CN113073105B - Polynucleotide sequence for expressing HPV56L1, expression vector, host cell and application thereof - Google Patents

Abstract

The invention provides a polynucleotide sequence for expressing HPV56L1, and an expression vector, a host cell and application thereof. The polynucleotide sequence is used for producing HPV56L1 protein with high yield. The HPV56L1 protein prepared by the method can be used for preparing vaccines for preventing HPV56 infection.

Description

Polynucleotide sequence for expressing HPV56L1, expression vector, host cell and application thereof

Technical Field

The invention relates to the field of biotechnology, relates to a method for producing HPV56L1 protein, and in particular relates to an expression HPV56L1 polynucleotide sequence, an expression vector, a host cell and application thereof.

Background

Human papillomaviruses (human papillomavirus, HPV) are non-enveloped, small double-stranded circular DNA viruses belonging to the papovaviridae family, members of the genus papilloma vacuole virus a. So far, the genotypes identified by HPV viruses have exceeded 200, and 13 of them may induce canceration after persistent infection, and are considered as high-risk HPVs (hrHPVs). HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59 have been demonstrated to be capable of transforming infected cells into malignant cells to initiate cervical cancer according to published data by the International cancer research institute (International Agency of Research on Cancer, IARC). [ iarc.biological agents: a review of human carcinogenic is.iarc Monogr Eval Carcinog Risks Hum 2012;100B. HPV, which is a virus particle composed of a capsid of about 8000 base pairs surrounded by a nucleic acid of about 60nm diameter when observed under electron microscopy, is a positive icosahedral symmetry. [ Knope, DM., howley, PM.fields virology.6th. Philadelphia, pa., wolters Kluwer/Lippincott Williams & Wilkins Health;2013] only one strand of the viral double stranded DNA genome was used as a transcription template, comprising ten open reading frames, three genomic regions encoding the strands, i.e. early regions (E) encoding 6 viral regulatory proteins (E1, E2, E4, E5, E6 and E7), late regions (L) encoding two viral capsid proteins L1 and L2 and long control regions (long control region, LCR) regulating viral genome replication, transcription, translation.

The antigen component of the prophylactic HPV vaccines currently on the market is mainly Virus-like particles (VLPs) composed of capsid proteins (L1). VLPs are recombinant proteins expressed by genetic engineering means, i.e. viral capsid proteins are produced by heterologous recombinant expression systems, and the expression products are purified to obtain virus-like particles having a spatial structure similar to that of the native virus, which do not contain viral nucleic acids. VLP vaccines, due to the lack of viral genetic material, do not have the ability to infect hosts, but their properties close to the natural viral structure can stimulate the body to produce effective humoral and cellular immunity, which has the effect of preventing infections and diseases. The vaccine produced by the strategy method has single and stable components, strong immunogenicity and higher safety. The global vaccine safety consultation committee (Global Advisory Committee on Vaccine Safety, GACVS) in cooperation with the world health organization (World Health Organisation, WHO) regularly organized the screening of HPV vaccine-related safety data, in the last screening of 20 th 7 th 2017, they summarized more than 2.7 billions of post-vaccination data, with the conclusion that: HPV vaccines are very safe, and there is no obvious evidence that HPV vaccines are associated with any serious side effects or significant medical conditions. [ GACVS.safety update of HPV vaccines.https:// www.who.int/vaccine_safety/com/traffic/topics/HPV/june_2017/en/; 2017.]

Numerous studies have shown that HPV major capsid protein L1 can be expressed in a variety of expression systems, and assembled into virus-like particles similar to the morphological structure of natural HPV without the aid of minor capsid protein L2. At present, three companies have marketed preventive HPV vaccines: bivalent vaccine of gram

(HPV 16, 18), tetravalent vaccine from moesadong corporation

(HPV 6, 11, 16, 18) and nine-valent vaccine->

9 ( HPV 6, 11, 16, 18, 31, 33, 45, 52, 58), divalent vaccine ++of Xiamen Wantai biological technology Co., ltd>

(HPV 16, 18). The three companies respectively adopt an insect cell-baculovirus expression system, a saccharomyces cerevisiae expression system and an escherichia coli expression system to prepare HPV L1 proteins, and the purified antigen adsorbs an adjuvant to prepare the VLP vaccine for preventing HPV infection.

However, HPV56 is not reported to be used as a high-risk HPV capable of inducing malignant tumors such as cervical cancer, and HPV56L1 protein is not utilized to assemble VLP.

Disclosure of Invention

The invention aims at providing a polynucleotide sequence for expressing HPV56L1, and an expression vector, a host cell and application thereof.

In one aspect, the invention provides a polynucleotide sequence for encoding HPV56L1 protein, wherein the polynucleotide sequence is as set forth in SEQ ID NO: 4.

Further, the amino acid sequence of the HPV56L1 protein is shown in SEQ ID NO: 1.

In a second aspect, the invention provides a recombinant expression vector comprising a polynucleotide sequence as described above.

Further, the recombinant expression vector is a vector which encodes a polypeptide as set forth in SEQ ID NO:4 into a plasmid. The plasmid may be a plasmid commonly used in the laboratory, for example, the plasmid provided in the examples of the present application is pMTZ.

Further, the recombinant expression vector also contains a promoter and a terminator.

Further, the promoter may be pMOX and the terminator may be MOX.

In a third aspect the present invention provides a host cell comprising or incorporating the recombinant expression vector described above.

Further, the host cell is a yeast.

Further, the yeast is selected from Hansenula polymorpha (Hansenula polymorpha).

In a fourth aspect, the present invention provides a method for preparing a recombinant hansenula species expressing HPV56L1 protein, comprising the steps of:

1) The nucleotide sequence SEQ ID NO:4, inserting the polynucleotide shown in the figure into an expression vector to construct a recombinant expression vector;

2) Transforming the recombinant expression vector obtained in the step 1) into Hansenula species;

3) Culturing the hansenula polymorpha strain obtained in the step 2) to obtain a recombinant hansenula polymorpha strain containing exogenous polynucleotides.

In a fifth aspect, the invention provides a method of producing HPV56L1 protein, the method comprising: construction of a nucleic acid comprising a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO:4, culturing recombinant hansenula polymorpha of the polynucleotide shown in the formula 4, collecting thalli, crushing the thalli to obtain lysate, and separating and purifying the lysate to obtain HPV56L1 protein.

Further, the polynucleotide sequence is integrated into a plasmid, and the Hansenula contains the plasmid.

Further, in the culture process, the pH is 5.0-7.0, the fermentation temperature is 30-37 ℃, the stirring rotation speed is less than or equal to 950rpm, the air flow is less than or equal to 2.0VVM, the tank pressure is less than or equal to 0.10MPa, and the dissolved oxygen is more than 10%.

Further, when the glycerol in the basal medium is consumed in the culture process, the thallus wet weight is greater than 100g/L, and the glycerol is added, so that the glycerol feeding speed is 200-600 g/h; when the thallus wet weight is greater than 200g/L, methanol is added at one time, the methanol is added into the thallus wet weight induction period, after the dissolved oxygen is increased to 80% after the methanol is completely consumed, the methanol is added, the methanol flow acceleration is gradually regulated along with the acceleration of the thallus by using the methanol, the dissolved oxygen is controlled to be more than 20% in the induction process, and the thallus wet weight is induced to be 300-400 g/L for 30-50 h, and then the fermentation is finished.

Further, the separation and purification means that the bacterial lysate is firstly passed through a cationic chromatographic column and then is passed through a chromatographic column CHT.

Further, the cation exchange chromatography packing is POROS HS, nanogel SP, and the like.

Another aspect of the invention provides

The beneficial technical effects are as follows: experiments of the invention find that the SEQ ID NO:4 encodes HPV56L1 protein in substantially higher yields than other nucleotide sequences. Hansenula polymorpha serving as eukaryotic unicellular organisms has the advantages of low culture cost, rapid growth, clear molecular biology background and the like, and meanwhile, compared with a prokaryotic expression system, the Hansenula polymorpha has a more perfect protein post-translational modification system, and an expression product does not contain endotoxin. In addition, compared with other eukaryotic expression systems (such as Saccharomyces cerevisiae), hansenula polymorpha has the advantages of stable genetic characters, high yield and more reasonable product glycosylation, and can avoid the problems of low integration copy number of exogenous genes of Pichia pastoris and the like.

Drawings

Fig. 1A: dot blotting (Dot blotting) to detect the expression of a recombinant Hansenula polymorpha engineering strain containing 56L1-1 coding sequence;

fig. 1B: dot blotting (Dot blotting) to detect the expression of the recombinant Hansenula polymorpha engineering strain containing 56L1-2 coding sequence;

FIG. 1C shows the detection of the expression of a recombinant Hansenula polymorpha engineered strain comprising the 56L1-3 coding sequence by Dot blotting (Dot blotting);

fig. 1D: dot blotting (Dot blotting) was performed to detect the expression of the recombinant Hansenula polymorpha engineering strain containing 56L1-4 coding sequences.

FIG. 2 SDS-PAGE detection of HPV56L1 protein expression during fermentation. M: a molecular weight standard; 1: before induction; 2: induction for 10 hours; 3: inducing for 20 hours; 4: inducing for 30 hours; 5: and (5) putting the fungus bodies into a tank.

FIG. 3 Western Blot detection of HPV56L1 protein expression during fermentation. M: a molecular weight standard; 1: before induction; 2: induction for 10 hours; 3: inducing for 20 hours; 4: inducing for 30 hours; 5: and (5) putting the fungus bodies into a tank.

FIG. 4 SDS-PAGE detection of purified HPV56L1 protein. M: a molecular weight standard; 1: HPV56L1 protein after purification.

FIG. 5 SEC-HPLC purity of purified HPV56L1 protein.

FIG. 6 is a transmission electron microscope observation of purified HPV56L1 protein.

FIG. 7 shows a schematic diagram of the structure of pMTZ vector.

FIG. 8 shows a schematic representation of the 56L1-1-pMTZ structure.

FIG. 9 shows a schematic representation of the structure of 56L 1-2-pMTZ.

FIG. 10 shows a schematic representation of the structure of 56L 1-3-pMTZ.

FIG. 11 shows a schematic representation of 56L1-4-pMTZ structure.

Detailed Description

In order to realize the high-efficiency expression of HPV56L1 protein in Hansenula polymorpha, the invention discloses a nucleotide sequence for encoding HPV56L1 protein and a preparation method of recombinant Hansenula polymorpha strain for expressing HPV56L1 protein, and discloses a fermentation process for ensuring the high-efficiency expression of HPV56L1 VLP. The expressed HPV56L1 protein sequentially passes through a cationic chromatographic column POROS HS and a chromatographic column CHT to be purified to obtain a high-purity target protein solution, and the target protein solution can be used as an antigen component of a monovalent recombinant HPV56L1 vaccine or a multivalent recombinant HPV vaccine, so that HPV56 infection is prevented, and cervical cancer and other related diseases (including but not limited to cervical cancer, vaginal cancer, vulvar cancer, endometrial cancer, anal cancer, penile cancer, head and neck cancer, lung cancer, bladder cancer, breast cancer, esophageal cancer, prostate cancer, ovarian cancer, colorectal adenoma and other cancers and precancerous lesions thereof) caused by HPV56 infection are prevented.

According to the amino acid sequence of HPV56L1 protein, 4 different DNA coding sequences are synthesized. The DNA sequences obtained by synthesis are respectively constructed on Hansenula polymorpha expression vectors to obtain 4 recombinant expression plasmids carrying HPV56L1 protein coding genes, and the 4 recombinant Hansenula polymorpha expression plasmids belong to intracellular expression plasmids. The recombinant plasmid is integrated into the genome of Hansenula polymorpha by a genetic engineering method, and is found by expression screening to contain SEQ ID NO:4 gene strain has HPV56L1 protein expression amount superior to other DNA coding sequence. Will contain the sequence of SEQ ID NO:4, fermenting and culturing the high-expression strain of the gene in a fermentation tank, purifying and carrying out chromatography to obtain high-purity HPV56L1 protein, and adsorbing the high-purity HPV56L1 protein by an aluminum adjuvant to obtain the HPV56L1 vaccine.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, the various methods set forth herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

EXAMPLE 1 construction of HPV56L1 protein engineering Strain

Selection of HPV56L1 amino acid sequence

The full-length HPV56L1 protein consists of 499 amino acids, and after GenBank searching and comparison analysis, a most representative conserved sequence (GenBank: ALT 54892.1) is selected as the amino acid sequence of HPV56L1, and the sequence information is shown in SEQ ID NO: 1.

