gacatcgaga tcacttgcgt ctactgcaag 1260

accgtgctgg agctgaccga ggtgttcgag aaggacctgt tcgtggtgta ccgggacagc 1320

atccctcacg ccgcttgcca caagtgtatc gatttctaca gccggatccg ggagctgaga 1380

cactacagcg acagcgtgta cggcgacaca ctggagaagc tgaccaacac cggcctgtac 1440

aacctgctga tccgctgcct ccgctgtcag aaacctctgc tgaggcacct gaacgagaag 1500

aggcggttcc acaacatcgc cggacactac aggggccagt gccacagttg ttgcaacagg 1560

gctaggcagg agaggctgca gagaaggaga gagacccagg tggcaggagc tcacggacct 1620

aaagctaccc tgcaggacat cgtgctgcat ctggagcctc agaacgagat ccccgtggat 1680

ctgctgggac acggacagct gtcagacagc gaagaggaga acgacgagat cgacggcgtg 1740

aaccaccagc atctgccagc cagaagagcc gaacctcagc ggcacaccat gctctgcatg 1800

tgttgcaagt gcgaggccag aatcgagctg gtggtggaaa gcagcgcaga cgatctgaga 1860

gccttccagc agctgttcct gaacaccctg agcttcgtct gcccttggtg cgcttctcag 1920

cag 1923

<210> 14

<211> 1914

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

auggacugga ccuggauucu guuccuggug gcugcugcca cuagagugca cagcgugggc 60

agcgaagugu ccgacaagag gacuugcgug ucccugacaa cccagagacu gccagugucc 120

cggaucaaga ccuacaccau caccgagggc agccugagag ccgugaucuu caucaccaag 180

cggggccuga aagucugugc cgauccucag gcuacuuggg ucagagacgu cgugaggagc 240

auggacagga agagcaacac ccggaacaac augauccaga ccaagcccac cggaacacag 300

cagagcacca acacagccgu gacccugaca ggaggaggag gaggaggaag cggaggagga 360

ggaggacacc agaagagaac agccauguuc caggacccuc aggagagcgg aaggaagcug 420

ccacagcucu guaccgagcu gcagaccacc auccacgaca ucauccucga gugcgucuac 480

ugcaagcagc agcugcugcg gagagaggug uacgacuucg ccuucaggga ccucugcauc 540

guguaccggg acggcaaucc cuacgccgug ggcgacaagu gccugaaguu cuacagcaag 600

aucagcgagu accggcacua uugcuacagc cuguacggaa ccacccugga gcagcaguac 660

aacaagcccc ucugcgaccu gcucauccgc ugcaucaacu gccagaagcc ccucuguccc 720

gaggaaaagc agaggcaccu ggacaagaag cagcgguucc acaacaccag gggcaggugg 780

accggccgcu gcaugaguug uuguaggagc agcaggacca gaagagagac ccagcuggca 840

ggagcccacg gagauacacc uacacugcac gaguacaugc uggaccugca gccagagacc 900

accgaucugu acggcuacgg acagcugaac gacagcagcg aggaggaaga cgagauugau 960

ggcccagccg gacaggcaga accagaucgg gcccacuaca acaucgucac cuucugcugc 1020

aagugcgaca gcacccugag acucugcgug cagucuaccc acguggacau caggacccug 1080

gaggaucugc ugaugggcac acugggcauc gucugcccca uuuguagcca gaagccagcc 1140

ggagccgcua gauuugagga uccuaccaga agcggcuaca agcugccaga ccuguguacc 1200

gagcugaaca ccagccugca ggacaucgag aucacuugcg ucuacugcaa gaccgugcug 1260

gagcugaccg agguguucga gaaggaccug uucguggugu accgggacag caucccucac 1320

gccgcuugcc acaaguguau cgauuucuac agccggaucc gggagcugag acacuacagc 1380

gacagcgugu acggcgacac acuggagaag cugaccaaca ccggccugua caaccugcug 1440

auccgcugcc uccgcuguca gaaaccucug cugaggcacc ugaacgagaa gaggcgguuc 1500

cacaacaucg ccggacacua caggggccag ugccacaguu guugcaacag ggcuaggcag 1560

gagaggcugc agagaaggag agagacccag guggcaggag cucacggacc uaaagcuacc 1620

cugcaggaca ucgugcugca ucuggagccu cagaacgaga uccccgugga ucugcuggga 1680

