CN110564751A

CN110564751A - Design and application of micro-ring DNA vaccine

Info

Publication number: CN110564751A
Application number: CN201910825875.8A
Authority: CN
Inventors: 谌平; 谢亦武; 陈志英
Original assignee: Shenzhen New Connaught Micro Ring Biological Technology Co Ltd
Current assignee: Shenzhen New Connaught Micro Ring Biological Technology Co Ltd
Priority date: 2019-09-03
Filing date: 2019-09-03
Publication date: 2019-12-13
Also published as: WO2021042947A1

Abstract

The application relates to design and application of a micro-ring DNA vaccine. In particular to a micro-ring DNA carrier for expressing a specific antigen or antigen fragment of pathogenic microorganism in vivo. The micro-ring DNA vector can mediate the high-efficiency expression of antigen protein in vivo, enhance the immunogenicity of DNA vaccine and simultaneously avoid the safety problem brought by resistance genes. The micro-ring DNA vector can be used for preventing and/or treating common infectious diseases and related cancers, and has the advantages of stronger antigen expression and higher safety.

Description

design and application of micro-ring DNA vaccine

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a Minicircle (MC) DNA vector, and more particularly relates to design and application of a Minicircle DNA vaccine.

Background

Vaccines are biological agents that stimulate the body to effectively produce adaptive immunity to a particular disease, and are generally composed of inactivated or attenuated pathogens, toxins, pathogenic proteins or polypeptides, and the like. The closest technology to the present invention is a plasmid-based DNA vaccine. With the development of immunology, researches show that the recombinant plasmid carrying the antigen coding gene is directly introduced into human or animal bodies, and the host cells are utilized to synthesize antigen protein, so that the body can be induced to generate antigen specific humoral and cellular immune response reactions, thereby preventing or treating diseases.

the DNA vaccine has distinct advantages and has more and more preclinical and clinical research reports. However, to date, there is no human DNA vaccine product approved for marketing. Compared with the traditional vaccine, the DNA vaccine has lower immunogenicity and is difficult to stimulate enough effective immune response. In addition, the plasmid carries a resistance screening gene, and thus the potential safety hazard such as resistance gene spreading is not negligible.

DNA itself is not or very little immunogenic. The source of immunogenicity of a DNA vaccine essentially refers to the encoded antigenic protein. The low transfection efficiency in plasmid body and limited antigen protein produced in host body are the root causes of low immunogenicity of DNA vaccine. Because the micro-ring DNA removes the prokaryotic skeleton DNA component in the plasmid, the vector is greatly reduced, and the transfection in vivo is facilitated. More importantly, due to the elimination of the "transgene silencing effect", the expression of the minicircle DNA in vivo is much higher than that of the plasmid.

The invention uses micro-ring (MC) DNA carrier to code antigen gene, which can mediate the high-efficiency expression of antigen protein in vivo, enhance the immunogenicity of DNA vaccine, and avoid the safety problem caused by resistance gene.

Disclosure of Invention

The present invention relates to a Minicircle (MC) DNA vector, and uses the Minicircle DNA vector to express antigen or antigen fragment in vivo, and stimulates the body to produce specific humoral immunity (antibody) and cellular immune response, thereby preventing or treating infectious diseases and related cancers.

The invention provides a recombinant gene vector for expressing an antigen or an antigen fragment, wherein the recombinant gene vector comprises a coding gene of the antigen or the antigen fragment.

In one aspect, the antigen is selected from a pathogenic microorganism specific antigen.

In one aspect, the recombinant gene vector is selected from a non-viral vector or a viral vector. Preferably, the non-viral vector is selected from a standard plasmid or other circular expression cassette, or the viral vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector and an adeno-associated viral vector.

In one aspect, the pathogenic microorganism-specific Antigen or Antigen fragment is selected from a bacillus anthracis Protective Antigen (PA), a Human Papilloma Virus (HPV) early protein (E6 or E7) Antigen, an HPV late protein (L1 or L2) Antigen, the non-viral vector is selected from a minicircle DNA vector, or the viral vector is selected from an adeno-associated viral vector.

In one aspect, the amino acid sequence of the PA antigen of bacillus anthracis is as set forth in SEQ ID NO: 2 is shown in the specification; the HPV early protein antigen is selected from HPV E6/E7 chimeric protein antigen, and the amino acid sequence of the HPV early protein antigen is shown as SEQ ID NO: 3 is shown in the specification; the HPV late protein antigen is selected from (1) HPV L1 protein antigen, and the amino acid sequence of the HPV late protein antigen is shown as SEQ ID NO: 4, (2) HPV L2 protein antigen, the amino acid sequence of which is shown as SEQ ID NO: 5, or (3) HPV L1/L2 chimeric protein antigen, wherein the amino acid sequence of the antigen is shown as SEQ ID NO: and 6. Since the HPV E6 and E7 genes are oncogenes, the complete E6, E7 cannot be directly designed into vaccines, requiring the misplacement of E6, E7 fragments (see fig. 2) to destroy their oncogenicity but retain their immunogenicity.

in one aspect, the bacillus anthracis PA antigen has a sequence identical to SEQ ID NO: 2, and has the same function with the previous bacillus anthracis PA antigen; the HPV E6/E7 chimeric protein antigen has the amino acid sequence similar to that of SEQ ID NO: 3, and has the same function with the prior HPV E6/E7 chimeric protein antigen; the HPV L1 protein antigen has a sequence similar to that of SEQ ID NO: 4, and has the same function with the previous HPV L1 protein antigen; the HPV L2 protein antigen has a sequence similar to that of SEQ ID NO: 5, and has the same function with the previous HPV L2 protein antigen; the HPV L1/L2 chimeric protein antigen has the amino acid sequence similar to that of SEQ ID NO: 6, and has the same function with the prior HPV L1/L2 chimeric protein antigen.

In one aspect, the nucleotide sequence of the gene encoding the PA antigen of bacillus anthracis is as set forth in SEQ ID NO: 7 is shown in the specification; the nucleotide sequence of the HPV E6/E7 chimeric protein antigen encoding gene is shown as SEQ ID NO: 8 is shown in the specification; the nucleotide sequence of the HPV L1 protein antigen encoding gene is shown as SEQ ID NO: 9 is shown in the figure; the nucleotide sequence of the HPV L2 protein antigen encoding gene is shown as SEQ ID NO: 10 is shown in the figure; the nucleotide sequence of the HPV L1/L2 chimeric protein antigen encoding gene is shown as SEQ ID NO: shown at 11.

In one aspect, the gene encoding the PA antigen of bacillus anthracis has a nucleotide sequence identical to SEQ ID NO: 7, and the encoded PA antigen of the bacillus anthracis has the same function as the PA antigen of the prior bacillus anthracis; the HPV E6/E7 chimeric protein antigen encoding gene has the sequence similar to that of SEQ ID NO: 8, and the encoded HPV E6/E7 chimeric protein antigen has the same functions as the prior HPV E6/E7 chimeric protein antigen; the HPV L1 protein antigen encoding gene has the sequence similar to that of SEQ ID NO: 9, and the encoded HPV L1 protein antigen has the same function with the prior HPV L1 protein antigen; the HPV L2 protein antigen encoding gene has the sequence similar to that of SEQ ID NO: 10, and the encoded HPV L2 protein antigen has the same function with the prior HPV L1 protein antigen; the HPV L1/L2 chimeric protein antigen encoding gene has the sequence similar to that of SEQ ID NO: 11, and the encoded HPV L1/L2 chimeric protein antigen has the same functions as the prior HPV L1/L2 chimeric protein antigen.

The application also provides a preparation method of the recombinant gene vector, which comprises the following specific steps:

(1) Respectively obtaining the coding gene sequences of the antigens from the prior art;

(2) Constructing a recombinant gene vector expressing the antigen according to the sequence in the step (1);

(3) Identifying the expression level of the recombinant gene vector in vitro;

Optionally, the step of (a) is carried out,

(4) Immunizing an animal and detecting the level of specific antibody and T cell immunity elicited;

The recombinant gene vector contains a coding gene of the antigen.

In one aspect, the antigen is selected from a pathogenic microorganism specific antigen or antigen fragment.

In one aspect, the pathogenic microorganism-specific antigen is selected from a PA antigen of bacillus anthracis, an HPV early protein (E6 or E7) antigen, an HPV late protein (L1 or L2) antigen, the non-viral vector is selected from a minicircle DNA vector, or the viral vector is selected from an adeno-associated viral vector.

in one aspect, the HPV early protein antigen is selected from the group consisting of an HPV E6/E7 chimeric protein antigen; the HPV late protein antigen is selected from (1) HPV L1 protein antigen, (2) HPV L2 protein antigen or (3) HPV L1/L2 chimeric protein antigen.