SEQ ID NO：1

MATWRPSENKVYLPPTPVSKVVATDSYVKRTSIFYHAGSSRLLAVGHPYYSVTKDNTKTNIP

KVSAYQYRVFRVRLPDPNKFGLPDTNIYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFN

RLDDTESSNLANNNVIEDSRDNISVDGKQTQLCIVGCTPAMGEHWTKGAVCKSTQVTTGD

CPPLALINTPIEDGDMIDTGFGAMDFKVLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDS

MWFYLRREQLFARHYFNRAGKVGETIPAELYLKGSNGREPPPSSVYVATPSGSMITSEAQLF

NKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMTISTATEQLSKYDARKINQYLRHVE

EYELQFVFQLCKITLSAEVMAYLHNMNANLLEDWNIGLSPPVATSLEDKYRYVRSTAITCQ

REQPPTEKQDPLAKYKFWDVNLQDSFSTDLDQFPLGRKFLMQLGTRSKPAVATSKKRSAPT

STSTPAKRKRR

Design and synthesis of HPV56L1 coding gene

For efficient expression of HPV56L1 protein in hansenula polymorpha, the sequence according to SEQ ID NO:1, optimizing the nucleotide sequence by adopting different software and codon optimization strategies, wherein the optimized nucleotide sequence is shown as SEQ ID NO:2, seq ID NO:3, SEQ ID NO:4, SEQ ID NO: shown at 5. Based on the above nucleotide sequences, the full-length gene was synthesized by Soujin Zhi Biotechnology Co., ltd, and the sequence of the synthesized gene was verified by sequencing.