cacggacagc ugucagacag cgaagaggag aacgacgaga ucgacggcgu gaaccaccag 1740

caucugccag ccagaagagc cgaaccucag cggcacacca ugcucugcau guguugcaag 1800

ugcgaggcca gaaucgagcu ggugguggaa agcagcgcag acgaucugag agccuuccag 1860

cagcuguucc ugaacacccu gagcuucguc ugcccuuggu gcgcuucuca gcag 1914

<210> 15

<211> 1602

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

auggacugga ccuggauucu guuccuggug gcugcugcca cuagagugca cagccaccag 60

aagagaacag ccauguucca ggacccucag gagagcggaa ggaagcugcc acagcucugu 120

accgagcugc agaccaccau ccacgacauc auccucgagu gcgucuacug caagcagcag 180

cugcugcgga gagaggugua cgacuucgcc uucagggacc ucugcaucgu guaccgggac 240

ggcaaucccu acgccguggg cgacaagugc cugaaguucu acagcaagau cagcgaguac 300

cggcacuauu gcuacagccu guacggaacc acccuggagc agcaguacaa caagccccuc 360

ugcgaccugc ucauccgcug caucaacugc cagaagcccc ucugucccga ggaaaagcag 420

aggcaccugg acaagaagca gcgguuccac aacaccaggg gcagguggac cggccgcugc 480

augaguuguu guaggagcag caggaccaga agagagaccc agcuggcagg agcccacgga 540

gauacaccua cacugcacga guacaugcug gaccugcagc cagagaccac cgaucuguac 600

ggcuacggac agcugaacga cagcagcgag gaggaagacg agauugaugg cccagccgga 660

caggcagaac cagaucgggc ccacuacaac aucgucaccu ucugcugcaa gugcgacagc 720

acccugagac ucugcgugca gucuacccac guggacauca ggacccugga ggaucugcug 780

augggcacac ugggcaucgu cugccccauu uguagccaga agccagccgg agccgcuaga 840

uuugaggauc cuaccagaag cggcuacaag cugccagacc uguguaccga gcugaacacc 900

agccugcagg acaucgagau cacuugcguc uacugcaaga ccgugcugga gcugaccgag 960

guguucgaga aggaccuguu cgugguguac cgggacagca ucccucacgc cgcuugccac 1020

aaguguaucg auuucuacag ccggauccgg gagcugagac acuacagcga cagcguguac 1080

ggcgacacac uggagaagcu gaccaacacc ggccuguaca accugcugau ccgcugccuc 1140

cgcugucaga aaccucugcu gaggcaccug aacgagaaga ggcgguucca caacaucgcc 1200

ggacacuaca ggggccagug ccacaguugu ugcaacaggg cuaggcagga gaggcugcag 1260

agaaggagag agacccaggu ggcaggagcu cacggaccua aagcuacccu gcaggacauc 1320

gugcugcauc uggagccuca gaacgagauc cccguggauc ugcugggaca cggacagcug 1380

ucagacagcg aagaggagaa cgacgagauc gacggcguga accaccagca ucugccagcc 1440

agaagagccg aaccucagcg gcacaccaug cucugcaugu guugcaagug cgaggccaga 1500

aucgagcugg ugguggaaag cagcgcagac gaucugagag ccuuccagca gcuguuccug 1560

aacacccuga gcuucgucug cccuuggugc gcuucucagc ag 1602

<210> 16

<211> 1923

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

augcgccugc ugauccuggc ccugcugggc aucugcagcc ugaccgccua caucguggag 60

ggcgugggca gcgaaguguc cgacaagagg acuugcgugu cccugacaac ccagagacug 120

ccaguguccc ggaucaagac cuacaccauc accgagggca gccugagagc cgugaucuuc 180

aucaccaagc ggggccugaa agucugugcc gauccucagg cuacuugggu cagagacguc 240

gugaggagca uggacaggaa gagcaacacc cggaacaaca ugauccagac caagcccacc 300

ggaacacagc agagcaccaa cacagccgug acccugacag gaggaggagg aggaggaagc 360

ggaggaggag gaggacacca gaagagaaca gccauguucc aggacccuca ggagagcgga 420

aggaagcugc cacagcucug uaccgagcug cagaccacca uccacgacau cauccucgag 480

ugcgucuacu gcaagcagca gcugcugcgg agagaggugu acgacuucgc cuucagggac 540