The present application also provides a host cell comprising the recombinant gene vector described previously.

In one aspect, the host cell comprises a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

The application also provides a pharmaceutical composition, which comprises the recombinant gene vector and a pharmaceutically acceptable carrier.

In one aspect, the pharmaceutical composition may be formulated into a pharmaceutical formulation according to conventional methods. In the preparation process, the recombinant gene vector is preferably mixed with a pharmaceutically acceptable carrier or diluted with a carrier. When the carrier serves as a diluent, it may be solid, semi-solid or liquid. The preparation is selected from tablet, pill, powder, capsule, suspension, emulsion, solution, aerosol, injectable solution, etc. Suitable carriers, excipients or diluents include water, lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, polyvinylpyrrolidone, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil and the like. The formulation may also include fillers, anticoagulants, lubricants, humectants, flavoring agents, emulsifiers, preservatives, and the like.

The application also provides the use of the recombinant gene vector, the host cell, or the pharmaceutical composition in the preparation of a medicament for treating diseases.

In one aspect, the disease is selected from an infectious disease or cancer. Preferably, the infectious disease is selected from bacillus anthracis infection or HPV infection, or the cancer is selected from cancer related to pathogenic microorganism, such as cervical cancer, liver cancer, nasopharyngeal cancer and the like.

The positive effects of the invention include: the micro-ring DNA carrier can mediate the high-efficiency expression of the antigen protein in vivo, enhance the immunogenicity of the DNA vaccine and simultaneously avoid the safety problem brought by the resistance gene.

Drawings

FIG. 1: anthrax bacillus PA antigen micro-ring DNA carrier map.

FIG. 2: HPV E6/E7 chimeric protein antigen micro-ring DNA vector map.

FIG. 3: HPV L1/L2 chimeric protein antigen micro-ring DNA vector map.

FIG. 4: the expression level of the bacillus anthracis PA antigen micro-loop DNA vector (MC.PA) detected by Western Blot is obviously higher than that of a plasmid (PL.PA).

FIG. 5: the bacillus anthracis PA antigen micro-ring DNA vector vaccine is used for immunizing a mouse and inducing the mouse to generate a PA antigen specific IgG antibody, and the level of the antibody is equivalent to that of a protein vaccine plus an aluminum adjuvant.

Detailed Description

example 1 micro-circular DNA Master plasmid construction

1) Synthesizing a full-length target gene DNA fragment, wherein the target gene is selected from coding gene sequences such as bacillus anthracis PA, HPV E6/E7 chimeric protein, HPV L1 protein, HPV L2 protein or HPV L1/L2 chimeric protein (such as SEQ ID NO: shown at 7-11).

2) The target gene fragment is subcloned between attB and attP sites of the micro-ring DNA empty vector to construct a micro-ring DNA mother plasmid. The minicircle DNA mother plasmid contains attB and attP recombination sites, 32 tandem repeats of an I-SceI cleavage site (I-SceI 32), a kana resistance gene, an SV40 DNA nuclear targeting sequence (DNA nuclear targeting sequence, DTS), a CMV promoter/enhancer, a chimeric intron (chimeric intron), a target gene, and a bovine growth factor (bGH) poly A signal.

Example 2 micro-Loop DNA preparation

1) minicircle DNA mother plasmid transformed genetically engineered E coli bacterium ZYCY10P3S2T (Nature Biotechnology 2010,28: 1287-9).

2) ZYCY10P3S2T containing minicircle mother plasmid is inoculated into TB culture medium containing kana, and shake culture is carried out at 37 ℃ for 12-16h, and a large amount of minicircle DNA mother plasmid is produced by thalli.

3) Adding an induction medium containing arabinose, and under the induction of the arabinose (shaking culture at 32 ℃ for 8h), ZYCY10P3S2T expresses a phi C31 recombinase and an endonuclease for recognizing an I-SceI site. The minicircle DNA mother plasmid undergoes DNA recombination at attB/attP recombination sites under the action of Φ C31 recombinase, forming two small circular DNA molecules: i) micro-ring DNA (only containing a target gene expression frame and 36-bp attR site); ii) a small loop consisting of plasmid backbone DNA (containing an I-SceI cleavage site). The small ring formed by the plasmid skeleton DNA and the non-recombined residual minicircle DNA mother plasmid are linearized under the action of I-SceI endonuclease, and then degraded by DNase, and only minicircle DNA is left in ZYCY10P3S2T bacteria.

4) the minicircle DNA was extracted using a plasmid extraction kit.

Example 3 Western Blot detection of protein (Bacillus anthracis PA antigen) expression

1. experimental procedure

1) preparing a protein sample: the micro-ring DNA or the equimolar micro-ring DNA mother plasmid is transfected into 293T cells by using a liposome transfection reagent (such as Lipofectamine 2000), a serum-free cell culture medium is replaced, and 293T cell culture supernatants are respectively collected after three days of culture in the serum-free medium.

2) Electrophoresis: adding a proper amount of loading buffer solution into a protein sample, heating for 3-5 minutes in boiling water to denature the protein, cooling, loading the protein into an SDS-PAGE gel loading hole, and performing electrophoresis at 80-100V for 1 hour.

3) Film transfer: proteins were transferred from SDS-PAGE gels to PVDF membranes using a wet transfer apparatus (Bio-Rad, USA) at 300mA for 1 hour.

4) And (3) sealing: after the PVDF membrane is cleaned, Western Blot blocking solution is added for blocking.

5) Primary antibody incubation: diluted anthrax PA antibody was added and incubated for 1 hour at room temperature.

6) And (3) secondary antibody incubation: diluted horseradish peroxidase (HRP) -labeled secondary antibody was added and incubated at room temperature for 1 hour.

7) Color development: proteins were detected using ECL chemiluminescence reagent (Cell Signaling, usa).

2. Results of the experiment

The WB results of fig. 4 illustrate: the minicircle mother plasmid (PL.PA) and the minicircle (MC.PA) can normally express the PA antigen of the bacillus anthracis, but the expression level of the equimolar MC.PA is obviously higher than that of the PL.PA. Blank: blank control; PA: PA protein positive control.

example 4 Bacillus anthracis PA antigen minicircle DNA (MC.PA) mouse immunization experiment

1. Mice were grouped (5 per group; N ═ 5) as follows:

Grouping	Immunization regimen
		Blank control group (N ═ 5)	intramuscular injection of PBS
Experimental group 1(N ═ 5)	Intramuscular injection of 20 μ g MC.PA
		Experimental group 2(N ═ 5)	Intramuscular injection of 20. mu.g MC.PA + EP (electrotransfer; 10ms)
Positive control group (N ═ 5)	0.5 μ gPA antigen (protein) +50 μ gAl (OH)₃(adjuvant)

2. After mice were immunized according to the group protocol, blood was collected periodically every 4 weeks (days 28, 56, 84), and the levels of anthrax PA-specific antibodies in the mouse serum were measured by ELISA.

Figure 5 the results of the mouse immunizations demonstrate: the intramuscular injection of MC.PA minicircle DNA (+ EP) can induce the organism to produce a large amount of Bacillus anthracis PA specific antibody, the antibody level is equivalent to that of a protein vaccine (+ aluminum hydroxide adjuvant), and the antibody types are mainly IgG1 and IgG2a subtypes.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be considered to be equivalent or modified within the technical scope of the present invention.