SEQ ID NO：2

ATGGCTACTTGGAGACCTTCCGAGAACAAGGTTTACCTGCCACCTACCCCAGTGTCTAA

GGTCGTTGCCACGGACTCCTACGTCAAGAGAACCTCGATCTTCTACCACGCAGGCTCGT

CCAGATTGCTGGCCGTTGGACACCCTTACTACTCTGTGACCAAGGACAACACCAAGAC

GAACATCCCAAAGGTTTCCGCCTACCAGTACAGAGTCTTCAGAGTTAGACTGCCAGAC

CCTAACAAGTTTGGTCTCCCTGACACCAACATCTACAACCCAGACCAGGAGAGACTTG

TGTGGGCATGTGTTGGTCTGGAGGTTGGCAGAGGACAGCCTTTGGGTGCTGGACTCTCT

GGCCACCCTCTGTTTAACAGACTCGACGATACTGAGTCGTCCAACTTGGCCAACAATA

ACGTCATTGAGGACTCCAGAGACAACATCTCTGTTGACGGCAAGCAGACCCAGTTGTG

CATTGTCGGATGTACTCCTGCTATGGGTGAACACTGGACGAAGGGTGCAGTGTGCAAG

TCCACCCAGGTTACCACAGGAGACTGCCCACCTCTTGCCTTGATCAACACGCCAATTGA

GGACGGTGACATGATCGACACCGGATTCGGTGCCATGGACTTCAAGGTGCTGCAGGAG

TCGAAGGCTGAGGTCCCTCTGGACATCGTTCAGTCCACCTGTAAGTACCCAGACTACCT

GAAGATGTCGGCAGACGCCTATGGTGACTCCATGTGGTTCTACTTGAGAAGAGAGCAG

CTCTTTGCCAGACACTACTTCAACAGAGCCGGCAAGGTCGGAGAGACCATTCCTGCAG

AGCTGTACCTCAAGGGCTCGAACGGTAGAGAACCACCTCCATCTTCCGTCTACGTTGCT

ACTCCTTCCGGATCTATGATCACGTCGGAGGCCCAGTTGTTCAACAAGCCATATTGGCT

GCAGCGTGCTCAAGGCCACAATAACGGCATCTGCTGGGGTAACCAACTGTTCGTGACT

GTTGTCGACACCACTAGATCCACCAACATGACGATTTCCACAGCTACCGAACAGCTGT

CGAAGTACGACGCCAGAAAGATCAACCAGTACCTCAGACACGTGGAGGAATACGAGT

TGCAGTTCGTCTTCCAGCTCTGCAAGATCACCCTTTCTGCCGAGGTTATGGCATACCTG

CACAACATGAATGCCAACTTGCTGGAGGACTGGAACATTGGATTGTCCCCTCCAGTTG

CCACCAGCCTCGAAGACAAGTACAGATACGTGAGATCGACTGCAATCACCTGTCAGAG

AGAGCAGCCACCTACGGAGAAGCAGGACCCACTTGCTAAGTACAAGTTCTGGGACGTC

AACCTGCAAGACTCCTTCTCGACCGACCTGGACCAGTTTCCTTTGGGCAGAAAGTTCCT

CATGCAACTGGGAACCAGATCTAAGCCAGCTGTCGCCACCTCCAAGAAGAGATCAGCA

CCTACTTCCACCTCGACGCCAGCCAAGAGAAAGCGCAGATAATAGSEQ ID NO：3

ATGGCAACCTGGAGACCATCTGAGAACAAGGTCTACTTGCCTCCAACTCCTGTTTCCAA

GGTTGTCGCAACCGACTCGTACGTGAAGAGAACGTCCATTTTCTACCACGCCGGTTCCT

CTAGACTGCTCGCTGTCGGTCACCCATATTACTCCGTTACGAAGGACAACACGAAGAC

CAACATCCCTAAGGTGTCTGCATACCAATACAGAGTTTTCAGAGTCAGACTCCCTGATC

CAAACAAGTTCGGCCTGCCAGACACGAACATCTACAACCCTGACCAAGAGAGACTGGT

CTGGGCTTGCGTGGGATTGGAGGTCGGTAGAGGCCAGCCACTGGGAGCCGGTCTTTCC

GGACACCCATTGTTCAACAGACTGGATGACACCGAATCCTCGAACCTGGCCAATAACA

ACGTGATCGAGGACAGTAGAGACAACATTTCGGTCGATGGTAAACAGACGCAGCTGTG

TATCGTTGGCTGCACCCCAGCCATGGGAGAGCACTGGACTAAGGGAGCCGTTTGTAAG

TCTACTCAGGTCACAACCGGCGACTGTCCTCCATTGGCTCTGATTAACACCCCTATCGA

GGACGGAGATATGATCGACACCGGTTTTGGAGCTATGGACTTCAAGGTTCTCCAGGAG

TCCAAGGCCGAGGTGCCATTGGACATCGTGCAGTCTACATGCAAGTACCCCGACTATTT

GAAGATGTCCGCCGATGCTTACGGAGACTCTATGTGGTTTTACCTTAGACGTGAGCAGT

TGTTCGCAAGACATTACTTCAACAGAGCCGGAAAGGTTGGTGAGACCATCCCAGCTGA

GTTGTACCTGAAGGGATCCAACGGCAGAGAGCCTCCACCTTCGTCTGTGTACGTCGCA

ACCCCAAGCGGTTCCATGATTACCTCTGAAGCACAGCTGTTTAACAAGCCTTACTGGCT

TCAGAGAGCCCAGGGTCACAACAATGGAATTTGTTGGGGCAACCAGCTCTTCGTTACC

GTCGTGGACACTACCAGATCTACTAACATGACCATCTCGACGGCCACTGAGCAGCTTTC

TAAGTACGACGCTAGAAAGATCAACCAATACCTGAGACACGTTGAAGAGTACGAGCTG

CAGTTTGTGTTCCAGCTGTGTAAGATTACCCTCTCCGCTGAGGTTATGGCCTACTTGCA

CAACATGAACGCAAACCTGTTGGAGGACTGGAACATCGGCTTGTCTCCACCTGTCGCA

ACCTCCCTGGAGGACAAGTACAGATACGTTAGATCTACCGCCATCACTTGCCAGAGAG

AGCAGCCTCCAACCGAGAAGCAGGACCCTCTGGCCAAGTACAAGTTCTGGGACGTTAA

CTTGCAGGACTCGTTCTCCACCGACCTCGACCAGTTCCCACTGGGTAGAAAGTTTTTGA

TGCAACTTGGCACCAGATCCAAGCCTGCCGTTGCTACTTCTAAGAAGAGATCCGCCCC

AACGTCGACTTCCACCCCTGCTAAAAGAAAGAGAAGATAATAGSEQ ID NO：4

ATGGCCACTTGGAGACCATCTGAAAACAAGGTTTACTTGCCACCTACTCCAGTCTCCAA

GGTTGTCGCTACTGACTCTTACGTTAAGAGAACTTCTATCTTCTACCACGCTGGATCTTC

CAGACTTTTGGCAGTGGGCCACCCATACTATTCGGTCACTAAGGACAACACTAAGACA

AACATCCCTAAAGTGTCTGCTTACCAGTACAGAGTCTTCAGAGTTCGTTTGCCTGACCC

AAACAAGTTCGGATTGCCAGACACTAACATCTACAACCCAGACCAGGAAAGATTAGTT

TGGGCCTGTGTCGGCCTCGAAGTTGGAAGAGGTCAGCCTCTTGGTGCTGGCTTGTCTGG

ACACCCACTCTTCAACAGATTGGACGATACTGAGTCATCCAACCTGGCTAACAACAAT

GTTATCGAAGACTCTAGAGATAACATTTCCGTTGACGGAAAGCAGACTCAGTTGTGTA

TTGTTGGTTGTACTCCAGCAATGGGCGAGCATTGGACCAAGGGTGCTGTTTGTAAGAG

CACTCAAGTTACCACTGGTGACTGCCCTCCACTGGCACTCATCAACACTCCAATCGAGG

ATGGTGACATGATCGACACCGGCTTTGGTGCTATGGACTTCAAGGTCTTGCAGGAGTCT

AAAGCCGAAGTTCCTTTAGACATTGTTCAATCCACCTGCAAGTACCCCGACTACTTGAA

GATGTCTGCTGATGCCTACGGTGACTCTATGTGGTTCTACTTGCGTAGAGAGCAGCTGT

TTGCTAGACACTACTTCAACAGAGCTGGTAAAGTCGGAGAAACGATTCCAGCCGAGTT

GTACTTGAAGGGTTCTAACGGAAGAGAACCTCCTCCATCCTCTGTCTACGTTGCCACTC

CTTCTGGTTCCATGATTACCTCTGAGGCTCAGCTCTTTAATAAGCCTTACTGGTTGCAGC

GTGCCCAAGGTCACAACAATGGAATCTGCTGGGGTAACCAGTTGTTCGTTACTGTCGTT

GACACCACTAGATCCACCAACATGACGATTTCTACAGCTACCGAACAGTTGTCCAAGT

ACGATGCCAGAAAGATCAACCAATACTTGAGACACGTTGAGGAATACGAGCTTCAGTT

CGTCTTTCAATTGTGCAAGATCACTTTGTCTGCCGAAGTTATGGCTTACTTGCACAACA

TGAATGCCAACCTTTTGGAGGACTGGAACATTGGATTGTCTCCTCCAGTTGCTACCAGT

TTGGAGGACAAGTACAGATATGTCAGATCCACTGCTATCACCTGTCAAAGAGAGCAGC

CACCTACTGAAAAGCAGGACCCACTGGCTAAATACAAGTTCTGGGATGTCAACTTGCA

AGACTCCTTCTCTACCGACCTTGATCAGTTCCCATTGGGTAGAAAGTTCCTTATGCAGT

TGGGAACCAGATCCAAACCTGCTGTTGCCACCTCCAAGAAGAGATCGGCTCCAACTTC

TACCTCCACTCCTGCCAAGAGAAAGCGTAGATAATAG

SEQ ID NO：5

ATGGCTACCTGGAGACCTTCCGAGAACAAGGTGTACCTCCCTCCAACCCCTGTGTCGA

AGGTCGTTGCTACCGACTCCTACGTCAAGAGAACCTCCATTTTCTACCACGCAGGCTCC

TCTAGATTGCTGGCCGTTGGACACCCTTATTACTCCGTTACCAAGGACAACACCAAGAC

TAACATCCCAAAGGTTTCCGCCTACCAATACAGAGTGTTTAGAGTCAGACTTCCAGACC

CTAACAAGTTCGGCTTGCCTGACACGAACATCTACAACCCTGACCAGGAGCGTCTAGT

CTGGGCTTGCGTTGGTCTGGAGGTCGGCAGAGGACAGCCATTGGGTGCAGGATTATCC

GGTCACCCTCTGTTTAACAGACTCGATGACACTGAATCTTCCAACTTGGCCAACAATAA

CGTGATTGAGGACTCCAGAGACAACATCTCTGTCGACGGTAAACAAACCCAGCTCTGC

ATCGTTGGATGCACTCCTGCTATGGGTGAACACTGGACTAAGGGAGCCGTTTGTAAGT

CTACCCAGGTTACTACCGGCGACTGTCCACCTTTGGCCTTGATTAACACCCCTATCGAG

GACGGAGACATGATCGATACTGGTTTCGGAGCAATGGACTTCAAGGTTCTTCAAGAGA

GTAAGGCTGAGGTTCCTTTGGACATCGTCCAGTCTACTTGTAAGTATCCAGACTACCTG

AAGATGTCCGCCGACGCTTACGGCGACTCCATGTGGTTCTACCTGAGAAGAGAGCAGT

TGTTCGCCAGACACTACTTCAACAGAGCCGGAAAGGTTGGTGAGACTATCCCTGCTGA

ACTGTACCTTAAGGGCTCCAACGGTAGAGAGCCACCACCTTCTTCAGTTTACGTCGCTA

CCCCATCCGGTTCTATGATCACTTCCGAAGCCCAACTGTTCAACAAGCCATACTGGCTC

CAGAGAGCACAGGGCCACAATAACGGTATTTGTTGGGGAAACCAGTTGTTCGTCACTG

TTGTGGACACTACGAGATCTACTAACATGACGATCTCCACCGCAACAGAGCAGCTTTCT

AAGTACGACGCTAGAAAGATCAACCAGTACCTGAGACACGTGGAAGAGTACGAGTTG

CAATTCGTTTTCCAGCTGTGTAAGATCACCTTGTCCGCTGAGGTCATGGCCTACCTGCA

CAACATGAACGCTAACTTGCTGGAAGACTGGAACATCGGCTTGTCCCCACCTGTCGCA

ACCTCTCTGGAGGACAAGTACAGATACGTTAGATCTACCGCAATTACTTGCCAGAGAG

AGCAACCTCCAACCGAGAAGCAAGACCCCCTTGCCAAGTACAAGTTCTGGGACGTTAA

CCTGCAGGACTCTTTCAGCACTGACCTGGACCAATTCCCTTTGGGAAGAAAGTTCTTGA

TGCAGTTAGGCACTAGATCTAAGCCAGCCGTCGCTACTTCTAAGAAGAGATCTGCCCCT

ACCTCGACTTCTACCCCAGCTAAGAGAAAGAGACGCTAATAG

Construction of recombinant expression vector for HPV56L1 protein

The Hansenula polymorpha expression vector applied in the invention is a pMTZ vector self-modified by the company. Cloning the optimized HPV56L1 coding sequence into a pMTZ vector through a BstBI enzyme cutting site at the 5 'end and a KpnI enzyme cutting site at the 3' end to respectively obtain expression vectors 56L1-1-pMTZ,56L1-2-pMTZ,56L1-3-pMTZ and 56L1-4-pMTZ. Transcription of the HPV56L1 coding sequence is regulated by the hansenula methanol oxidase promoter pMOX and MOX transcription termination region.

pMTZ vector sequence (SEQ ID NO: 6):

AGATCTGTCGACGCGGAGAACGATCTCCTCGAGCTGCTCGCGGATCAGCTTGTGGC

CCGGTAATGGAACCAGGCCGACGCGACGCTCCTTGCGGACCACGGTGGCTGGCGAGCC

CAGTTTGTGAACGAGGTCGTTTAGAACGTCCTCCGCAAAGTCCAGTGTCAGATGAATGT

CCTCCTCGGACCAATTCAGCATGTTCTCGAGCAGCCATCTGTCTTTGGAGTAGAAGCGTA

ATCTCTGCTCCTCGTTACTGTACCGGAAGAGGTAGTTTGCCTCGCCGCCCATAATGAACA

GGTTCTCTTTCTGGTGGCCTGTGAGCAGCGGGGACGTCTGGACGGCGTCGATGAGGCCC

TTGAGGCGCTCGTAGTACTTGTTCCGTCGCTGTAGCCGGCCGCGGTGACGATACCCACAT

AGAGGTCCTTGGCCATTAGTTTGATGAGGTGGGGCAGGATGGGCGACTCGGCATCGAAA

TTTTTGCCGTCGTCGTACAGTGTGATGTCACCATCGAATGTAATGAGCTGCAGCTTGCGA

TCTCGGATGGTTTTGGAATGGAAGAACCGCGACATCTCCAACAGCTGGGCCGTGTTGAG

AATGAGCCGGACGTCGTTGAACGAGGGGGCCACAAGCCGGCGTTTGCTGATGGCGCGG

CGCTCGTCCTCGATGTACAAGGCCTTTTCCAGAGGCAGTCTCGTGAAGAAGCTGCCAAC

GCTCGGAACCAGCTGCACGAGCCGAGACAATTCGGGGGTGCCGGCTTTGGTCATTTCAA

TCTTGTCGTCGATGAGGAGTTCGAGGTCGTGGAAGATTTCCGCGTAGCGGCGTTTTGCC

TCAGAGTTTACCATGAGGTCGTCCACTGCAGAGATGCCGTTGCTCTTCACCGCGTACAG

GACCAACGGCGTCGCCAGCAGGCCCTTGATCCATTCTATGAGGCCATCTCGACGGTGTT

CCTTGAGTGCGTACTCCACTCTGTAGCGACTGGACATCTCGAGACTGGGCTTGCTGTGC

TCGATGCACCAATTAATTGTTGCCGCATGCATCCTTGCACCGCAAGTTTTTAAAACCCAC

TCGCTTTAGCCGTCGCGTAAAACTTGTGAATCTGGCAACTGAGGGGGTTCTGCAGCCGC

AACCGAACTTTTCGCTTCGAGGACGCAGCTGCATGGTGTCATGTGAGGCTCTGTTTGCT

GGCGTAGCCTACAACGTGACCTTGCCTAACCGGACGGCGCTACCCACTGCTGTCTGTGC

CTGCTACCAGAAAATCACCAGAGCAGCAGAGGCCCGATGTGGCAACTGGTGGGGTGTC

GGACAGGCTGTTTCTCCACAGTGCAAATGCGGGTGAACCGGCCAGAAAGTAAATTCTTA

TGCTACCGTGCAGCGACTCCGACATCCCCAGTTTTTGCCCTACTTGATCACAGATGGGGT

CAGCGCTGCCGCTAAGTGTACCCAACCGTGCCCACACGGTCCATCTATAAATACTGCTGC

CAGTGCACGGTGGTGACATCAATCTAAAGTACAAAAACAAATTCGAAACGAGGAATTC

ACGTGGCCCAGCCGGCCGTCTCGGATCGGTACCGGAGACGTGGAAGGACATACCGCTTT

TGAGAAGCGTGTTTGAAAATAGTTCTTTTTCTGGTTTATATCGTTTATGAAGTGATGAGAT

GAAAAGCTGAAATAGCGAGTATAGGAAAATTTAATGAAAATTAAATTAAATATTTTCTTA

GGCTATTAGTCACCTTCAAAATGCCGGCCGCTTCTAAGAACGTTGTCATGATCGACAACT

ACGACTCGTTTACCTGGAACCTGTACGAGTACCTGTGTCAGGAGGGAGCCAATGTCGAG

GTTTTCAGGAACGATCAGATCACCATTCCGGAGATTGAGCAGCTCAAGCCGGACGTTGT

GGTGATATCCCCTGGTCCTGGCCATCCAAGAACAGACTCGGGAATATCTCGCGACGTGAT

CAGCCATTTTAAAGGCAAGATTCCTGTCTTTGGTGTCTGTATGGGCCAGCAGTGTATCTT

CGAGGAGTTTGGCGGAGACGTCGAGTATGCGGGCGAGATTGTCCATGGAAAAACGTCC

ACTGTTAAGCACGACAACAAGGGAATGTTCAAAAACGTTCCGCAAGATGTTGCTGTCAC

CAGATACCACTCGCTGGCCGGAACGCTCAAGTCGCTTCCGGACTGTCTAGAGATCACTG

CTCGCACAGACAACGGGATCATTATGGGTGTGAGACACAAGAAGTACACCATCGAGGG

CGTCCAGTTTCATCCAGAGAGCATTCTGACCGAGGAGGGCCATCTGATGATCCAGAATAT

CCTCAACGTTTCCGGTGGTTACTGGGAGGAAAATGCCAACGGCGCGGCTCAGAGAAAG

GAAAGCATATTGGAGAAAATATACGCGCAGAGACGAAAAGACTACGAGTTTGAGATGA

ACAGACCGGGGCGCAGATTTGCTGATCTAGAACTGTACTTGTCCATGGGACTGCACCGC

CGCTAATCAATTTTTACGACAGATTGGAGCAGAACATCAGCGCCGGCAAGGTTGCAATT

CTCAGCGAAATCAAGAGAGCGTCGCCTTCTAAAGGCGTCATCGACGGAGACGCTAACG

CTGCCAAACAGGCCCTCAACTACGCCAAGGCTGGAGTTGCCACAATTTCTGTTTTGACC

GAGCCAACCTGGTTTAAAGGAAATATCCAGGACCTGGAGGTGGCCAGAAAAGCCATTG

ACTCTGTGGCCAATAGACCGTGTATTTTGCGGAAGGAGTTTATCTTCAACAAGTACCAAA

TTCTAGAGGCCCGACTGGCGGGAGCAGACACGGTTCTGCTGATTGTCAAGATGCTGAGC

TCGGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATT

TTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAAT

TTTCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAA

AAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACG

TTTCTTTTTCTTGAAATTTTTTTTTTTAGTTTTTTTCTCTTTCAGTGACCTCCATTGATATTT

AAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAA

CTTTTTTTACTTCTTGTTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGGGGCGGTGTT

GACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAA

ACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAG

CGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTT

CGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTG

GTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCG

AGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGA

GATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGC

GTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTCCGACGGCGGCCCACGGGTCCCA

GGCCTCGGAGATCCGTCCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCT

TACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCT

GAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTT

CAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCT

TGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATG

TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTT

TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGG

CGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCG

CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAG

CGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTC

CAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA

ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACT

GGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG

GCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGT

TACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG

GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGAT

CCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGAT

TTTGGTCATGAGATC

56L1-1-pMTZ(SEQ ID NO：7)