cucugcaucg uguaccggga cggcaauccc uacgccgugg gcgacaagug ccugaaguuc 600

uacagcaaga ucagcgagua ccggcacuau ugcuacagcc uguacggaac cacccuggag 660

cagcaguaca acaagccccu cugcgaccug cucauccgcu gcaucaacug ccagaagccc 720

cucugucccg aggaaaagca gaggcaccug gacaagaagc agcgguucca caacaccagg 780

ggcaggugga ccggccgcug caugaguugu uguaggagca gcaggaccag aagagagacc 840

cagcuggcag gagcccacgg agauacaccu acacugcacg aguacaugcu ggaccugcag 900

ccagagacca ccgaucugua cggcuacgga cagcugaacg acagcagcga ggaggaagac 960

gagauugaug gcccagccgg acaggcagaa ccagaucggg cccacuacaa caucgucacc 1020

uucugcugca agugcgacag cacccugaga cucugcgugc agucuaccca cguggacauc 1080

aggacccugg aggaucugcu gaugggcaca cugggcaucg ucugccccau uuguagccag 1140

aagccagccg gagccgcuag auuugaggau ccuaccagaa gcggcuacaa gcugccagac 1200

cuguguaccg agcugaacac cagccugcag gacaucgaga ucacuugcgu cuacugcaag 1260

accgugcugg agcugaccga gguguucgag aaggaccugu ucguggugua ccgggacagc 1320

aucccucacg ccgcuugcca caaguguauc gauuucuaca gccggauccg ggagcugaga 1380

cacuacagcg acagcgugua cggcgacaca cuggagaagc ugaccaacac cggccuguac 1440

aaccugcuga uccgcugccu ccgcugucag aaaccucugc ugaggcaccu gaacgagaag 1500

aggcgguucc acaacaucgc cggacacuac aggggccagu gccacaguug uugcaacagg 1560

gcuaggcagg agaggcugca gagaaggaga gagacccagg uggcaggagc ucacggaccu 1620

aaagcuaccc ugcaggacau cgugcugcau cuggagccuc agaacgagau ccccguggau 1680

cugcugggac acggacagcu gucagacagc gaagaggaga acgacgagau cgacggcgug 1740

aaccaccagc aucugccagc cagaagagcc gaaccucagc ggcacaccau gcucugcaug 1800

uguugcaagu gcgaggccag aaucgagcug gugguggaaa gcagcgcaga cgaucugaga 1860

gccuuccagc agcuguuccu gaacacccug agcuucgucu gcccuuggug cgcuucucag 1920

cag 1923

<210> 17

<211> 1017

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca 60

ttaccgccat gttgacattg attattgact agttattaat agtaatcaat tacggggtca 120

ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 180

ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 240

acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 300

ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 360

aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 420

tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 480

gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 540

gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 600

ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 660

ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 720

caccgggacc gatccagcct ccatcggctc gcatctctcc ttcacgcgcc cgccgcccta 780

cctgaggccg ccatccacgc cggttgagtc gcgttctgcc gcctcccgcc tgtggtgcct 840

cctgaactgc gtccgccgtc taggtaagtt taaagctcag gtcgagaccg ggcctttgtc 900

cggcgctccc ttggagccta cctagactca gccggctctc cacgctttgc ctgaccctgc 960

ttgctcaact ctactctggc taactagaga acccactgct tactggctta tcgaaat 1017

<210> 18

<211> 3388

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gactcttcgc gatgtacggg ccagatatac gcgttccatt gcatacgttg tatccatatc 60