Sequence listing

SEQ ID NO: tissue-type plasminogen activator (tPA) signal peptide 1

MDAMKRGLCCVLLLCGAVFVSPS

SEQ ID NO: 2 Anthrax bacterium PA antigen (underlined: tPA signal peptide)

MDAMKRGLCCVLLLCGAVFVSNSEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG

SEQ ID NO: 3HPV E6/E7 chimeric protein antigen (underlined: tPA signal peptide)

MDAMKRGLCCVLLLCGAVFVSPSMHQKRTAMFQDPQERPRKLPHLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRYMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLDKCLKFYSKISEYRYYCYSVYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPMARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRYYSDSVMYGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCFYSRIRELRYYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHKIAGHYRGQCHSCCNRARQERLQRRRETQVARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLSTLSFVCPWCASQQ

SEQ ID NO: 4HPV L1 protein antigen (underlined: tPA signal peptide)

MDAMKRGLCCVLLLCGAVFVSPSMSLWLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNNKILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGVGISGHPLLNKLDDTENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDDLYIKGSGSTANLASSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADVMTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTTAKRKKRKL

SEQ ID NO: 5HPV L2 protein antigen (underlined: tPA signal peptide)

MDAMKRGLCCVLLLCGAVFVSPSMKRASATQLYKTCKQAGTCPPDIISKVEGKTIADQILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLGTRPPTATDTLAPVRPPLTVDPVGPSDPSIVSLVEETSFIDAGAPTSVPSIPPDVSGFSITTSTDTTPAILDINNTVTTVTTHNNPTFTDPSVLQPPTPAETGGHFTLSSSTISTHNYEEIPMDT

SEQ ID NO: 6HPV L1/L2 chimeric protein antigen (underlined: tPA signal peptide; double wavy line: HPV L1; double transverse line: HPV L211-200)

MDAMKRGLCCVLLLCGAVFVSPS

SEQ ID NO: 7 anthrax bacillus PA antigen coding gene

atggatgcaatgaagagagggctctgctgtgtgctgctgctgtgtggagcagtcttcgtttcgaacagcgaggtgaagcaggagaacagactgctgaatgaatcggagtcatcgagtcaggggttgctcggttactacttctccgatctcaatttccaggcccccatggtagtcacttcctccacaacaggggatctgagtatcccctcctcggagctggagaacatcccttccgaaaaccagtatttccagagcgcaatctggtccggcttcatcaaggtaaagaaatccgacgagtataccttcgccacttccgcagataaccatgtcaccatgtgggtggacgaccaggaggtcatcaacaaggcctccaactctaacaagatccgcctcgagaaaggccgtttgtaccagattaagatccagtaccagagagaaaaccctactgagaagggcctcgacttcaagctgtactggaccgactcgcagaacaagaaggaagtgatcagttccgataacctgcagctgccggaattgaagcagaagtcatccaacagtcgcaagaagcgctcgacaagtgccggtcctaccgtgccagatcgggacaacgacggcatccccgattctctcgaggtggaggggtacaccgtggatgtgaaaaacaagaggaccttcctctccccatggatctcgaacatccacgagaagaaggggctgaccaagtacaaatcgtcccccgagaagtggagtacggcttccgacccctactcggatttcgagaaggtgaccggcagaattgataagaacgtgtcgcccgaggcccgccatcccctcgtcgccgcctacccgattgtgcacgtcgatatggaaaacatcatcttaagtaagaacgaggaccagtccacccagaacaccgacagccagacccgtactatctccaagaatactagcacatccagaacccatacgtcggaagtgcacggcaacgccgaggtgcacgcaagtttctttgatattggcggctctgtgtcggccggattcagtaactcaaactcgagcaccgtcgctatcgatcacagcctgtcgttggcgggcgagcgaacatgggcggaaaccatgggcctcaataccgccgacaccgcccgactgaacgctaacatccgatacgtgaacacgggtaccgcccctatctacaacgtgctgcccaccacgtccctggtcctcggaaaaaatcagaccttggcgaccatcaaggccaaggagaaccagctatcccagatcctcgcccccaacaactactacccctcgaagaacctggcccccattgccctcaacgcgcaggacgacttcagcagtactcccattactatgaactacaaccagttcctcgagctggaaaagaccaagcagttgcgcctggacacagaccaggtgtacgggaatatcgccacatacaacttcgagaacggtcgcgtgcgggtggataccggctcgaactggtccgaggtcttaccccaaatccaggagaccacagccaggatcatcttcaacggcaaggatctgaacctcgtcgagcgccgcatcgcggcggtgaacccctctgaccctcttgagacgaccaagcctgatatgacgctgaaggaggcgctgaagatcgctttcgggttcaacgagcccaacggcaacctccagtaccaggggaaggacataactgagttcgacttcaatttcgatcagcagacatcccagaatatcaagaaccagctcgccgagctgaacgcaaccaacatttacaccgtgctggataagatcaagctgaacgccaagatgaacatcctgatccgcgacaaaaggttccactatgaccggaacaacatcgctgtgggcgccgatgagtccgtcgtcaaggaagcccatcgagaagtgatcaactcatcgaccgaggggctccttctgaacatcgacaaggatatcagaaagatcctctccgggtacatcgtggagatcgaggataccgagggcctgaaggaagtcattaacgaccgctacgatatgctgaacatctcgtcgctacgccaggacggcaagacctttatcgacttcaagaagtacaacgacaaactgccactctacatctctaacccaaactacaaagtgaatgtgtacgccgtcacgaaggagaacactatcatcaatccctctgagaacggtgacacctcgaccaacgggatcaaaaagatcctcattttctcgaagaagggctacgaaatcgggtaa

SEQ ID NO: 8HPV E6/E7 chimeric protein antigen encoding gene

atggatgctatgaaacggggcctgtgctgcgtgctgctcctgtgcggcgctgtgtttgtgagccctagcatgcaccagaagagaaccgccatgttccaggaccctcaggagagacctaggaagctgcctcacctgtgtacagagctccagacaaccatccacgacatcatcctggagtgcgtgtactgtaagcagcagctgctgagaagagaggtgtacgacttcgccttcagagacctgtgcatcgtgtacagagacggcaacccttacgccgtgtgcgataagtgtctgaagttctattccaaaatctccgaatataggtacatgcacggcgacacccctaccctgcacgagtacatgctggacctccagcctgagaccacagacctgtactgctacgagcagctgaacgacagctctgaggaagaggacgagattgacggacctgctggccaggccgagcctgacagagcccactacaatatcgtgacattctgttgcaaatgcgactccacactggacaagtgcctgaagttctacagcaagatctctgagtacagatactactgctactctgtgtacggcaccacactggagcagcagtacaacaagcctctgtgcgacctcctgatccgctgcatcaactgccagaagcctctgtgccctgaggagaagcagagacacctggacaagaagcagcggttccacaacatcagaggcagatggaccggcaggtgcatgtcctgctgtagatcctccagaaccagacgggagacccagctgcactacaacatcgtgaccttctgctgcaagtgcgactctaccctgagactgtgcgtgcagtctacccacgtggacatcagaaccctggaggacctgctgatgggcaccctgggcatcgtgtgccctatctgctctcagaagcctatggccaggttcgaggaccctaccagaagaccctacaagctgcctgacctgtgcaccgagctgaacacctctctgcaagacatcgagatcacctgcgtgtactgcaagaccgtgctggagctgaccgaggtgttcgagttcgccttcaaggacctgttcgtggtgtacagagacagcatccctcacgctgcctgccacaagtgcatcgacttctattccaggatcagggagctgcgctattactccgactctgtgatgtacggccccaaggccaccctccaggacatcgtgctgcacctggagcctcagaacgagatccccgtggacctgctgtgccacgagcagctgtctgactctgaagaggagaacgacgagatcgacggcgtgaaccaccagcacctgcctgccaggagagctgaaccccagcggcataccatgctgtgtatgtgcttctactctaggatcagagagctgaggtactactctgactctgtgtacggcgacaccctggagaagctgaccaacaccggcctgtacaacctgctgatccggtgcctgaggtgccagaagcctctgaaccctgccgagaagctgagacacctgaacgagaagagaagattccacaagatcgctggccactacagaggccagtgccactcttgctgcaacagagccagacaggagagactccagcggagaagggagacccaggtggccagaagagccgagcctcagagacacaccatgctgtgcatgtgctgcaagtgcgaggccagaatcgagctggtggtggagagctctgccgacgacctgagagccttccagcagctgttcctgtctaccctgagcttcgtgtgcccttggtgcgcctctcagcagtag