AGATCTGTCGACGCGGAGAACGATCTCCTCGAGCTGCTCGCGGATCAGCTTGTGGCCCG

GTAATGGAACCAGGCCGACGCGACGCTCCTTGCGGACCACGGTGGCTGGCGAGCCCAG

TTTGTGAACGAGGTCGTTTAGAACGTCCTCCGCAAAGTCCAGTGTCAGATGAATGTCCT

CCTCGGACCAATTCAGCATGTTCTCGAGCAGCCATCTGTCTTTGGAGTAGAAGCGTAATC

TCTGCTCCTCGTTACTGTACCGGAAGAGGTAGTTTGCCTCGCCGCCCATAATGAACAGGT

TCTCTTTCTGGTGGCCTGTGAGCAGCGGGGACGTCTGGACGGCGTCGATGAGGCCCTTG

AGGCGCTCGTAGTACTTGTTCCGTCGCTGTAGCCGGCCGCGGTGACGATACCCACATAG

AGGTCCTTGGCCATTAGTTTGATGAGGTGGGGCAGGATGGGCGACTCGGCATCGAAATT

TTTGCCGTCGTCGTACAGTGTGATGTCACCATCGAATGTAATGAGCTGCAGCTTGCGATC

TCGGATGGTTTTGGAATGGAAGAACCGCGACATCTCCAACAGCTGGGCCGTGTTGAGAA

TGAGCCGGACGTCGTTGAACGAGGGGGCCACAAGCCGGCGTTTGCTGATGGCGCGGCG

CTCGTCCTCGATGTACAAGGCCTTTTCCAGAGGCAGTCTCGTGAAGAAGCTGCCAACGC

TCGGAACCAGCTGCACGAGCCGAGACAATTCGGGGGTGCCGGCTTTGGTCATTTCAATC

TTGTCGTCGATGAGGAGTTCGAGGTCGTGGAAGATTTCCGCGTAGCGGCGTTTTGCCTC

AGAGTTTACCATGAGGTCGTCCACTGCAGAGATGCCGTTGCTCTTCACCGCGTACAGGA

CCAACGGCGTCGCCAGCAGGCCCTTGATCCATTCTATGAGGCCATCTCGACGGTGTTCCT

TGAGTGCGTACTCCACTCTGTAGCGACTGGACATCTCGAGACTGGGCTTGCTGTGCTCG

ATGCACCAATTAATTGTTGCCGCATGCATCCTTGCACCGCAAGTTTTTAAAACCCACTCG

CTTTAGCCGTCGCGTAAAACTTGTGAATCTGGCAACTGAGGGGGTTCTGCAGCCGCAAC

CGAACTTTTCGCTTCGAGGACGCAGCTGCATGGTGTCATGTGAGGCTCTGTTTGCTGGC

GTAGCCTACAACGTGACCTTGCCTAACCGGACGGCGCTACCCACTGCTGTCTGTGCCTG

CTACCAGAAAATCACCAGAGCAGCAGAGGCCCGATGTGGCAACTGGTGGGGTGTCGGA

CAGGCTGTTTCTCCACAGTGCAAATGCGGGTGAACCGGCCAGAAAGTAAATTCTTATGC

TACCGTGCAGCGACTCCGACATCCCCAGTTTTTGCCCTACTTGATCACAGATGGGGTCAG

CGCTGCCGCTAAGTGTACCCAACCGTGCCCACACGGTCCATCTATAAATACTGCTGCCAG

TGCACGGTGGTGACATCAATCTAAAGTACAAAAACAAATTCGAAACGATGGCTACTTGG

AGACCTTCCGAGAACAAGGTTTACCTGCCACCTACCCCAGTGTCTAAGGTCGTTGCCAC

GGACTCCTACGTCAAGAGAACCTCGATCTTCTACCACGCAGGCTCGTCCAGATTGCTGG

CCGTTGGACACCCTTACTACTCTGTGACCAAGGACAACACCAAGACGAACATCCCAAA

GGTTTCCGCCTACCAGTACAGAGTCTTCAGAGTTAGACTGCCAGACCCTAACAAGTTTG

GTCTCCCTGACACCAACATCTACAACCCAGACCAGGAGAGACTTGTGTGGGCATGTGTT

GGTCTGGAGGTTGGCAGAGGACAGCCTTTGGGTGCTGGACTCTCTGGCCACCCTCTGTT

TAACAGACTCGACGATACTGAGTCGTCCAACTTGGCCAACAATAACGTCATTGAGGACT

CCAGAGACAACATCTCTGTTGACGGCAAGCAGACCCAGTTGTGCATTGTCGGATGTACT

CCTGCTATGGGTGAACACTGGACGAAGGGTGCAGTGTGCAAGTCCACCCAGGTTACCA

CAGGAGACTGCCCACCTCTTGCCTTGATCAACACGCCAATTGAGGACGGTGACATGATC

GACACCGGATTCGGTGCCATGGACTTCAAGGTGCTGCAGGAGTCGAAGGCTGAGGTCC

CTCTGGACATCGTTCAGTCCACCTGTAAGTACCCAGACTACCTGAAGATGTCGGCAGAC

GCCTATGGTGACTCCATGTGGTTCTACTTGAGAAGAGAGCAGCTCTTTGCCAGACACTA

CTTCAACAGAGCCGGCAAGGTCGGAGAGACCATTCCTGCAGAGCTGTACCTCAAGGGC

TCGAACGGTAGAGAACCACCTCCATCTTCCGTCTACGTTGCTACTCCTTCCGGATCTATG

ATCACGTCGGAGGCCCAGTTGTTCAACAAGCCATATTGGCTGCAGCGTGCTCAAGGCCA

CAATAACGGCATCTGCTGGGGTAACCAACTGTTCGTGACTGTTGTCGACACCACTAGAT

CCACCAACATGACGATTTCCACAGCTACCGAACAGCTGTCGAAGTACGACGCCAGAAA

GATCAACCAGTACCTCAGACACGTGGAGGAATACGAGTTGCAGTTCGTCTTCCAGCTCT

GCAAGATCACCCTTTCTGCCGAGGTTATGGCATACCTGCACAACATGAATGCCAACTTGC

TGGAGGACTGGAACATTGGATTGTCCCCTCCAGTTGCCACCAGCCTCGAAGACAAGTAC

AGATACGTGAGATCGACTGCAATCACCTGTCAGAGAGAGCAGCCACCTACGGAGAAGC

AGGACCCACTTGCTAAGTACAAGTTCTGGGACGTCAACCTGCAAGACTCCTTCTCGACC

GACCTGGACCAGTTTCCTTTGGGCAGAAAGTTCCTCATGCAACTGGGAACCAGATCTAA

GCCAGCTGTCGCCACCTCCAAGAAGAGATCAGCACCTACTTCCACCTCGACGCCAGCCA

AGAGAAAGCGCAGATAATAGGTACCGGAGACGTGGAAGGACATACCGCTTTTGAGAAG

CGTGTTTGAAAATAGTTCTTTTTCTGGTTTATATCGTTTATGAAGTGATGAGATGAAAAGC

TGAAATAGCGAGTATAGGAAAATTTAATGAAAATTAAATTAAATATTTTCTTAGGCTATTA

GTCACCTTCAAAATGCCGGCCGCTTCTAAGAACGTTGTCATGATCGACAACTACGACTC

GTTTACCTGGAACCTGTACGAGTACCTGTGTCAGGAGGGAGCCAATGTCGAGGTTTTCA

GGAACGATCAGATCACCATTCCGGAGATTGAGCAGCTCAAGCCGGACGTTGTGGTGATA

TCCCCTGGTCCTGGCCATCCAAGAACAGACTCGGGAATATCTCGCGACGTGATCAGCCA

TTTTAAAGGCAAGATTCCTGTCTTTGGTGTCTGTATGGGCCAGCAGTGTATCTTCGAGGA

GTTTGGCGGAGACGTCGAGTATGCGGGCGAGATTGTCCATGGAAAAACGTCCACTGTTA

AGCACGACAACAAGGGAATGTTCAAAAACGTTCCGCAAGATGTTGCTGTCACCAGATA

CCACTCGCTGGCCGGAACGCTCAAGTCGCTTCCGGACTGTCTAGAGATCACTGCTCGCA

CAGACAACGGGATCATTATGGGTGTGAGACACAAGAAGTACACCATCGAGGGCGTCCA

GTTTCATCCAGAGAGCATTCTGACCGAGGAGGGCCATCTGATGATCCAGAATATCCTCAA

CGTTTCCGGTGGTTACTGGGAGGAAAATGCCAACGGCGCGGCTCAGAGAAAGGAAAGC

ATATTGGAGAAAATATACGCGCAGAGACGAAAAGACTACGAGTTTGAGATGAACAGACC

GGGGCGCAGATTTGCTGATCTAGAACTGTACTTGTCCATGGGACTGCACCGCCGCTAATC

AATTTTTACGACAGATTGGAGCAGAACATCAGCGCCGGCAAGGTTGCAATTCTCAGCGA

AATCAAGAGAGCGTCGCCTTCTAAAGGCGTCATCGACGGAGACGCTAACGCTGCCAAA

CAGGCCCTCAACTACGCCAAGGCTGGAGTTGCCACAATTTCTGTTTTGACCGAGCCAAC

CTGGTTTAAAGGAAATATCCAGGACCTGGAGGTGGCCAGAAAAGCCATTGACTCTGTGG

CCAATAGACCGTGTATTTTGCGGAAGGAGTTTATCTTCAACAAGTACCAAATTCTAGAGG

CCCGACTGGCGGGAGCAGACACGGTTCTGCTGATTGTCAAGATGCTGAGCTCGGATCCC

CCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGAC

TCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTTCCCTCTT

TCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGAC

CGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTC

TTGAAATTTTTTTTTTTAGTTTTTTTCTCTTTCAGTGACCTCCATTGATATTTAAGTTAATA

AACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTAC

TTCTTGTTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGGGGCGGTGTTGACAATTAA

TCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCC

AAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGT

TCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGT

GGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGAC

AACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGG

AGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGA

GCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTC

GTGGCCGAGGAGCAGGACTGACACGTCCGACGGCGGCCCACGGGTCCCAGGCCTCGG

AGATCCGTCCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATTCAC

GCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAG

GTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTC

TTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGA

AGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATGTGAGCAAA

AGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGG

CTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACC

CGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCT

GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCG

CTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG

GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGT

CTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG

GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT

ACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG

GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTT

TTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT

CTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCA

TGAGATC

56L1-2-pMTZ(SEQ ID NO：8)