ataatatgta catttatatt ggctcatgtc caacattacc gccatgttga cattgattat 120

tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 180

tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 240

cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 300

gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 360

tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 420

agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 480

ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac 540

ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 600

aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc 660

gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga 720

gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccatc 780

ggctcgcatc tctccttcac gcgcccgccg ccctacctga ggccgccatc cacgccggtt 840

gagtcgcgtt ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg ccgtctaggt 900

aagtttaaag ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga gcctacctag 960

actcagccgg ctctccacgc tttgcctgac cctgcttgct caactctact ctggctaact 1020

agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa 1080

gctggctagc gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt 1140

ggaattctgc agatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac 1200

ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 1260

cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 1320

aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 1380

cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 1440

ggcttctact gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc 1500

tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttctcgcc gccaaggatc 1560

tgatggcgca ggggatcaag ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt 1620

gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat 1680

gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag 1740

gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac 1800

gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac 1860

gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc 1920

ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg 1980

ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag 2040

cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat 2100

caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag 2160

gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc 2220

ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg 2280

ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg 2340

ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag 2400

ttcttctgaa ttattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg 2460

tgcggtattt cacaccgcat acaggtggca cttttcgggg aaatgtgcgc ggaaccccta 2520

tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 2580

aaatgcttca ataatagcac gtgctaaaac ttcattttta atttaaaagg atctaggtga 2640

agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 2700

cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 2760

tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 2820

agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 2880

tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 2940

acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 3000

ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 3060

gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 3120

gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 3180

gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 3240

tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 3300

caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctgggct 3360

tttgctggcc ttttgctcac atgttctt 3388

Claims

1. A fusion protein, which is a protein derived from a human,

it includes: signal peptides and HPV viral antigen proteins;

wherein the content of the first and second substances,

the signal peptide is XCL1 signal peptide of an amino acid sequence shown in SEQ ID NO. 7 or IgE signal peptide of an amino acid sequence shown in SEQ ID NO. 5;

the amino acid sequence of the XCL1 chemokine is shown as SEQ ID NO. 6;

the HPV virus antigen protein is selected from at least one of the following proteins:

4, and the HPV18 virus E7 protein.

2. The fusion protein of claim 1, wherein the amino acid sequence is as set forth in any one of SEQ ID NOs 8-10.

3. A nucleic acid encoding the fusion protein of claim 1 or 2.

4. The nucleic acid of claim 3, wherein the nucleotide sequence is as shown in any one of SEQ ID NOS 11 to 13 or 14 to 16.

5. A recombinant vector comprising a backbone vector and the nucleic acid of claim 3 or 4.

6. The recombinant vector according to claim 5, wherein the backbone vector is selected from the group consisting of pVAX1 series vectors and pVR series vectors.

7. A recombinant host transformed or transfected with the recombinant vector of any one of claims 5-6.

8. The recombinant host according to claim 7, wherein the host cell is a bacterial or mammalian cell.

9. The recombinant host according to claim 8, wherein the bacterium is Escherichia coli; the mammalian cell HEK293T cell.

10. The method for producing the fusion protein according to claim 1 or 2, wherein a culture containing the fusion protein is obtained by culturing the recombinant host according to any one of claims 7 to 9.

11. The method for producing a nucleic acid according to claim 3 or 4, wherein a culture containing the nucleic acid is obtained by culturing the recombinant host according to any one of claims 7 to 9.

12. Use of the fusion protein according to claim 1 or 2, the nucleic acid according to claim 3 or 4, the recombinant vector according to claim 5 or 6, the recombinant host according to any one of claims 7 to 9, the fusion protein produced by the production method according to claim 10, and/or the nucleic acid produced by the production method according to claim 11 for the production of a vaccine; the vaccine is used for preventing and treating diseases caused by HPV virus infection.

13. A vaccine for the prevention and treatment of diseases caused by HPV viral infection, comprising: the fusion protein according to claim 1 or 2, the nucleic acid according to claim 3 or 4, the recombinant vector according to claim 5 or 6, the fusion protein produced by the production method according to claim 10, and/or the nucleic acid produced by the production method according to claim 11.