SEQ ID NO: 9HPV L1 protein antigen encoding gene

atggatgctatgaaacggggcctgtgctgcgtgctgctcctgtgcggcgctgtgtttgtgagccctagcatgagcctgtggctgcccagcgaggccaccgtgtacctgccccccgtgcccgtgagcaaggtggtgagcaccgacgagtacgtggccaggaccaacatctactaccacgccggcaccagcaggctgctggccgtgggccacccctacttccccatcaagaagcccaacaacaacaagatcctggtgcccaaggtgagcggcctgcagtacagggtgttcaggatccacctgcccgaccccaacaagttcggcttccccgacaccagcttctacaaccccgacacccagaggctggtgtgggcctgcgtgggcgtggaggtgggcaggggccagcccctgggcgtgggcatcagcggccaccccctgctgaacaagctggacgacaccgagaacgccagcgcctacgccgccaacgccggcgtggacaacagggagtgcatcagcatggactacaagcagacccagctgtgcctgatcggctgcaagccccccatcggcgagcactggggcaagggcagcccctgcaccaacgtggccgtgaaccccggcgactgcccccccctggagctgatcaacaccgtgatccaggacggcgacatggtggacaccggcttcggcgccatggacttcaccaccctgcaggccaacaagagcgaggtgcccctggacatctgcaccagcatctgcaagtaccccgactacatcaagatggtgagcgagccctacggcgacagcctgttcttctacctgaggagggagcagatgttcgtgaggcacctgttcaacagggccggcgccgtgggcgagaacgtgcccgacgacctgtacatcaagggcagcggcagcaccgccaacctggccagcagcaactacttccccacccccagcggcagcatggtgaccagcgacgcccagatcttcaacaagccctactggctgcagagggcccagggccacaacaacggcatctgctggggcaaccagctgttcgtgaccgtggtggacaccaccaggagcaccaacatgagcctgtgcgccgccatcagcaccagcgagaccacctacaagaacaccaacttcaaggagtacctgaggcacggcgaggagtacgacctgcagttcatcttccagctgtgcaagatcaccctgaccgccgacgtgatgacctacatccacagcatgaacagcaccatcctggaggactggaacttcggcctgcagcccccccccggcggcaccctggaggacacctacaggttcgtgaccagccaggccatcgcctgccagaagcacaccccccccgcccccaaggaggaccccctgaagaagtacaccttctgggaggtgaacctgaaggagaagttcagcgccgacctggaccagttccccctgggcaggaagttcctgctgcaggccggcctgaaggccaagcccaagttcaccctgggcaagaggaaggccacccccaccaccagcagcaccagcaccaccgccaagaggaagaagaggaagctgTAG

SEQ ID NO: 10HPV L2 protein antigen encoding gene

atggatgctatgaaacggggcctgtgctgcgtgctgctcctgtgcggcgctgtgtttgtgagccctagcatgaagagggccagcgccacccagctgtacaagacctgcaagcaggccggcacctgcccccccgacatcatctccaaggtggagggcaagaccatcgccgaccagatcctgcagtacggcagcatgggcgtgttcttcggcggcctgggcatcggcaccggcagcggcaccggcggcaggaccggctacatccccctgggcaccaggccccccaccgccaccgacaccctggcccccgtgaggccccccctgaccgtggaccccgtgggccccagcgaccccagcatcgtgagcctggtggaggagaccagcttcatcgacgccggcgcccccaccagcgtgcccagcatcccccccgacgtgagcggcttcagcatcaccaccagcaccgacaccacccccgccatcctggacatcaacaacaccgtgaccaccgtgaccacccacaacaaccccaccttcaccgaccccagcgtgctgcagccccccacccccgccgagaccggcggccacttcaccctgagcagcagcaccatcagcacccacaactacgaggagatccccatggacaccTAG

SEQ ID NO: 11HPV L1/L2 chimeric protein antigen encoding gene

atggatgctatgaaacggggcctgtgctgcgtgctgctcctgtgcggcgctgtgtttgtgagccctagcatgagcctgtggctgcccagcgaggccaccgtgtacctgccccccgtgcccgtgagcaaggtggtgagcaccgacgagtacgtggccaggaccaacatctactaccacgccggcaccagcaggctgctggccgtgggccacccctacttccccatcaagaagcccaacaacaacaagatcctggtgcccaaggtgagcggcctgcagtacagggtgttcaggatccacctgcccgaccccaacaagttcggcttccccgacaccagcttctacaaccccgacacccagaggctggtgtgggcctgcgtgggcgtggaggtgggcaggggccagcccctgggcgtgggcatcagcggccaccccctgctgaacaagctggacgacaccgagaacgccagcgcctacgccgccaacgccggcgtggacaacagggagtgcatcagcatggactacaagcagacccagctgtgcctgatcggctgcaagccccccatcggcgagcactggggcaagggcagcccctgcaccaacgtggccgtgaaccccggcgactgcccccccctggagctgatcaacaccgtgatccaggacggcgacatggtggacaccggcttcggcgccatggacttcaccaccctgcaggccaacaagagcgaggtgcccctggacatctgcaccagcatctgcaagtaccccgactacatcaagatggtgagcgagccctacggcgacagcctgttcttctacctgaggagggagcagatgttcgtgaggcacctgttcaacagggccggcgccgtgggcgagaacgtgcccgacgacctgtacatcaagggcagcggcagcaccgccaacctggccagcagcaactacttccccacccccagcggcagcatggtgaccagcgacgcccagatcttcaacaagccctactggctgcagagggcccagggccacaacaacggcatctgctggggcaaccagctgttcgtgaccgtggtggacaccaccaggagcaccaacatgagcctgtgcgccgccatcagcaccagcgagaccacctacaagaacaccaacttcaaggagtacctgaggcacggcgaggagtacgacctgcagttcatcttccagctgtgcaagatcaccctgaccgccgacgtgatgacctacatccacagcatgaacagcaccatcctggaggactggaacttcggcctgcagcccccccccggcggcaccctggaggacacctacaggttcgtgaccagccaggccatcgcctgccagaagcacaccccccccgcccccaaggaggaccccctgaagaagtacaccttctgggaggtgaacctgaaggagaagttcagcgccgacctggaccagttccccctgggcaggaagttcctgctgcaggccggcctgaaggccaagcccaagttcaccctgggcaagaggaaggccacccccaccaccagcagcaccagcaccaccgccaagaggaagaagaggaagctgatgaagagggccagcgccacccagctgtacaagacctgcaagcaggccggcacctgcccccccgacatcatctccaaggtggagggcaagaccatcgccgaccagatcctgcagtacggcagcatgggcgtgttcttcggcggcctgggcatcggcaccggcagcggcaccggcggcaggaccggctacatccccctgggcaccaggccccccaccgccaccgacaccctggcccccgtgaggccccccctgaccgtggaccccgtgggccccagcgaccccagcatcgtgagcctggtggaggagaccagcttcatcgacgccggcgcccccaccagcgtgcccagcatcccccccgacgtgagcggcttcagcatcaccaccagcaccgacaccacccccgccatcctggacatcaacaacaccgtgaccaccgtgaccacccacaacaaccccaccttcaccgaccccagcgtgctgcagccccccacccccgccgagaccggcggccacttcaccctgagcagcagcaccatcagcacccacaactacgaggagatccccatggacaccTAG

SEQUENCE LISTING

<110> Shenzhen Xinnuo micro-ring Biotech Limited

<120> design and application of micro-ring DNA vaccine

<130> CP11901189C

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 23

<212> PRT

<213> tissue-type plasminogen activator (tPA) signal peptide

<400> 1

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser

20

<210> 2

<211> 758

<212> PRT

<213> Bacillus anthracis PA antigen (underlined: tPA signal peptide)

<400> 2

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Asn Ser Glu Val Lys Gln Glu Asn Arg Leu Leu