AGATCTGTCGACGCGGAGAACGATCTCCTCGAGCTGCTCGCGGATCAGCTTGTGGC

CCGGTAATGGAACCAGGCCGACGCGACGCTCCTTGCGGACCACGGTGGCTGGCGAGCC

CAGTTTGTGAACGAGGTCGTTTAGAACGTCCTCCGCAAAGTCCAGTGTCAGATGAATGT

CCTCCTCGGACCAATTCAGCATGTTCTCGAGCAGCCATCTGTCTTTGGAGTAGAAGCGTA

ATCTCTGCTCCTCGTTACTGTACCGGAAGAGGTAGTTTGCCTCGCCGCCCATAATGAACA

GGTTCTCTTTCTGGTGGCCTGTGAGCAGCGGGGACGTCTGGACGGCGTCGATGAGGCCC

TTGAGGCGCTCGTAGTACTTGTTCCGTCGCTGTAGCCGGCCGCGGTGACGATACCCACAT

AGAGGTCCTTGGCCATTAGTTTGATGAGGTGGGGCAGGATGGGCGACTCGGCATCGAAA

TTTTTGCCGTCGTCGTACAGTGTGATGTCACCATCGAATGTAATGAGCTGCAGCTTGCGA

TCTCGGATGGTTTTGGAATGGAAGAACCGCGACATCTCCAACAGCTGGGCCGTGTTGAG

AATGAGCCGGACGTCGTTGAACGAGGGGGCCACAAGCCGGCGTTTGCTGATGGCGCGG

CGCTCGTCCTCGATGTACAAGGCCTTTTCCAGAGGCAGTCTCGTGAAGAAGCTGCCAAC

GCTCGGAACCAGCTGCACGAGCCGAGACAATTCGGGGGTGCCGGCTTTGGTCATTTCAA

TCTTGTCGTCGATGAGGAGTTCGAGGTCGTGGAAGATTTCCGCGTAGCGGCGTTTTGCC

TCAGAGTTTACCATGAGGTCGTCCACTGCAGAGATGCCGTTGCTCTTCACCGCGTACAG

GACCAACGGCGTCGCCAGCAGGCCCTTGATCCATTCTATGAGGCCATCTCGACGGTGTT

CCTTGAGTGCGTACTCCACTCTGTAGCGACTGGACATCTCGAGACTGGGCTTGCTGTGC

TCGATGCACCAATTAATTGTTGCCGCATGCATCCTTGCACCGCAAGTTTTTAAAACCCAC

TCGCTTTAGCCGTCGCGTAAAACTTGTGAATCTGGCAACTGAGGGGGTTCTGCAGCCGC

AACCGAACTTTTCGCTTCGAGGACGCAGCTGCATGGTGTCATGTGAGGCTCTGTTTGCT

GGCGTAGCCTACAACGTGACCTTGCCTAACCGGACGGCGCTACCCACTGCTGTCTGTGC

CTGCTACCAGAAAATCACCAGAGCAGCAGAGGCCCGATGTGGCAACTGGTGGGGTGTC

GGACAGGCTGTTTCTCCACAGTGCAAATGCGGGTGAACCGGCCAGAAAGTAAATTCTTA

TGCTACCGTGCAGCGACTCCGACATCCCCAGTTTTTGCCCTACTTGATCACAGATGGGGT

CAGCGCTGCCGCTAAGTGTACCCAACCGTGCCCACACGGTCCATCTATAAATACTGCTGC

CAGTGCACGGTGGTGACATCAATCTAAAGTACAAAAACAAATTCGAAACGATGGCAAC

CTGGAGACCATCTGAGAACAAGGTCTACTTGCCTCCAACTCCTGTTTCCAAGGTTGTCG

CAACCGACTCGTACGTGAAGAGAACGTCCATTTTCTACCACGCCGGTTCCTCTAGACTG

CTCGCTGTCGGTCACCCATATTACTCCGTTACGAAGGACAACACGAAGACCAACATCCC

TAAGGTGTCTGCATACCAATACAGAGTTTTCAGAGTCAGACTCCCTGATCCAAACAAGT

TCGGCCTGCCAGACACGAACATCTACAACCCTGACCAAGAGAGACTGGTCTGGGCTTG

CGTGGGATTGGAGGTCGGTAGAGGCCAGCCACTGGGAGCCGGTCTTTCCGGACACCCA

TTGTTCAACAGACTGGATGACACCGAATCCTCGAACCTGGCCAATAACAACGTGATCGA

GGACAGTAGAGACAACATTTCGGTCGATGGTAAACAGACGCAGCTGTGTATCGTTGGCT

GCACCCCAGCCATGGGAGAGCACTGGACTAAGGGAGCCGTTTGTAAGTCTACTCAGGTC

ACAACCGGCGACTGTCCTCCATTGGCTCTGATTAACACCCCTATCGAGGACGGAGATATG

ATCGACACCGGTTTTGGAGCTATGGACTTCAAGGTTCTCCAGGAGTCCAAGGCCGAGGT

GCCATTGGACATCGTGCAGTCTACATGCAAGTACCCCGACTATTTGAAGATGTCCGCCGA

TGCTTACGGAGACTCTATGTGGTTTTACCTTAGACGTGAGCAGTTGTTCGCAAGACATTA

CTTCAACAGAGCCGGAAAGGTTGGTGAGACCATCCCAGCTGAGTTGTACCTGAAGGGA

TCCAACGGCAGAGAGCCTCCACCTTCGTCTGTGTACGTCGCAACCCCAAGCGGTTCCAT

GATTACCTCTGAAGCACAGCTGTTTAACAAGCCTTACTGGCTTCAGAGAGCCCAGGGTC

ACAACAATGGAATTTGTTGGGGCAACCAGCTCTTCGTTACCGTCGTGGACACTACCAGA

TCTACTAACATGACCATCTCGACGGCCACTGAGCAGCTTTCTAAGTACGACGCTAGAAA

GATCAACCAATACCTGAGACACGTTGAAGAGTACGAGCTGCAGTTTGTGTTCCAGCTGT

GTAAGATTACCCTCTCCGCTGAGGTTATGGCCTACTTGCACAACATGAACGCAAACCTGT

TGGAGGACTGGAACATCGGCTTGTCTCCACCTGTCGCAACCTCCCTGGAGGACAAGTAC

AGATACGTTAGATCTACCGCCATCACTTGCCAGAGAGAGCAGCCTCCAACCGAGAAGCA

GGACCCTCTGGCCAAGTACAAGTTCTGGGACGTTAACTTGCAGGACTCGTTCTCCACCG

ACCTCGACCAGTTCCCACTGGGTAGAAAGTTTTTGATGCAACTTGGCACCAGATCCAAG

CCTGCCGTTGCTACTTCTAAGAAGAGATCCGCCCCAACGTCGACTTCCACCCCTGCTAA

AAGAAAGAGAAGATAATAGGTACCGGAGACGTGGAAGGACATACCGCTTTTGAGAAGC

GTGTTTGAAAATAGTTCTTTTTCTGGTTTATATCGTTTATGAAGTGATGAGATGAAAAGCT

GAAATAGCGAGTATAGGAAAATTTAATGAAAATTAAATTAAATATTTTCTTAGGCTATTAG

TCACCTTCAAAATGCCGGCCGCTTCTAAGAACGTTGTCATGATCGACAACTACGACTCG

TTTACCTGGAACCTGTACGAGTACCTGTGTCAGGAGGGAGCCAATGTCGAGGTTTTCAG

GAACGATCAGATCACCATTCCGGAGATTGAGCAGCTCAAGCCGGACGTTGTGGTGATAT

CCCCTGGTCCTGGCCATCCAAGAACAGACTCGGGAATATCTCGCGACGTGATCAGCCAT

TTTAAAGGCAAGATTCCTGTCTTTGGTGTCTGTATGGGCCAGCAGTGTATCTTCGAGGAG

TTTGGCGGAGACGTCGAGTATGCGGGCGAGATTGTCCATGGAAAAACGTCCACTGTTAA

GCACGACAACAAGGGAATGTTCAAAAACGTTCCGCAAGATGTTGCTGTCACCAGATACC

ACTCGCTGGCCGGAACGCTCAAGTCGCTTCCGGACTGTCTAGAGATCACTGCTCGCACA

GACAACGGGATCATTATGGGTGTGAGACACAAGAAGTACACCATCGAGGGCGTCCAGTT

TCATCCAGAGAGCATTCTGACCGAGGAGGGCCATCTGATGATCCAGAATATCCTCAACGT

TTCCGGTGGTTACTGGGAGGAAAATGCCAACGGCGCGGCTCAGAGAAAGGAAAGCATA

TTGGAGAAAATATACGCGCAGAGACGAAAAGACTACGAGTTTGAGATGAACAGACCGG

GGCGCAGATTTGCTGATCTAGAACTGTACTTGTCCATGGGACTGCACCGCCGCTAATCAA

TTTTTACGACAGATTGGAGCAGAACATCAGCGCCGGCAAGGTTGCAATTCTCAGCGAAA

TCAAGAGAGCGTCGCCTTCTAAAGGCGTCATCGACGGAGACGCTAACGCTGCCAAACA

GGCCCTCAACTACGCCAAGGCTGGAGTTGCCACAATTTCTGTTTTGACCGAGCCAACCT

GGTTTAAAGGAAATATCCAGGACCTGGAGGTGGCCAGAAAAGCCATTGACTCTGTGGCC

AATAGACCGTGTATTTTGCGGAAGGAGTTTATCTTCAACAAGTACCAAATTCTAGAGGCC

CGACTGGCGGGAGCAGACACGGTTCTGCTGATTGTCAAGATGCTGAGCTCGGATCCCCC

ACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTC

CGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTTCCCTCTTTC

TTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCG

CCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTT

GAAATTTTTTTTTTTAGTTTTTTTCTCTTTCAGTGACCTCCATTGATATTTAAGTTAATAAA

CGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTT

CTTGTTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGGGGCGGTGTTGACAATTAATC

ATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAA

GTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTC

TGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGG

TCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAA

CACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAG

GTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGC

AGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGT

GGCCGAGGAGCAGGACTGACACGTCCGACGGCGGCCCACGGGTCCCAGGCCTCGGAG

ATCCGTCCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATTCACGC

CCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGT

CCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTT

TTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAG

GTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATGTGAGCAAAAG

GCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCT

CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCG

ACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTT

CCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT

TCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGC

TGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT

GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGAT

TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACG

GCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAA

AAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTT

GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT

TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGA

GATC

56L1-3-pMTZ(SEQ ID NO：9)

AGATCTGTCGACGCGGAGAACGATCTCCTCGAGCTGCTCGCGGATCAGCTTGTGGC

CCGGTAATGGAACCAGGCCGACGCGACGCTCCTTGCGGACCACGGTGGCTGGCGAGCC

CAGTTTGTGAACGAGGTCGTTTAGAACGTCCTCCGCAAAGTCCAGTGTCAGATGAATGT

CCTCCTCGGACCAATTCAGCATGTTCTCGAGCAGCCATCTGTCTTTGGAGTAGAAGCGTA

ATCTCTGCTCCTCGTTACTGTACCGGAAGAGGTAGTTTGCCTCGCCGCCCATAATGAACA

GGTTCTCTTTCTGGTGGCCTGTGAGCAGCGGGGACGTCTGGACGGCGTCGATGAGGCCC

TTGAGGCGCTCGTAGTACTTGTTCCGTCGCTGTAGCCGGCCGCGGTGACGATACCCACAT

AGAGGTCCTTGGCCATTAGTTTGATGAGGTGGGGCAGGATGGGCGACTCGGCATCGAAA

TTTTTGCCGTCGTCGTACAGTGTGATGTCACCATCGAATGTAATGAGCTGCAGCTTGCGA

TCTCGGATGGTTTTGGAATGGAAGAACCGCGACATCTCCAACAGCTGGGCCGTGTTGAG

AATGAGCCGGACGTCGTTGAACGAGGGGGCCACAAGCCGGCGTTTGCTGATGGCGCGG

CGCTCGTCCTCGATGTACAAGGCCTTTTCCAGAGGCAGTCTCGTGAAGAAGCTGCCAAC

GCTCGGAACCAGCTGCACGAGCCGAGACAATTCGGGGGTGCCGGCTTTGGTCATTTCAA

TCTTGTCGTCGATGAGGAGTTCGAGGTCGTGGAAGATTTCCGCGTAGCGGCGTTTTGCC

TCAGAGTTTACCATGAGGTCGTCCACTGCAGAGATGCCGTTGCTCTTCACCGCGTACAG

GACCAACGGCGTCGCCAGCAGGCCCTTGATCCATTCTATGAGGCCATCTCGACGGTGTT

CCTTGAGTGCGTACTCCACTCTGTAGCGACTGGACATCTCGAGACTGGGCTTGCTGTGC

TCGATGCACCAATTAATTGTTGCCGCATGCATCCTTGCACCGCAAGTTTTTAAAACCCAC

TCGCTTTAGCCGTCGCGTAAAACTTGTGAATCTGGCAACTGAGGGGGTTCTGCAGCCGC

AACCGAACTTTTCGCTTCGAGGACGCAGCTGCATGGTGTCATGTGAGGCTCTGTTTGCT

GGCGTAGCCTACAACGTGACCTTGCCTAACCGGACGGCGCTACCCACTGCTGTCTGTGC

CTGCTACCAGAAAATCACCAGAGCAGCAGAGGCCCGATGTGGCAACTGGTGGGGTGTC

GGACAGGCTGTTTCTCCACAGTGCAAATGCGGGTGAACCGGCCAGAAAGTAAATTCTTA

TGCTACCGTGCAGCGACTCCGACATCCCCAGTTTTTGCCCTACTTGATCACAGATGGGGT

CAGCGCTGCCGCTAAGTGTACCCAACCGTGCCCACACGGTCCATCTATAAATACTGCTGC

CAGTGCACGGTGGTGACATCAATCTAAAGTACAAAAACAAATTCGAAACGATGGCCACT

TGGAGACCATCTGAAAACAAGGTTTACTTGCCACCTACTCCAGTCTCCAAGGTTGTCGC

TACTGACTCTTACGTTAAGAGAACTTCTATCTTCTACCACGCTGGATCTTCCAGACTTTTG

GCAGTGGGCCACCCATACTATTCGGTCACTAAGGACAACACTAAGACAAACATCCCTAA

AGTGTCTGCTTACCAGTACAGAGTCTTCAGAGTTCGTTTGCCTGACCCAAACAAGTTCG

GATTGCCAGACACTAACATCTACAACCCAGACCAGGAAAGATTAGTTTGGGCCTGTGTC

GGCCTCGAAGTTGGAAGAGGTCAGCCTCTTGGTGCTGGCTTGTCTGGACACCCACTCTT

CAACAGATTGGACGATACTGAGTCATCCAACCTGGCTAACAACAATGTTATCGAAGACT

CTAGAGATAACATTTCCGTTGACGGAAAGCAGACTCAGTTGTGTATTGTTGGTTGTACTC

CAGCAATGGGCGAGCATTGGACCAAGGGTGCTGTTTGTAAGAGCACTCAAGTTACCACT

GGTGACTGCCCTCCACTGGCACTCATCAACACTCCAATCGAGGATGGTGACATGATCGA

CACCGGCTTTGGTGCTATGGACTTCAAGGTCTTGCAGGAGTCTAAAGCCGAAGTTCCTT

TAGACATTGTTCAATCCACCTGCAAGTACCCCGACTACTTGAAGATGTCTGCTGATGCCT

ACGGTGACTCTATGTGGTTCTACTTGCGTAGAGAGCAGCTGTTTGCTAGACACTACTTCA

ACAGAGCTGGTAAAGTCGGAGAAACGATTCCAGCCGAGTTGTACTTGAAGGGTTCTAA

CGGAAGAGAACCTCCTCCATCCTCTGTCTACGTTGCCACTCCTTCTGGTTCCATGATTAC

CTCTGAGGCTCAGCTCTTTAATAAGCCTTACTGGTTGCAGCGTGCCCAAGGTCACAACA

ATGGAATCTGCTGGGGTAACCAGTTGTTCGTTACTGTCGTTGACACCACTAGATCCACCA

ACATGACGATTTCTACAGCTACCGAACAGTTGTCCAAGTACGATGCCAGAAAGATCAAC

CAATACTTGAGACACGTTGAGGAATACGAGCTTCAGTTCGTCTTTCAATTGTGCAAGATC

ACTTTGTCTGCCGAAGTTATGGCTTACTTGCACAACATGAATGCCAACCTTTTGGAGGAC

TGGAACATTGGATTGTCTCCTCCAGTTGCTACCAGTTTGGAGGACAAGTACAGATATGTC

AGATCCACTGCTATCACCTGTCAAAGAGAGCAGCCACCTACTGAAAAGCAGGACCCAC

TGGCTAAATACAAGTTCTGGGATGTCAACTTGCAAGACTCCTTCTCTACCGACCTTGATC

AGTTCCCATTGGGTAGAAAGTTCCTTATGCAGTTGGGAACCAGATCCAAACCTGCTGTT

GCCACCTCCAAGAAGAGATCGGCTCCAACTTCTACCTCCACTCCTGCCAAGAGAAAGC

GTAGATAATAGGTACCGGAGACGTGGAAGGACATACCGCTTTTGAGAAGCGTGTTTGAA

AATAGTTCTTTTTCTGGTTTATATCGTTTATGAAGTGATGAGATGAAAAGCTGAAATAGCG

AGTATAGGAAAATTTAATGAAAATTAAATTAAATATTTTCTTAGGCTATTAGTCACCTTCA

AAATGCCGGCCGCTTCTAAGAACGTTGTCATGATCGACAACTACGACTCGTTTACCTGG

AACCTGTACGAGTACCTGTGTCAGGAGGGAGCCAATGTCGAGGTTTTCAGGAACGATCA

GATCACCATTCCGGAGATTGAGCAGCTCAAGCCGGACGTTGTGGTGATATCCCCTGGTC

CTGGCCATCCAAGAACAGACTCGGGAATATCTCGCGACGTGATCAGCCATTTTAAAGGC

AAGATTCCTGTCTTTGGTGTCTGTATGGGCCAGCAGTGTATCTTCGAGGAGTTTGGCGGA

GACGTCGAGTATGCGGGCGAGATTGTCCATGGAAAAACGTCCACTGTTAAGCACGACA

ACAAGGGAATGTTCAAAAACGTTCCGCAAGATGTTGCTGTCACCAGATACCACTCGCTG

GCCGGAACGCTCAAGTCGCTTCCGGACTGTCTAGAGATCACTGCTCGCACAGACAACG

GGATCATTATGGGTGTGAGACACAAGAAGTACACCATCGAGGGCGTCCAGTTTCATCCA

GAGAGCATTCTGACCGAGGAGGGCCATCTGATGATCCAGAATATCCTCAACGTTTCCGG

TGGTTACTGGGAGGAAAATGCCAACGGCGCGGCTCAGAGAAAGGAAAGCATATTGGAG

AAAATATACGCGCAGAGACGAAAAGACTACGAGTTTGAGATGAACAGACCGGGGCGCA

GATTTGCTGATCTAGAACTGTACTTGTCCATGGGACTGCACCGCCGCTAATCAATTTTTAC

GACAGATTGGAGCAGAACATCAGCGCCGGCAAGGTTGCAATTCTCAGCGAAATCAAGA

GAGCGTCGCCTTCTAAAGGCGTCATCGACGGAGACGCTAACGCTGCCAAACAGGCCCT

CAACTACGCCAAGGCTGGAGTTGCCACAATTTCTGTTTTGACCGAGCCAACCTGGTTTA

AAGGAAATATCCAGGACCTGGAGGTGGCCAGAAAAGCCATTGACTCTGTGGCCAATAG

ACCGTGTATTTTGCGGAAGGAGTTTATCTTCAACAAGTACCAAATTCTAGAGGCCCGACT

GGCGGGAGCAGACACGGTTCTGCTGATTGTCAAGATGCTGAGCTCGGATCCCCCACACA

CCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGC

ATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTTCCCTCTTTCTTCCT

CTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCG

TTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAT

TTTTTTTTTTAGTTTTTTTCTCTTTCAGTGACCTCCATTGATATTTAAGTTAATAAACGGTC

TTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGT

TCATTAGAAAGAAAGCATAGCAATCTAATCTAAGGGGCGGTGTTGACAATTAATCATCGG

CATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGA

CCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGAC

CGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGG

GACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCC

TGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGT

GTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCG

TGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCG

AGGAGCAGGACTGACACGTCCGACGGCGGCCCACGGGTCCCAGGCCTCGGAGATCCGT

CCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCC

CCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTAT

TTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTT

CTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTG

GGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATGTGAGCAAAAGGCCAGC

AAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCC

CCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGAC

TATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCC

TGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAAT

GCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGC

ACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA

ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA

GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACAC

TAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAG

TTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCA

AGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACG

GGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATC

56L1-4-pMTZ(SEQ ID NO：10)

AGATCTGTCGACGCGGAGAACGATCTCCTCGAGCTGCTCGCGGATCAGCTTGTGGC

CCGGTAATGGAACCAGGCCGACGCGACGCTCCTTGCGGACCACGGTGGCTGGCGAGCC

CAGTTTGTGAACGAGGTCGTTTAGAACGTCCTCCGCAAAGTCCAGTGTCAGATGAATGT

CCTCCTCGGACCAATTCAGCATGTTCTCGAGCAGCCATCTGTCTTTGGAGTAGAAGCGTA

ATCTCTGCTCCTCGTTACTGTACCGGAAGAGGTAGTTTGCCTCGCCGCCCATAATGAACA

GGTTCTCTTTCTGGTGGCCTGTGAGCAGCGGGGACGTCTGGACGGCGTCGATGAGGCCC

TTGAGGCGCTCGTAGTACTTGTTCCGTCGCTGTAGCCGGCCGCGGTGACGATACCCACAT

AGAGGTCCTTGGCCATTAGTTTGATGAGGTGGGGCAGGATGGGCGACTCGGCATCGAAA

TTTTTGCCGTCGTCGTACAGTGTGATGTCACCATCGAATGTAATGAGCTGCAGCTTGCGA

TCTCGGATGGTTTTGGAATGGAAGAACCGCGACATCTCCAACAGCTGGGCCGTGTTGAG

AATGAGCCGGACGTCGTTGAACGAGGGGGCCACAAGCCGGCGTTTGCTGATGGCGCGG

CGCTCGTCCTCGATGTACAAGGCCTTTTCCAGAGGCAGTCTCGTGAAGAAGCTGCCAAC

GCTCGGAACCAGCTGCACGAGCCGAGACAATTCGGGGGTGCCGGCTTTGGTCATTTCAA

TCTTGTCGTCGATGAGGAGTTCGAGGTCGTGGAAGATTTCCGCGTAGCGGCGTTTTGCC

TCAGAGTTTACCATGAGGTCGTCCACTGCAGAGATGCCGTTGCTCTTCACCGCGTACAG

GACCAACGGCGTCGCCAGCAGGCCCTTGATCCATTCTATGAGGCCATCTCGACGGTGTT

CCTTGAGTGCGTACTCCACTCTGTAGCGACTGGACATCTCGAGACTGGGCTTGCTGTGC

TCGATGCACCAATTAATTGTTGCCGCATGCATCCTTGCACCGCAAGTTTTTAAAACCCAC

TCGCTTTAGCCGTCGCGTAAAACTTGTGAATCTGGCAACTGAGGGGGTTCTGCAGCCGC

AACCGAACTTTTCGCTTCGAGGACGCAGCTGCATGGTGTCATGTGAGGCTCTGTTTGCT

GGCGTAGCCTACAACGTGACCTTGCCTAACCGGACGGCGCTACCCACTGCTGTCTGTGC

CTGCTACCAGAAAATCACCAGAGCAGCAGAGGCCCGATGTGGCAACTGGTGGGGTGTC

GGACAGGCTGTTTCTCCACAGTGCAAATGCGGGTGAACCGGCCAGAAAGTAAATTCTTA

TGCTACCGTGCAGCGACTCCGACATCCCCAGTTTTTGCCCTACTTGATCACAGATGGGGT

CAGCGCTGCCGCTAAGTGTACCCAACCGTGCCCACACGGTCCATCTATAAATACTGCTGC

CAGTGCACGGTGGTGACATCAATCTAAAGTACAAAAACAAATTCGAAACGATGGCTACC

TGGAGACCTTCCGAGAACAAGGTGTACCTCCCTCCAACCCCTGTGTCGAAGGTCGTTGC

TACCGACTCCTACGTCAAGAGAACCTCCATTTTCTACCACGCAGGCTCCTCTAGATTGCT

GGCCGTTGGACACCCTTATTACTCCGTTACCAAGGACAACACCAAGACTAACATCCCAA

AGGTTTCCGCCTACCAATACAGAGTGTTTAGAGTCAGACTTCCAGACCCTAACAAGTTC

GGCTTGCCTGACACGAACATCTACAACCCTGACCAGGAGCGTCTAGTCTGGGCTTGCGT

TGGTCTGGAGGTCGGCAGAGGACAGCCATTGGGTGCAGGATTATCCGGTCACCCTCTGT

TTAACAGACTCGATGACACTGAATCTTCCAACTTGGCCAACAATAACGTGATTGAGGAC

TCCAGAGACAACATCTCTGTCGACGGTAAACAAACCCAGCTCTGCATCGTTGGATGCAC

TCCTGCTATGGGTGAACACTGGACTAAGGGAGCCGTTTGTAAGTCTACCCAGGTTACTA

CCGGCGACTGTCCACCTTTGGCCTTGATTAACACCCCTATCGAGGACGGAGACATGATC

GATACTGGTTTCGGAGCAATGGACTTCAAGGTTCTTCAAGAGAGTAAGGCTGAGGTTCC

TTTGGACATCGTCCAGTCTACTTGTAAGTATCCAGACTACCTGAAGATGTCCGCCGACGC

TTACGGCGACTCCATGTGGTTCTACCTGAGAAGAGAGCAGTTGTTCGCCAGACACTACT

TCAACAGAGCCGGAAAGGTTGGTGAGACTATCCCTGCTGAACTGTACCTTAAGGGCTCC

AACGGTAGAGAGCCACCACCTTCTTCAGTTTACGTCGCTACCCCATCCGGTTCTATGATC

ACTTCCGAAGCCCAACTGTTCAACAAGCCATACTGGCTCCAGAGAGCACAGGGCCACA

ATAACGGTATTTGTTGGGGAAACCAGTTGTTCGTCACTGTTGTGGACACTACGAGATCTA

CTAACATGACGATCTCCACCGCAACAGAGCAGCTTTCTAAGTACGACGCTAGAAAGATC

AACCAGTACCTGAGACACGTGGAAGAGTACGAGTTGCAATTCGTTTTCCAGCTGTGTAA

GATCACCTTGTCCGCTGAGGTCATGGCCTACCTGCACAACATGAACGCTAACTTGCTGG

AAGACTGGAACATCGGCTTGTCCCCACCTGTCGCAACCTCTCTGGAGGACAAGTACAGA

TACGTTAGATCTACCGCAATTACTTGCCAGAGAGAGCAACCTCCAACCGAGAAGCAAGA

CCCCCTTGCCAAGTACAAGTTCTGGGACGTTAACCTGCAGGACTCTTTCAGCACTGACC

TGGACCAATTCCCTTTGGGAAGAAAGTTCTTGATGCAGTTAGGCACTAGATCTAAGCCA

GCCGTCGCTACTTCTAAGAAGAGATCTGCCCCTACCTCGACTTCTACCCCAGCTAAGAG

AAAGAGACGCTAATAGGTACCGGAGACGTGGAAGGACATACCGCTTTTGAGAAGCGTG

TTTGAAAATAGTTCTTTTTCTGGTTTATATCGTTTATGAAGTGATGAGATGAAAAGCTGAA

ATAGCGAGTATAGGAAAATTTAATGAAAATTAAATTAAATATTTTCTTAGGCTATTAGTCA

CCTTCAAAATGCCGGCCGCTTCTAAGAACGTTGTCATGATCGACAACTACGACTCGTTTA

CCTGGAACCTGTACGAGTACCTGTGTCAGGAGGGAGCCAATGTCGAGGTTTTCAGGAA

CGATCAGATCACCATTCCGGAGATTGAGCAGCTCAAGCCGGACGTTGTGGTGATATCCC

CTGGTCCTGGCCATCCAAGAACAGACTCGGGAATATCTCGCGACGTGATCAGCCATTTTA

AAGGCAAGATTCCTGTCTTTGGTGTCTGTATGGGCCAGCAGTGTATCTTCGAGGAGTTTG

GCGGAGACGTCGAGTATGCGGGCGAGATTGTCCATGGAAAAACGTCCACTGTTAAGCA

CGACAACAAGGGAATGTTCAAAAACGTTCCGCAAGATGTTGCTGTCACCAGATACCACT

CGCTGGCCGGAACGCTCAAGTCGCTTCCGGACTGTCTAGAGATCACTGCTCGCACAGAC

AACGGGATCATTATGGGTGTGAGACACAAGAAGTACACCATCGAGGGCGTCCAGTTTCA

TCCAGAGAGCATTCTGACCGAGGAGGGCCATCTGATGATCCAGAATATCCTCAACGTTTC

CGGTGGTTACTGGGAGGAAAATGCCAACGGCGCGGCTCAGAGAAAGGAAAGCATATTG

GAGAAAATATACGCGCAGAGACGAAAAGACTACGAGTTTGAGATGAACAGACCGGGGC

GCAGATTTGCTGATCTAGAACTGTACTTGTCCATGGGACTGCACCGCCGCTAATCAATTT

TTACGACAGATTGGAGCAGAACATCAGCGCCGGCAAGGTTGCAATTCTCAGCGAAATCA

AGAGAGCGTCGCCTTCTAAAGGCGTCATCGACGGAGACGCTAACGCTGCCAAACAGGC

CCTCAACTACGCCAAGGCTGGAGTTGCCACAATTTCTGTTTTGACCGAGCCAACCTGGT

TTAAAGGAAATATCCAGGACCTGGAGGTGGCCAGAAAAGCCATTGACTCTGTGGCCAAT

AGACCGTGTATTTTGCGGAAGGAGTTTATCTTCAACAAGTACCAAATTCTAGAGGCCCG

ACTGGCGGGAGCAGACACGGTTCTGCTGATTGTCAAGATGCTGAGCTCGGATCCCCCAC

ACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCG

CGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTTCCCTCTTTCTT

CCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCC

TCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGA

AATTTTTTTTTTTAGTTTTTTTCTCTTTCAGTGACCTCCATTGATATTTAAGTTAATAAACG

GTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCT

TGTTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGGGGCGGTGTTGACAATTAATCAT

CGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGT

TGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTG

GACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTC

CGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACA

CCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGT

CGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAG

CCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGG

CCGAGGAGCAGGACTGACACGTCCGACGGCGGCCCACGGGTCCCAGGCCTCGGAGATC

CGTCCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCT

CCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCC

TATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTT

TTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTT

TTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATGTGAGCAAAAGGCC

AGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCG

CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACA

GGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCG

ACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCT

CAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT

GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAG

TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAG

CAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT

ACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAA

AGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGT

TTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT

CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGA

TC

construction of recombinant expression Strain of HPV56L1 protein

The Hansenula polymorpha host used in the present invention was derived from the wild-type Hansenula polymorpha CBS4732 strain (ATCC 34438) and purchased from American type culture Collection (American type culture collection, ATCC). 56L1-1-pMTZ,56L1-2-pMTZ,56L1-3-pMTZ,56L1-4-pMTZ recombinant expression plasmids were linearized with ScaI enzyme, electrotransferred to Hansenula polymorpha, under conditions of 1500V,120Ω, 50. Mu.F, respectively. After electrotransformation, the bacterial liquid was coated on YPD plates (200. Mu.g/mL Zeocin) and cultured for 1-2 days at 37℃in an inverted manner.