20 25 30

Asn Glu Ser Glu Ser Ser Ser Gln Gly Leu Leu Gly Tyr Tyr Phe Ser

35 40 45

Asp Leu Asn Phe Gln Ala Pro Met Val Val Thr Ser Ser Thr Thr Gly

50 55 60

Asp Leu Ser Ile Pro Ser Ser Glu Leu Glu Asn Ile Pro Ser Glu Asn

65 70 75 80

Gln Tyr Phe Gln Ser Ala Ile Trp Ser Gly Phe Ile Lys Val Lys Lys

85 90 95

Ser Asp Glu Tyr Thr Phe Ala Thr Ser Ala Asp Asn His Val Thr Met

100 105 110

Trp Val Asp Asp Gln Glu Val Ile Asn Lys Ala Ser Asn Ser Asn Lys

115 120 125

Ile Arg Leu Glu Lys Gly Arg Leu Tyr Gln Ile Lys Ile Gln Tyr Gln

130 135 140

Arg Glu Asn Pro Thr Glu Lys Gly Leu Asp Phe Lys Leu Tyr Trp Thr

145 150 155 160

Asp Ser Gln Asn Lys Lys Glu Val Ile Ser Ser Asp Asn Leu Gln Leu

165 170 175

Pro Glu Leu Lys Gln Lys Ser Ser Asn Ser Arg Lys Lys Arg Ser Thr

180 185 190

Ser Ala Gly Pro Thr Val Pro Asp Arg Asp Asn Asp Gly Ile Pro Asp

195 200 205

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

210 215 220

Phe Leu Ser Pro Trp Ile Ser Asn Ile His Glu Lys Lys Gly Leu Thr

225 230 235 240

Lys Tyr Lys Ser Ser Pro Glu Lys Trp Ser Thr Ala Ser Asp Pro Tyr

245 250 255

Ser Asp Phe Glu Lys Val Thr Gly Arg Ile Asp Lys Asn Val Ser Pro

260 265 270

Glu Ala Arg His Pro Leu Val Ala Ala Tyr Pro Ile Val His Val Asp

275 280 285

Met Glu Asn Ile Ile Leu Ser Lys Asn Glu Asp Gln Ser Thr Gln Asn

290 295 300

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

305 310 315 320

Thr His Thr Ser Glu Val His Gly Asn Ala Glu Val His Ala Ser Phe

325 330 335

Phe Asp Ile Gly Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser

340 345 350

Ser Thr Val Ala Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg Thr

355 360 365

Trp Ala Glu Thr Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu Asn

370 375 380

Ala Asn Ile Arg Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn Val

385 390 395 400

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

405 410 415

Ile Lys Ala Lys Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn Asn

420 425 430

Tyr Tyr Pro Ser Lys Asn Leu Ala Pro Ile Ala Leu Asn Ala Gln Asp

435 440 445

Asp Phe Ser Ser Thr Pro Ile Thr Met Asn Tyr Asn Gln Phe Leu Glu

450 455 460

Leu Glu Lys Thr Lys Gln Leu Arg Leu Asp Thr Asp Gln Val Tyr Gly

465 470 475 480

Asn Ile Ala Thr Tyr Asn Phe Glu Asn Gly Arg Val Arg Val Asp Thr

485 490 495

Gly Ser Asn Trp Ser Glu Val Leu Pro Gln Ile Gln Glu Thr Thr Ala

500 505 510

Arg Ile Ile Phe Asn Gly Lys Asp Leu Asn Leu Val Glu Arg Arg Ile

515 520 525

Ala Ala Val Asn Pro Ser Asp Pro Leu Glu Thr Thr Lys Pro Asp Met

530 535 540

Thr Leu Lys Glu Ala Leu Lys Ile Ala Phe Gly Phe Asn Glu Pro Asn

545 550 555 560

Gly Asn Leu Gln Tyr Gln Gly Lys Asp Ile Thr Glu Phe Asp Phe Asn

565 570 575

Phe Asp Gln Gln Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala Glu Leu

580 585 590

Asn Ala Thr Asn Ile Tyr Thr Val Leu Asp Lys Ile Lys Leu Asn Ala

595 600 605

Lys Met Asn Ile Leu Ile Arg Asp Lys Arg Phe His Tyr Asp Arg Asn

610 615 620

Asn Ile Ala Val Gly Ala Asp Glu Ser Val Val Lys Glu Ala His Arg

625 630 635 640

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

645 650 655

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

660 665 670

Glu Gly Leu Lys Glu Val Ile Asn Asp Arg Tyr Asp Met Leu Asn Ile

675 680 685

Ser Ser Leu Arg Gln Asp Gly Lys Thr Phe Ile Asp Phe Lys Lys Tyr

690 695 700

Asn Asp Lys Leu Pro Leu Tyr Ile Ser Asn Pro Asn Tyr Lys Val Asn

705 710 715 720

Val Tyr Ala Val Thr Lys Glu Asn Thr Ile Ile Asn Pro Ser Glu Asn

725 730 735

Gly Asp Thr Ser Thr Asn Gly Ile Lys Lys Ile Leu Ile Phe Ser Lys

740 745 750

Lys Gly Tyr Glu Ile Gly

755

<210> 3

<211> 602

<212> PRT

<213> HPV E6/E7 chimeric protein antigen (underlined: tPA signal peptide)

<400> 3

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser Met His Gln Lys Arg Thr Ala Met Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg Tyr Met His Gly Asp

100 105 110

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

115 120 125

Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Val Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp

195 200 205

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

210 215 220

Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly

225 230 235 240

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

245 250 255

Arg Glu Thr Gln Leu His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Val Phe Glu Phe Ala Phe Lys Asp Leu Phe Val Val Tyr Arg Asp Ser

355 360 365

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

370 375 380

Arg Glu Leu Arg Tyr Tyr Ser Asp Ser Val Met Tyr Gly Pro Lys Ala

385 390 395 400

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

405 410 415

Val Asp Leu Leu Cys His Glu Gln Leu Ser Asp Ser Glu Glu Glu Asn

420 425 430

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

435 440 445

Glu Pro Gln Arg His Thr Met Leu Cys Met Cys Phe Tyr Ser Arg Ile

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Phe Val Cys Pro Trp Cys Ala Ser Gln Gln

595 600

<210> 4

<211> 528

<212> PRT

<213> HPV L1 protein antigen (underlined: tPA signal peptide)

<400> 4

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser Met Ser Leu Trp Leu Pro Ser Glu Ala

20 25 30

Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val Val Ser Thr Asp

35 40 45

Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr His Ala Gly Thr Ser Arg

50 55 60

Leu Leu Ala Val Gly His Pro Tyr Phe Pro Ile Lys Lys Pro Asn Asn

65 70 75 80

Asn Lys Ile Leu Val Pro Lys Val Ser Gly Leu Gln Tyr Arg Val Phe

85 90 95

Arg Ile His Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser

100 105 110

Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys Val Gly Val

115 120 125

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

130 135 140

Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Ala Ser Ala Tyr Ala Ala

145 150 155 160

Asn Ala Gly Val Asp Asn Arg Glu Cys Ile Ser Met Asp Tyr Lys Gln

165 170 175

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

180 185 190

Gly Lys Gly Ser Pro Cys Thr Asn Val Ala Val Asn Pro Gly Asp Cys

195 200 205

Pro Pro Leu Glu Leu Ile Asn Thr Val Ile Gln Asp Gly Asp Met Val

210 215 220

Asp Thr Gly Phe Gly Ala Met Asp Phe Thr Thr Leu Gln Ala Asn Lys

225 230 235 240

Ser Glu Val Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys Tyr Pro Asp

245 250 255

Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp Ser Leu Phe Phe Tyr

260 265 270

Leu Arg Arg Glu Gln Met Phe Val Arg His Leu Phe Asn Arg Ala Gly

275 280 285

Ala Val Gly Glu Asn Val Pro Asp Asp Leu Tyr Ile Lys Gly Ser Gly

290 295 300

Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr Phe Pro Thr Pro Ser Gly

305 310 315 320

Ser Met Val Thr Ser Asp Ala Gln Ile Phe Asn Lys Pro Tyr Trp Leu

325 330 335

Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu

340 345 350

Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met Ser Leu Cys

355 360 365

Ala Ala Ile Ser Thr Ser Glu Thr Thr Tyr Lys Asn Thr Asn Phe Lys

370 375 380

Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln

385 390 395 400

Leu Cys Lys Ile Thr Leu Thr Ala Asp Val Met Thr Tyr Ile His Ser

405 410 415

Met Asn Ser Thr Ile Leu Glu Asp Trp Asn Phe Gly Leu Gln Pro Pro

420 425 430

Pro Gly Gly Thr Leu Glu Asp Thr Tyr Arg Phe Val Thr Ser Gln Ala

435 440 445

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

450 455 460

Lys Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser Ala

465 470 475 480

Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly

485 490 495

Leu Lys Ala Lys Pro Lys Phe Thr Leu Gly Lys Arg Lys Ala Thr Pro

500 505 510

Thr Thr Ser Ser Thr Ser Thr Thr Ala Lys Arg Lys Lys Arg Lys Leu

515 520 525

<210> 5

<211> 214

<212> PRT

<213> HPV L2 protein antigen (underlined: tPA signal peptide)

<400> 5

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser Met Lys Arg Ala Ser Ala Thr Gln Leu

20 25 30

Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Ser

35 40 45

Lys Val Glu Gly Lys Thr Ile Ala Asp Gln Ile Leu Gln Tyr Gly Ser

50 55 60

Met Gly Val Phe Phe Gly Gly Leu Gly Ile Gly Thr Gly Ser Gly Thr

65 70 75 80

Gly Gly Arg Thr Gly Tyr Ile Pro Leu Gly Thr Arg Pro Pro Thr Ala

85 90 95

Thr Asp Thr Leu Ala Pro Val Arg Pro Pro Leu Thr Val Asp Pro Val

100 105 110

Gly Pro Ser Asp Pro Ser Ile Val Ser Leu Val Glu Glu Thr Ser Phe

115 120 125

Ile Asp Ala Gly Ala Pro Thr Ser Val Pro Ser Ile Pro Pro Asp Val

130 135 140

Ser Gly Phe Ser Ile Thr Thr Ser Thr Asp Thr Thr Pro Ala Ile Leu

145 150 155 160

Asp Ile Asn Asn Thr Val Thr Thr Val Thr Thr His Asn Asn Pro Thr

165 170 175

Phe Thr Asp Pro Ser Val Leu Gln Pro Pro Thr Pro Ala Glu Thr Gly

180 185 190

Gly His Phe Thr Leu Ser Ser Ser Thr Ile Ser Thr His Asn Tyr Glu

195 200 205

Glu Ile Pro Met Asp Thr

210

<210> 6

<211> 719

<212> PRT

<213> HPV L1/L2 chimeric protein antigen (underlined: tPA signal peptide; double wavy line: HPV L1; double transverse line: HPV L211-200)

<400> 6

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser Met Ser Leu Trp Leu Pro Ser Glu Ala