EXAMPLE 2 expression screening of HPV56L1 recombinant engineering Strain

A plurality of recombinant Hansenula polymorpha single colonies were randomly picked from the electrotransformed YPD plates and inoculated into YPD liquid medium for overnight culture at 37 ℃. And (3) taking part of bacterial liquid, centrifuging, discarding YPD culture medium, adding an induction culture medium BMMY, and carrying out induction at 37 ℃ for 48 hours, and collecting bacterial cells. The acid-treated glass beads are subjected to vigorous shaking to break thalli, the broken fungus supernatant is collected after centrifugation, and the expression condition of HPV56L1 protein in the broken fungus liquid is detected by a Dot blotting method (Dot blotting).

The Dot blotting results show (figure 1) that recombinant engineering strains containing different HPV56L1 coding sequences are clearly expressed, but the HPV56L1 proteins of different coding sequences have certain differences in expression. In contrast, the expression effect of the recombinant engineering bacteria containing 56L1-3 coding sequences (FIG. 1-C) is obviously better than that of the recombinant engineering bacteria containing 56L1-1, 56L1-2 and 56L1-4 coding sequences (FIGS. 1-A, B and D).

To further verify the expression advantage of the 56L1-3 coding sequences, 3 strains were each selected from the engineering strains containing 56L1-1, 56L1-2, 56L1-3 and 56L1-4 coding sequences for expression, and the expression of HPV56L1 protein in the bacterial supernatant was quantitatively detected by enzyme-linked immunosorbent assay (ELISA). ELISA detection results are shown in the following table.

TABLE 1ELISA detection of antigen expression levels of 56L1 protein in the supernatants of different strains

EXAMPLE 3 fermentation Process of HPV56L1 recombinant Hansenula polymorpha expression Strain

Seed liquid preparation: 1 recombinant Hansenula polymorpha engineering strain containing 56L1-3 coding sequences was inoculated into sterilized 1000mL shake flask YPG medium in a clean bench (under aseptic conditions). The shake flask was placed in a constant temperature shaker for cultivation at 37℃and shaking table rotation speed 190rpm for cultivation time 24h. When the seed liquid OD ₆₀₀ When the value reaches 2.0, the shake flask culture is stopped, and the fermentation seed liquid can be stored at 4 ℃ for use after verification.

Fermenting in a fermentation tank: preparing 20L of basic culture medium according to the formula of BSM1, and sterilizing for 30min at 121 ℃ for later use. Inoculating the cultured qualified fermentation seed liquid into a 30L fermentation tank according to the proportion of 5% under the protection of flame. In the fermentation culture process, the pH is controlled at 5.0, the fermentation temperature is 37 ℃, the stirring speed is less than or equal to 950rpm, the air flow is less than or equal to 2.0VVM, the tank pressure is less than or equal to 0.10MPa, and the dissolved oxygen is more than 10%. When the glycerol in the basal medium is consumed, the wet weight of the thalli is about 100g/L, and the feeding of the glycerol is started, and the glycerol feeding speed is 200-600 g/h. When the thallus wet weight is greater than 200g/L, methanol is fed into the thallus to enter a methanol induction period, the methanol flow acceleration is gradually regulated along with the acceleration of the thallus by using the methanol, the dissolved oxygen is controlled to be more than 20% in the induction process, and the thallus is induced to finish fermentation after 30 hours. The thalli are stored at-20 ℃ for purification after high-speed centrifugation. The fermentation supernatants at different times were subjected to SDS-PAGE and Western Blot identification (FIGS. 2, 3).

The result shows that the expression condition of HPV56L1 protein is continuously increased along with the extension of the induction time, and the fermentation expression quantity meets the requirement of mass production.

In the culture process, the inventor searches to find that the pH is 5.0-7.0, the fermentation temperature is 30-37 ℃, the stirring rotation speed is less than or equal to 950rpm, the air flow is less than or equal to 2.0VVM, the tank pressure is less than or equal to 0.10MPa, and the dissolved oxygen is more than 10%; in the culture process, when the glycerol in the basic culture medium is consumed, the thallus wet weight is greater than 100g/L, and the glycerol is added at the feeding speed of 200-600 g/h; when the thallus wet weight is greater than 200g/L, methanol is added at one time, the methanol is added into the thallus wet weight induction period, after the dissolved oxygen is increased to 80% after the methanol is completely consumed, the methanol is added, the methanol flow acceleration is gradually regulated along with the acceleration of the thallus by using the methanol, the dissolved oxygen is controlled to be more than 20% in the induction process, and the thallus wet weight is induced to be 300-400 g/L for 30-50 h, and then the fermentation is finished. The above conditions are suitable for culturing strains.

EXAMPLE 4 purification Process of HPV56L1 recombinant protein

And (3) thallus crushing: taking HPV56L1 fermentation tank-placing wet thalli stored at the temperature of minus 20 ℃, adding a bacteria breaking buffer solution (containing 0.4mol/L sodium chloride and 0.1mol/L MOPS) according to the proportion of 1:4, re-suspending and uniformly stirring the thalli, filtering the bacterial suspension by using a screen, cooling the filtered bacterial suspension to the temperature of 4 ℃ by using an ice bath, crushing the bacterial suspension in the ice bath for 5 times under the pressure of 1500bar, and checking that the bacterial breaking rate is more than or equal to 80 percent by using a microscope. The crushed solution was centrifuged at 8500 for 20min at 4℃and the supernatant was collected.

Column chromatography: loading the clarified liquid to a cation chromatography column POROS HS for preliminary purification, eluting with 1.5mol/L sodium chloride solution, and collecting the eluate of the preliminary purification; and (3) loading the primarily purified protein solution to a chromatographic column CHT for refining and purifying, eluting by using 200mol/L phosphate buffer solution, and obtaining the high-purity HPV56L1 protein, wherein the SEC-HPLC purity is more than or equal to 95%. SDS-PAGE detection results of purified HPV56L1 protein are shown in FIG. 4, and SEC-HPLC detection results are shown in FIG. 5.

EXAMPLE 5 Transmission Electron microscopy of HPV56L1 recombinant protein

The purified HPV56L1 protein was dropped onto a clean plastic plate to form droplets. The copper mesh was inserted into the middle of the droplet with forceps to allow the lower surface of the copper mesh to be immersed with the liquid, and after standing at room temperature for 20 minutes, the copper mesh was taken out with forceps, and the liquid was sucked dry from the edge of the copper mesh with filter paper. And (3) placing the copper mesh adsorbed with the sample on the surface of the dye liquor, dyeing for 10 seconds at room temperature, taking out the copper mesh, sucking the redundant liquid by using filter paper, and airing. The morphology of the virus-like particles was observed by using a transmission electron microscope observation (JEM-2100, japanese electronics Co., ltd.). Transmission electron microscopy of HPV56L1 protein is shown in fig. 6.

EXAMPLE 6 preparation of HPV56L1 protein-containing vaccine

Diluting the HPV56L1 protein stock solution prepared according to the embodiment 1-4 to 250 mug/mL by using stock solution dilution buffer solution, adding 250 mug/mL aluminum phosphate adjuvant into 1mL diluted protein solution, mixing, adsorbing for 1-3 h, and obtaining HPV56L1 protein vaccine, and preserving at 4 ℃ in a dark place.

EXAMPLE 7 immunogenicity of HPV56L1 protein vaccine

Different doses of HPV56L1 vaccine are respectively administered into mice, the positive conversion rate of specific antibodies in serum is measured by an enzyme-linked immunosorbent assay (ELISA), the percentage of positive serum in each dose group is calculated, and the ED is calculated by using SPSS software ₅₀ (half effective dose) value, whereby the immunogenicity of the vaccine is assessed.

1. Immunization of animals

60 Balb/c females, 6-8 weeks old, were randomly divided into 6 groups of 10 mice per dose group. The appropriate dosage range is selected according to the antigen content of the sample, and dilution is performed by using blank aluminum adjuvant diluent according to the following table, and the sample dilution and immunization of animals need to be completely and uniformly mixed. Five injections of 0.5 mL/mouse were performed subcutaneously, 1 needle was immunized for 0 day, the orbit was sampled after 28 days, and serum was separated for detection of the positive transfer rate of neutralizing antibodies.

Animals were grouped as follows:

group of	Test article	Dosage (μg/0.5 mL)	Immunization program	A mouse
					1	HPV56L1 vaccine	0.04000	One needle for 0 day	10
2	HPV56L1 vaccine	0.01000	One needle for 0 day	10
					3	HPV56L1 vaccine	0.00250	One needle for 0 day	10
4	HPV56L1 vaccine	0.00063	One needle for 0 day	10
					5	HPV56L1 vaccine	0.00016	One needle for 0 day	10
6	Physiological saline	/	One needle for 0 day	10

ELISA method for detecting positive transfer rate of antibody in serum

The test steps are as follows: 1) Coating: the HPV 11L1 stock solution, HPV 16L1 stock solution and HPV 18L1 stock solution were diluted with phosphate buffer (0.01 mol/mL, pH 7.4), respectively, and the ELISA plates were added at 100. Mu.L/well and incubated at 4℃overnight or 37℃for 2 hours. 2) Closing: the plate was washed 6 times with 300. Mu.L/well of wash solution, 200. Mu.L of blocking solution was added to each well, and the plate was blocked at 37℃for 2 hours. 3) PBST dilutions containing 2.0% skim milk powder were used at 1: serum was diluted 1000-fold, 100 μl/well was added to the elisa plate, double well assay, incubated at 37 ℃ for 1 hour, and positive and blank controls were set. 4) Adding enzyme-labeled secondary antibodies: plates were washed 6 times with 300 μl/well of wash solution, diluted 1: the goat anti-mouse-HRP was diluted with 10000, and an ELISA plate was added at 100. Mu.L/well, and incubated at 37℃for 1 hour. 5) Color development: the plate was washed 6 times with 300. Mu.L/well of washing solution, and 100. Mu.L/well of freshly prepared developing solution was added thereto and developed at 37℃for 10 minutes. 6) Terminating the reading: the stop solution was mixed with 50. Mu.L/Kong Jiazhi plate and slightly shaken, and then read with an ELISA reader, the measurement wavelength was 450nm, and the reference wavelength was 620nm.

3. In vivo efficacy ED ₅₀ Calculation of (2)

In vivo efficacy ED of HPV56L1 vaccine calculated from antibody positive conversion results of mouse serum at different dose levels ₅₀ The value of (2) is 0.00072. Mu.g, showing that HPV56L1 vaccine has good immunogenicity.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, many modifications and variations of the methods and compositions of the invention set forth herein will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Sequence listing

<110> Chongqing Bowei Baitai biopharmaceutical Co., ltd

<120> a polynucleotide sequence for expressing HPV56L1, expression vector, host cell and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 499

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Met Ala Thr Trp Arg Pro Ser Glu Asn Lys Val Tyr Leu Pro Pro Thr