20 25 30

Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val Val Ser Thr Asp

35 40 45

Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr His Ala Gly Thr Ser Arg

50 55 60

Leu Leu Ala Val Gly His Pro Tyr Phe Pro Ile Lys Lys Pro Asn Asn

65 70 75 80

Asn Lys Ile Leu Val Pro Lys Val Ser Gly Leu Gln Tyr Arg Val Phe

85 90 95

Arg Ile His Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser

100 105 110

Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys Val Gly Val

115 120 125

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

130 135 140

Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Ala Ser Ala Tyr Ala Ala

145 150 155 160

Asn Ala Gly Val Asp Asn Arg Glu Cys Ile Ser Met Asp Tyr Lys Gln

165 170 175

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

180 185 190

Gly Lys Gly Ser Pro Cys Thr Asn Val Ala Val Asn Pro Gly Asp Cys

195 200 205

Pro Pro Leu Glu Leu Ile Asn Thr Val Ile Gln Asp Gly Asp Met Val

210 215 220

Asp Thr Gly Phe Gly Ala Met Asp Phe Thr Thr Leu Gln Ala Asn Lys

225 230 235 240

Ser Glu Val Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys Tyr Pro Asp

245 250 255

Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp Ser Leu Phe Phe Tyr

260 265 270

Leu Arg Arg Glu Gln Met Phe Val Arg His Leu Phe Asn Arg Ala Gly

275 280 285

Ala Val Gly Glu Asn Val Pro Asp Asp Leu Tyr Ile Lys Gly Ser Gly

290 295 300

Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr Phe Pro Thr Pro Ser Gly

305 310 315 320

Ser Met Val Thr Ser Asp Ala Gln Ile Phe Asn Lys Pro Tyr Trp Leu

325 330 335

Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu

340 345 350

Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met Ser Leu Cys

355 360 365

Ala Ala Ile Ser Thr Ser Glu Thr Thr Tyr Lys Asn Thr Asn Phe Lys

370 375 380

Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln

385 390 395 400

Leu Cys Lys Ile Thr Leu Thr Ala Asp Val Met Thr Tyr Ile His Ser

405 410 415

Met Asn Ser Thr Ile Leu Glu Asp Trp Asn Phe Gly Leu Gln Pro Pro

420 425 430

Pro Gly Gly Thr Leu Glu Asp Thr Tyr Arg Phe Val Thr Ser Gln Ala

435 440 445

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

450 455 460

Lys Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser Ala

465 470 475 480

Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly

485 490 495

Leu Lys Ala Lys Pro Lys Phe Thr Leu Gly Lys Arg Lys Ala Thr Pro

500 505 510

Thr Thr Ser Ser Thr Ser Thr Thr Ala Lys Arg Lys Lys Arg Lys Leu

515 520 525

Met Lys Arg Ala Ser Ala Thr Gln Leu Tyr Lys Thr Cys Lys Gln Ala

530 535 540

Gly Thr Cys Pro Pro Asp Ile Ile Ser Lys Val Glu Gly Lys Thr Ile

545 550 555 560

Ala Asp Gln Ile Leu Gln Tyr Gly Ser Met Gly Val Phe Phe Gly Gly

565 570 575

Leu Gly Ile Gly Thr Gly Ser Gly Thr Gly Gly Arg Thr Gly Tyr Ile

580 585 590

Pro Leu Gly Thr Arg Pro Pro Thr Ala Thr Asp Thr Leu Ala Pro Val

595 600 605

Arg Pro Pro Leu Thr Val Asp Pro Val Gly Pro Ser Asp Pro Ser Ile

610 615 620

Val Ser Leu Val Glu Glu Thr Ser Phe Ile Asp Ala Gly Ala Pro Thr

625 630 635 640

Ser Val Pro Ser Ile Pro Pro Asp Val Ser Gly Phe Ser Ile Thr Thr

645 650 655

Ser Thr Asp Thr Thr Pro Ala Ile Leu Asp Ile Asn Asn Thr Val Thr

660 665 670

Thr Val Thr Thr His Asn Asn Pro Thr Phe Thr Asp Pro Ser Val Leu

675 680 685

Gln Pro Pro Thr Pro Ala Glu Thr Gly Gly His Phe Thr Leu Ser Ser

690 695 700

Ser Thr Ile Ser Thr His Asn Tyr Glu Glu Ile Pro Met Asp Thr

705 710 715

<210> 7

<211> 2277

<212> DNA

<213> Bacillus anthracis PA antigen encoding gene

<400> 7

atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60

tcgaacagcg aggtgaagca ggagaacaga ctgctgaatg aatcggagtc atcgagtcag 120

gggttgctcg gttactactt ctccgatctc aatttccagg cccccatggt agtcacttcc 180

tccacaacag gggatctgag tatcccctcc tcggagctgg agaacatccc ttccgaaaac 240

cagtatttcc agagcgcaat ctggtccggc ttcatcaagg taaagaaatc cgacgagtat 300

accttcgcca cttccgcaga taaccatgtc accatgtggg tggacgacca ggaggtcatc 360

aacaaggcct ccaactctaa caagatccgc ctcgagaaag gccgtttgta ccagattaag 420

atccagtacc agagagaaaa ccctactgag aagggcctcg acttcaagct gtactggacc 480

gactcgcaga acaagaagga agtgatcagt tccgataacc tgcagctgcc ggaattgaag 540

cagaagtcat ccaacagtcg caagaagcgc tcgacaagtg ccggtcctac cgtgccagat 600

cgggacaacg acggcatccc cgattctctc gaggtggagg ggtacaccgt ggatgtgaaa 660

aacaagagga ccttcctctc cccatggatc tcgaacatcc acgagaagaa ggggctgacc 720

aagtacaaat cgtcccccga gaagtggagt acggcttccg acccctactc ggatttcgag 780

aaggtgaccg gcagaattga taagaacgtg tcgcccgagg cccgccatcc cctcgtcgcc 840

gcctacccga ttgtgcacgt cgatatggaa aacatcatct taagtaagaa cgaggaccag 900

tccacccaga acaccgacag ccagacccgt actatctcca agaatactag cacatccaga 960

acccatacgt cggaagtgca cggcaacgcc gaggtgcacg caagtttctt tgatattggc 1020

ggctctgtgt cggccggatt cagtaactca aactcgagca ccgtcgctat cgatcacagc 1080

ctgtcgttgg cgggcgagcg aacatgggcg gaaaccatgg gcctcaatac cgccgacacc 1140

gcccgactga acgctaacat ccgatacgtg aacacgggta ccgcccctat ctacaacgtg 1200

ctgcccacca cgtccctggt cctcggaaaa aatcagacct tggcgaccat caaggccaag 1260

gagaaccagc tatcccagat cctcgccccc aacaactact acccctcgaa gaacctggcc 1320

cccattgccc tcaacgcgca ggacgacttc agcagtactc ccattactat gaactacaac 1380

cagttcctcg agctggaaaa gaccaagcag ttgcgcctgg acacagacca ggtgtacggg 1440

aatatcgcca catacaactt cgagaacggt cgcgtgcggg tggataccgg ctcgaactgg 1500

tccgaggtct taccccaaat ccaggagacc acagccagga tcatcttcaa cggcaaggat 1560

ctgaacctcg tcgagcgccg catcgcggcg gtgaacccct ctgaccctct tgagacgacc 1620

aagcctgata tgacgctgaa ggaggcgctg aagatcgctt tcgggttcaa cgagcccaac 1680

ggcaacctcc agtaccaggg gaaggacata actgagttcg acttcaattt cgatcagcag 1740

acatcccaga atatcaagaa ccagctcgcc gagctgaacg caaccaacat ttacaccgtg 1800

ctggataaga tcaagctgaa cgccaagatg aacatcctga tccgcgacaa aaggttccac 1860

tatgaccgga acaacatcgc tgtgggcgcc gatgagtccg tcgtcaagga agcccatcga 1920

gaagtgatca actcatcgac cgaggggctc cttctgaaca tcgacaagga tatcagaaag 1980

atcctctccg ggtacatcgt ggagatcgag gataccgagg gcctgaagga agtcattaac 2040

gaccgctacg atatgctgaa catctcgtcg ctacgccagg acggcaagac ctttatcgac 2100

ttcaagaagt acaacgacaa actgccactc tacatctcta acccaaacta caaagtgaat 2160

gtgtacgccg tcacgaagga gaacactatc atcaatccct ctgagaacgg tgacacctcg 2220

accaacggga tcaaaaagat cctcattttc tcgaagaagg gctacgaaat cgggtaa 2277

<210> 8

<211> 1809

<212> DNA

<213> HPV E6/E7 chimeric protein antigen encoding gene

<400> 8

atggatgcta tgaaacgggg cctgtgctgc gtgctgctcc tgtgcggcgc tgtgtttgtg 60

agccctagca tgcaccagaa gagaaccgcc atgttccagg accctcagga gagacctagg 120

aagctgcctc acctgtgtac agagctccag acaaccatcc acgacatcat cctggagtgc 180

gtgtactgta agcagcagct gctgagaaga gaggtgtacg acttcgcctt cagagacctg 240

tgcatcgtgt acagagacgg caacccttac gccgtgtgcg ataagtgtct gaagttctat 300

tccaaaatct ccgaatatag gtacatgcac ggcgacaccc ctaccctgca cgagtacatg 360

ctggacctcc agcctgagac cacagacctg tactgctacg agcagctgaa cgacagctct 420

gaggaagagg acgagattga cggacctgct ggccaggccg agcctgacag agcccactac 480

aatatcgtga cattctgttg caaatgcgac tccacactgg acaagtgcct gaagttctac 540

agcaagatct ctgagtacag atactactgc tactctgtgt acggcaccac actggagcag 600

cagtacaaca agcctctgtg cgacctcctg atccgctgca tcaactgcca gaagcctctg 660

tgccctgagg agaagcagag acacctggac aagaagcagc ggttccacaa catcagaggc 720

agatggaccg gcaggtgcat gtcctgctgt agatcctcca gaaccagacg ggagacccag 780

ctgcactaca acatcgtgac cttctgctgc aagtgcgact ctaccctgag actgtgcgtg 840

cagtctaccc acgtggacat cagaaccctg gaggacctgc tgatgggcac cctgggcatc 900

gtgtgcccta tctgctctca gaagcctatg gccaggttcg aggaccctac cagaagaccc 960

tacaagctgc ctgacctgtg caccgagctg aacacctctc tgcaagacat cgagatcacc 1020

tgcgtgtact gcaagaccgt gctggagctg accgaggtgt tcgagttcgc cttcaaggac 1080

ctgttcgtgg tgtacagaga cagcatccct cacgctgcct gccacaagtg catcgacttc 1140

tattccagga tcagggagct gcgctattac tccgactctg tgatgtacgg ccccaaggcc 1200

accctccagg acatcgtgct gcacctggag cctcagaacg agatccccgt ggacctgctg 1260

tgccacgagc agctgtctga ctctgaagag gagaacgacg agatcgacgg cgtgaaccac 1320

cagcacctgc ctgccaggag agctgaaccc cagcggcata ccatgctgtg tatgtgcttc 1380

tactctagga tcagagagct gaggtactac tctgactctg tgtacggcga caccctggag 1440

aagctgacca acaccggcct gtacaacctg ctgatccggt gcctgaggtg ccagaagcct 1500

ctgaaccctg ccgagaagct gagacacctg aacgagaaga gaagattcca caagatcgct 1560

ggccactaca gaggccagtg ccactcttgc tgcaacagag ccagacagga gagactccag 1620

cggagaaggg agacccaggt ggccagaaga gccgagcctc agagacacac catgctgtgc 1680

atgtgctgca agtgcgaggc cagaatcgag ctggtggtgg agagctctgc cgacgacctg 1740

agagccttcc agcagctgtt cctgtctacc ctgagcttcg tgtgcccttg gtgcgcctct 1800

cagcagtag 1809

<210> 9

<211> 1587

<212> DNA

<213> HPV L1 protein antigen-encoding gene

<400> 9

atggatgcta tgaaacgggg cctgtgctgc gtgctgctcc tgtgcggcgc tgtgtttgtg 60

agccctagca tgagcctgtg gctgcccagc gaggccaccg tgtacctgcc ccccgtgccc 120

gtgagcaagg tggtgagcac cgacgagtac gtggccagga ccaacatcta ctaccacgcc 180

ggcaccagca ggctgctggc cgtgggccac ccctacttcc ccatcaagaa gcccaacaac 240

aacaagatcc tggtgcccaa ggtgagcggc ctgcagtaca gggtgttcag gatccacctg 300

cccgacccca acaagttcgg cttccccgac accagcttct acaaccccga cacccagagg 360

ctggtgtggg cctgcgtggg cgtggaggtg ggcaggggcc agcccctggg cgtgggcatc 420

agcggccacc ccctgctgaa caagctggac gacaccgaga acgccagcgc ctacgccgcc 480

aacgccggcg tggacaacag ggagtgcatc agcatggact acaagcagac ccagctgtgc 540

ctgatcggct gcaagccccc catcggcgag cactggggca agggcagccc ctgcaccaac 600

gtggccgtga accccggcga ctgccccccc ctggagctga tcaacaccgt gatccaggac 660

ggcgacatgg tggacaccgg cttcggcgcc atggacttca ccaccctgca ggccaacaag 720

agcgaggtgc ccctggacat ctgcaccagc atctgcaagt accccgacta catcaagatg 780

gtgagcgagc cctacggcga cagcctgttc ttctacctga ggagggagca gatgttcgtg 840

aggcacctgt tcaacagggc cggcgccgtg ggcgagaacg tgcccgacga cctgtacatc 900

aagggcagcg gcagcaccgc caacctggcc agcagcaact acttccccac ccccagcggc 960

agcatggtga ccagcgacgc ccagatcttc aacaagccct actggctgca gagggcccag 1020

ggccacaaca acggcatctg ctggggcaac cagctgttcg tgaccgtggt ggacaccacc 1080

aggagcacca acatgagcct gtgcgccgcc atcagcacca gcgagaccac ctacaagaac 1140

accaacttca aggagtacct gaggcacggc gaggagtacg acctgcagtt catcttccag 1200

ctgtgcaaga tcaccctgac cgccgacgtg atgacctaca tccacagcat gaacagcacc 1260

atcctggagg actggaactt cggcctgcag cccccccccg gcggcaccct ggaggacacc 1320

tacaggttcg tgaccagcca ggccatcgcc tgccagaagc acaccccccc cgcccccaag 1380

gaggaccccc tgaagaagta caccttctgg gaggtgaacc tgaaggagaa gttcagcgcc 1440

gacctggacc agttccccct gggcaggaag ttcctgctgc aggccggcct gaaggccaag 1500

cccaagttca ccctgggcaa gaggaaggcc acccccacca ccagcagcac cagcaccacc 1560

gccaagagga agaagaggaa gctgtag 1587

<210> 10

<211> 645

<212> DNA

<213> HPV L2 protein antigen-encoding gene

<400> 10

atggatgcta tgaaacgggg cctgtgctgc gtgctgctcc tgtgcggcgc tgtgtttgtg 60

agccctagca tgaagagggc cagcgccacc cagctgtaca agacctgcaa gcaggccggc 120

acctgccccc ccgacatcat ctccaaggtg gagggcaaga ccatcgccga ccagatcctg 180

cagtacggca gcatgggcgt gttcttcggc ggcctgggca tcggcaccgg cagcggcacc 240

ggcggcagga ccggctacat ccccctgggc accaggcccc ccaccgccac cgacaccctg 300

gcccccgtga ggccccccct gaccgtggac cccgtgggcc ccagcgaccc cagcatcgtg 360

agcctggtgg aggagaccag cttcatcgac gccggcgccc ccaccagcgt gcccagcatc 420

ccccccgacg tgagcggctt cagcatcacc accagcaccg acaccacccc cgccatcctg 480

gacatcaaca acaccgtgac caccgtgacc acccacaaca accccacctt caccgacccc 540

agcgtgctgc agccccccac ccccgccgag accggcggcc acttcaccct gagcagcagc 600

accatcagca cccacaacta cgaggagatc cccatggaca cctag 645

<210> 11

<211> 2160

<212> DNA

<213> HPV L1/L2 chimeric protein antigen encoding gene

<400> 11

atggatgcta tgaaacgggg cctgtgctgc gtgctgctcc tgtgcggcgc tgtgtttgtg 60

agccctagca tgagcctgtg gctgcccagc gaggccaccg tgtacctgcc ccccgtgccc 120

gtgagcaagg tggtgagcac cgacgagtac gtggccagga ccaacatcta ctaccacgcc 180

ggcaccagca ggctgctggc cgtgggccac ccctacttcc ccatcaagaa gcccaacaac 240

aacaagatcc tggtgcccaa ggtgagcggc ctgcagtaca gggtgttcag gatccacctg 300

cccgacccca acaagttcgg cttccccgac accagcttct acaaccccga cacccagagg 360

ctggtgtggg cctgcgtggg cgtggaggtg ggcaggggcc agcccctggg cgtgggcatc 420

agcggccacc ccctgctgaa caagctggac gacaccgaga acgccagcgc ctacgccgcc 480

aacgccggcg tggacaacag ggagtgcatc agcatggact acaagcagac ccagctgtgc 540

ctgatcggct gcaagccccc catcggcgag cactggggca agggcagccc ctgcaccaac 600

gtggccgtga accccggcga ctgccccccc ctggagctga tcaacaccgt gatccaggac 660

ggcgacatgg tggacaccgg cttcggcgcc atggacttca ccaccctgca ggccaacaag 720

agcgaggtgc ccctggacat ctgcaccagc atctgcaagt accccgacta catcaagatg 780

gtgagcgagc cctacggcga cagcctgttc ttctacctga ggagggagca gatgttcgtg 840

aggcacctgt tcaacagggc cggcgccgtg ggcgagaacg tgcccgacga cctgtacatc 900

aagggcagcg gcagcaccgc caacctggcc agcagcaact acttccccac ccccagcggc 960

agcatggtga ccagcgacgc ccagatcttc aacaagccct actggctgca gagggcccag 1020

ggccacaaca acggcatctg ctggggcaac cagctgttcg tgaccgtggt ggacaccacc 1080

aggagcacca acatgagcct gtgcgccgcc atcagcacca gcgagaccac ctacaagaac 1140

accaacttca aggagtacct gaggcacggc gaggagtacg acctgcagtt catcttccag 1200

ctgtgcaaga tcaccctgac cgccgacgtg atgacctaca tccacagcat gaacagcacc 1260

atcctggagg actggaactt cggcctgcag cccccccccg gcggcaccct ggaggacacc 1320

tacaggttcg tgaccagcca ggccatcgcc tgccagaagc acaccccccc cgcccccaag 1380

gaggaccccc tgaagaagta caccttctgg gaggtgaacc tgaaggagaa gttcagcgcc 1440

gacctggacc agttccccct gggcaggaag ttcctgctgc aggccggcct gaaggccaag 1500

cccaagttca ccctgggcaa gaggaaggcc acccccacca ccagcagcac cagcaccacc 1560

gccaagagga agaagaggaa gctgatgaag agggccagcg ccacccagct gtacaagacc 1620

tgcaagcagg ccggcacctg cccccccgac atcatctcca aggtggaggg caagaccatc 1680

gccgaccaga tcctgcagta cggcagcatg ggcgtgttct tcggcggcct gggcatcggc 1740

accggcagcg gcaccggcgg caggaccggc tacatccccc tgggcaccag gccccccacc 1800

gccaccgaca ccctggcccc cgtgaggccc cccctgaccg tggaccccgt gggccccagc 1860

gaccccagca tcgtgagcct ggtggaggag accagcttca tcgacgccgg cgcccccacc 1920

agcgtgccca gcatcccccc cgacgtgagc ggcttcagca tcaccaccag caccgacacc 1980

acccccgcca tcctggacat caacaacacc gtgaccaccg tgaccaccca caacaacccc 2040

accttcaccg accccagcgt gctgcagccc cccacccccg ccgagaccgg cggccacttc 2100

accctgagca gcagcaccat cagcacccac aactacgagg agatccccat ggacacctag 2160

Claims

1. A recombinant gene vector expressing an antigen or an antigen fragment, wherein the recombinant gene vector comprises a gene encoding the antigen or the antigen fragment;

Preferably, the antigen is selected from antigens specific to a pathogenic microorganism;

preferably, the recombinant gene vector is selected from a non-viral vector or a viral vector; more preferably, the non-viral vector is selected from a standard plasmid or other circular expression cassette, or the viral vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector and an adeno-associated viral vector.

2. The recombinant gene vector according to claim 1, wherein the pathogenic microorganism-specific Antigen or Antigen fragment is selected from Protective Antigen of Bacillus anthracis (PA), Human Papilloma Virus (HPV) early protein (E6 or E7) Antigen, HPV late protein (L1 or L2) Antigen, the non-viral vector is selected from minicircle DNA vector, or the viral vector is selected from adeno-associated viral vector.

3. The recombinant gene vector of claim 1 or 2, wherein the amino acid sequence of the PA antigen of Bacillus anthracis is as set forth in SEQ ID NO: 2 is shown in the specification; the HPV early protein antigen is selected from HPV E6/E7 chimeric protein antigen, and the amino acid sequence of the HPV early protein antigen is shown as SEQ ID NO: 3 is shown in the specification; the HPV late protein antigen is selected from (1) HPV L1 protein antigen, and the amino acid sequence of the HPV late protein antigen is shown as SEQ ID NO: 4, (2) HPV L2 protein antigen, the amino acid sequence of which is shown as SEQ ID NO: 5, or (3) HPV L1/L2 chimeric protein antigen, the amino acid sequence of which is shown as SEQ ID NO: and 6.

4. The recombinant gene vector of claim 3, wherein the nucleotide sequence of the PA antigen encoding gene of Bacillus anthracis is as shown in SEQ ID NO: 7 is shown in the specification; the nucleotide sequence of the HPV E6/E7 chimeric protein antigen encoding gene is shown as SEQ ID NO: 8 is shown in the specification; the nucleotide sequence of the HPV L1 protein antigen encoding gene is shown as SEQ ID NO: 9 is shown in the figure; the nucleotide sequence of the HPV L2 protein antigen encoding gene is shown as SEQ ID NO: 10 is shown in the figure; the nucleotide sequence of the HPV L1/L2 chimeric protein antigen encoding gene is shown as SEQ ID NO: shown at 11.

5. The method for preparing the recombinant gene vector according to any one of claims 1 to 4, comprising the steps of:

(3) identifying the expression level of the recombinant gene vector in vitro;

Optionally, the step of (a) is carried out,

the recombinant gene vector comprises a coding gene of the antigen;

Preferably, the antigen is selected from a pathogenic microorganism specific antigen or antigen fragment;

6. The process of claim 5, wherein the pathogenic microorganism-specific antigen is selected from PA antigen of Bacillus anthracis, HPV early protein (E6 or E7) antigen, HPV late protein (L1 or L2) antigen, the non-viral vector is selected from minicircle DNA vector, or the viral vector is selected from adeno-associated viral vector.

7. The method according to claim 6, wherein the HPV early protein antigen is selected from the group consisting of HPV E6/E7 chimeric protein antigen; the HPV late protein antigen is selected from (1) HPV L1 protein antigen, (2) HPV L2 protein antigen or (3) HPV L1/L2 chimeric protein antigen.

8. A host cell comprising the recombinant gene vector of any one of claims 1-4; preferably, the host cell comprises a bacterial cell, a yeast cell, an insect cell or a mammalian cell.

9. A pharmaceutical composition comprising the recombinant gene vector of any one of claims 1-4, and a pharmaceutically acceptable carrier.

10. Use of the recombinant gene vector of any one of claims 1-4, the host cell of claim 8, or the pharmaceutical composition of claim 9 in the manufacture of a medicament for the treatment of a disease;

Preferably, the disease is selected from infectious diseases or cancer; more preferably, the infectious disease is selected from bacillus anthracis infection or HPV infection, or the cancer is selected from cancer related to pathogenic microorganism, such as cervical cancer, liver cancer, nasopharyngeal cancer and the like.