1 5 10 15

Pro Val Ser Lys Val Val Ala Thr Asp Ser Tyr Val Lys Arg Thr Ser

20 25 30

Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Ala Val Gly His Pro

35 40 45

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

50 55 60

Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn

65 70 75 80

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

85 90 95

Leu Val Trp Ala Cys Val Gly Leu Glu Val Gly Arg Gly Gln Pro Leu

100 105 110

Gly Ala Gly Leu Ser Gly His Pro Leu Phe Asn Arg Leu Asp Asp Thr

115 120 125

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

130 135 140

Asn Ile Ser Val Asp Gly Lys Gln Thr Gln Leu Cys Ile Val Gly Cys

145 150 155 160

Thr Pro Ala Met Gly Glu His Trp Thr Lys Gly Ala Val Cys Lys Ser

165 170 175

Thr Gln Val Thr Thr Gly Asp Cys Pro Pro Leu Ala Leu Ile Asn Thr

180 185 190

Pro Ile Glu Asp Gly Asp Met Ile Asp Thr Gly Phe Gly Ala Met Asp

195 200 205

Phe Lys Val Leu Gln Glu Ser Lys Ala Glu Val Pro Leu Asp Ile Val

210 215 220

Gln Ser Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ser Ala Asp Ala

225 230 235 240

Tyr Gly Asp Ser Met Trp Phe Tyr Leu Arg Arg Glu Gln Leu Phe Ala

245 250 255

Arg His Tyr Phe Asn Arg Ala Gly Lys Val Gly Glu Thr Ile Pro Ala

260 265 270

Glu Leu Tyr Leu Lys Gly Ser Asn Gly Arg Glu Pro Pro Pro Ser Ser

275 280 285

Val Tyr Val Ala Thr Pro Ser Gly Ser Met Ile Thr Ser Glu Ala Gln

290 295 300

Leu Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn

305 310 315 320

Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr

325 330 335

Arg Ser Thr Asn Met Thr Ile Ser Thr Ala Thr Glu Gln Leu Ser Lys

340 345 350

Tyr Asp Ala Arg Lys Ile Asn Gln Tyr Leu Arg His Val Glu Glu Tyr

355 360 365

Glu Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Ser Ala Glu

370 375 380

Val Met Ala Tyr Leu His Asn Met Asn Ala Asn Leu Leu Glu Asp Trp

385 390 395 400

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

405 410 415

Arg Tyr Val Arg Ser Thr Ala Ile Thr Cys Gln Arg Glu Gln Pro Pro

420 425 430

Thr Glu Lys Gln Asp Pro Leu Ala Lys Tyr Lys Phe Trp Asp Val Asn

435 440 445

Leu Gln Asp Ser Phe Ser Thr Asp Leu Asp Gln Phe Pro Leu Gly Arg

450 455 460

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

465 470 475 480

Ser Lys Lys Arg Ser Ala Pro Thr Ser Thr Ser Thr Pro Ala Lys Arg

485 490 495

Lys Arg Arg

<210> 2

<211> 1503

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atggctactt ggagaccttc cgagaacaag gtttacctgc cacctacccc agtgtctaag 60

gtcgttgcca cggactccta cgtcaagaga acctcgatct tctaccacgc aggctcgtcc 120

agattgctgg ccgttggaca cccttactac tctgtgacca aggacaacac caagacgaac 180

atcccaaagg tttccgccta ccagtacaga gtcttcagag ttagactgcc agaccctaac 240

aagtttggtc tccctgacac caacatctac aacccagacc aggagagact tgtgtgggca 300

tgtgttggtc tggaggttgg cagaggacag cctttgggtg ctggactctc tggccaccct 360

ctgtttaaca gactcgacga tactgagtcg tccaacttgg ccaacaataa cgtcattgag 420

gactccagag acaacatctc tgttgacggc aagcagaccc agttgtgcat tgtcggatgt 480

actcctgcta tgggtgaaca ctggacgaag ggtgcagtgt gcaagtccac ccaggttacc 540

acaggagact gcccacctct tgccttgatc aacacgccaa ttgaggacgg tgacatgatc 600

gacaccggat tcggtgccat ggacttcaag gtgctgcagg agtcgaaggc tgaggtccct 660

ctggacatcg ttcagtccac ctgtaagtac ccagactacc tgaagatgtc ggcagacgcc 720

tatggtgact ccatgtggtt ctacttgaga agagagcagc tctttgccag acactacttc 780

aacagagccg gcaaggtcgg agagaccatt cctgcagagc tgtacctcaa gggctcgaac 840

ggtagagaac cacctccatc ttccgtctac gttgctactc cttccggatc tatgatcacg 900

tcggaggccc agttgttcaa caagccatat tggctgcagc gtgctcaagg ccacaataac 960

ggcatctgct ggggtaacca actgttcgtg actgttgtcg acaccactag atccaccaac 1020

atgacgattt ccacagctac cgaacagctg tcgaagtacg acgccagaaa gatcaaccag 1080

tacctcagac acgtggagga atacgagttg cagttcgtct tccagctctg caagatcacc 1140

ctttctgccg aggttatggc atacctgcac aacatgaatg ccaacttgct ggaggactgg 1200

aacattggat tgtcccctcc agttgccacc agcctcgaag acaagtacag atacgtgaga 1260

tcgactgcaa tcacctgtca gagagagcag ccacctacgg agaagcagga cccacttgct 1320

aagtacaagt tctgggacgt caacctgcaa gactccttct cgaccgacct ggaccagttt 1380

cctttgggca gaaagttcct catgcaactg ggaaccagat ctaagccagc tgtcgccacc 1440

tccaagaaga gatcagcacc tacttccacc tcgacgccag ccaagagaaa gcgcagataa 1500

tag 1503

<210> 3

<211> 1503

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atggcaacct ggagaccatc tgagaacaag gtctacttgc ctccaactcc tgtttccaag 60

gttgtcgcaa ccgactcgta cgtgaagaga acgtccattt tctaccacgc cggttcctct 120

agactgctcg ctgtcggtca cccatattac tccgttacga aggacaacac gaagaccaac 180

atccctaagg tgtctgcata ccaatacaga gttttcagag tcagactccc tgatccaaac 240

aagttcggcc tgccagacac gaacatctac aaccctgacc aagagagact ggtctgggct 300

tgcgtgggat tggaggtcgg tagaggccag ccactgggag ccggtctttc cggacaccca 360

ttgttcaaca gactggatga caccgaatcc tcgaacctgg ccaataacaa cgtgatcgag 420

gacagtagag acaacatttc ggtcgatggt aaacagacgc agctgtgtat cgttggctgc 480

accccagcca tgggagagca ctggactaag ggagccgttt gtaagtctac tcaggtcaca 540

accggcgact gtcctccatt ggctctgatt aacaccccta tcgaggacgg agatatgatc 600

gacaccggtt ttggagctat ggacttcaag gttctccagg agtccaaggc cgaggtgcca 660

ttggacatcg tgcagtctac atgcaagtac cccgactatt tgaagatgtc cgccgatgct 720

tacggagact ctatgtggtt ttaccttaga cgtgagcagt tgttcgcaag acattacttc 780

aacagagccg gaaaggttgg tgagaccatc ccagctgagt tgtacctgaa gggatccaac 840

ggcagagagc ctccaccttc gtctgtgtac gtcgcaaccc caagcggttc catgattacc 900

tctgaagcac agctgtttaa caagccttac tggcttcaga gagcccaggg tcacaacaat 960

ggaatttgtt ggggcaacca gctcttcgtt accgtcgtgg acactaccag atctactaac 1020

atgaccatct cgacggccac tgagcagctt tctaagtacg acgctagaaa gatcaaccaa 1080

tacctgagac acgttgaaga gtacgagctg cagtttgtgt tccagctgtg taagattacc 1140

ctctccgctg aggttatggc ctacttgcac aacatgaacg caaacctgtt ggaggactgg 1200

aacatcggct tgtctccacc tgtcgcaacc tccctggagg acaagtacag atacgttaga 1260

tctaccgcca tcacttgcca gagagagcag cctccaaccg agaagcagga ccctctggcc 1320

aagtacaagt tctgggacgt taacttgcag gactcgttct ccaccgacct cgaccagttc 1380

ccactgggta gaaagttttt gatgcaactt ggcaccagat ccaagcctgc cgttgctact 1440

tctaagaaga gatccgcccc aacgtcgact tccacccctg ctaaaagaaa gagaagataa 1500

tag 1503

<210> 4

<211> 1503

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

atggccactt ggagaccatc tgaaaacaag gtttacttgc cacctactcc agtctccaag 60

gttgtcgcta ctgactctta cgttaagaga acttctatct tctaccacgc tggatcttcc 120

agacttttgg cagtgggcca cccatactat tcggtcacta aggacaacac taagacaaac 180

atccctaaag tgtctgctta ccagtacaga gtcttcagag ttcgtttgcc tgacccaaac 240

aagttcggat tgccagacac taacatctac aacccagacc aggaaagatt agtttgggcc 300

tgtgtcggcc tcgaagttgg aagaggtcag cctcttggtg ctggcttgtc tggacaccca 360

ctcttcaaca gattggacga tactgagtca tccaacctgg ctaacaacaa tgttatcgaa 420

gactctagag ataacatttc cgttgacgga aagcagactc agttgtgtat tgttggttgt 480

actccagcaa tgggcgagca ttggaccaag ggtgctgttt gtaagagcac tcaagttacc 540

actggtgact gccctccact ggcactcatc aacactccaa tcgaggatgg tgacatgatc 600

gacaccggct ttggtgctat ggacttcaag gtcttgcagg agtctaaagc cgaagttcct 660

ttagacattg ttcaatccac ctgcaagtac cccgactact tgaagatgtc tgctgatgcc 720

tacggtgact ctatgtggtt ctacttgcgt agagagcagc tgtttgctag acactacttc 780

aacagagctg gtaaagtcgg agaaacgatt ccagccgagt tgtacttgaa gggttctaac 840

ggaagagaac ctcctccatc ctctgtctac gttgccactc cttctggttc catgattacc 900

tctgaggctc agctctttaa taagccttac tggttgcagc gtgcccaagg tcacaacaat 960

ggaatctgct ggggtaacca gttgttcgtt actgtcgttg acaccactag atccaccaac 1020

atgacgattt ctacagctac cgaacagttg tccaagtacg atgccagaaa gatcaaccaa 1080

tacttgagac acgttgagga atacgagctt cagttcgtct ttcaattgtg caagatcact 1140

ttgtctgccg aagttatggc ttacttgcac aacatgaatg ccaacctttt ggaggactgg 1200

aacattggat tgtctcctcc agttgctacc agtttggagg acaagtacag atatgtcaga 1260

tccactgcta tcacctgtca aagagagcag ccacctactg aaaagcagga cccactggct 1320

aaatacaagt tctgggatgt caacttgcaa gactccttct ctaccgacct tgatcagttc 1380

ccattgggta gaaagttcct tatgcagttg ggaaccagat ccaaacctgc tgttgccacc 1440

tccaagaaga gatcggctcc aacttctacc tccactcctg ccaagagaaa gcgtagataa 1500

tag 1503

<210> 5

<211> 1503

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atggctacct ggagaccttc cgagaacaag gtgtacctcc ctccaacccc tgtgtcgaag 60

gtcgttgcta ccgactccta cgtcaagaga acctccattt tctaccacgc aggctcctct 120

agattgctgg ccgttggaca cccttattac tccgttacca aggacaacac caagactaac 180

atcccaaagg tttccgccta ccaatacaga gtgtttagag tcagacttcc agaccctaac 240

aagttcggct tgcctgacac gaacatctac aaccctgacc aggagcgtct agtctgggct 300

tgcgttggtc tggaggtcgg cagaggacag ccattgggtg caggattatc cggtcaccct 360

ctgtttaaca gactcgatga cactgaatct tccaacttgg ccaacaataa cgtgattgag 420

gactccagag acaacatctc tgtcgacggt aaacaaaccc agctctgcat cgttggatgc 480

actcctgcta tgggtgaaca ctggactaag ggagccgttt gtaagtctac ccaggttact 540

accggcgact gtccaccttt ggccttgatt aacaccccta tcgaggacgg agacatgatc 600

gatactggtt tcggagcaat ggacttcaag gttcttcaag agagtaaggc tgaggttcct 660

ttggacatcg tccagtctac ttgtaagtat ccagactacc tgaagatgtc cgccgacgct 720

tacggcgact ccatgtggtt ctacctgaga agagagcagt tgttcgccag acactacttc 780

aacagagccg gaaaggttgg tgagactatc cctgctgaac tgtaccttaa gggctccaac 840

ggtagagagc caccaccttc ttcagtttac gtcgctaccc catccggttc tatgatcact 900

tccgaagccc aactgttcaa caagccatac tggctccaga gagcacaggg ccacaataac 960

ggtatttgtt ggggaaacca gttgttcgtc actgttgtgg acactacgag atctactaac 1020

atgacgatct ccaccgcaac agagcagctt tctaagtacg acgctagaaa gatcaaccag 1080

tacctgagac acgtggaaga gtacgagttg caattcgttt tccagctgtg taagatcacc 1140

ttgtccgctg aggtcatggc ctacctgcac aacatgaacg ctaacttgct ggaagactgg 1200

aacatcggct tgtccccacc tgtcgcaacc tctctggagg acaagtacag atacgttaga 1260

tctaccgcaa ttacttgcca gagagagcaa cctccaaccg agaagcaaga cccccttgcc 1320

aagtacaagt tctgggacgt taacctgcag gactctttca gcactgacct ggaccaattc 1380

cctttgggaa gaaagttctt gatgcagtta ggcactagat ctaagccagc cgtcgctact 1440

tctaagaaga gatctgcccc tacctcgact tctaccccag ctaagagaaa gagacgctaa 1500

tag 1503

<210> 6

<211> 4753

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60

taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120

gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180

ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240

ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300

tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360

ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420

ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480

tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540

gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600

acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660

atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720

tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780

aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840

aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900

agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960

actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020

gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080

aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140

aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200

cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260

agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320

caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380

tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440

accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500

taaagtacaa aaacaaattc gaaacgagga attcacgtgg cccagccggc cgtctcggat 1560

cggtaccgga gacgtggaag gacataccgc ttttgagaag cgtgtttgaa aatagttctt 1620

tttctggttt atatcgttta tgaagtgatg agatgaaaag ctgaaatagc gagtatagga 1680

aaatttaatg aaaattaaat taaatatttt cttaggctat tagtcacctt caaaatgccg 1740

gccgcttcta agaacgttgt catgatcgac aactacgact cgtttacctg gaacctgtac 1800

gagtacctgt gtcaggaggg agccaatgtc gaggttttca ggaacgatca gatcaccatt 1860

ccggagattg agcagctcaa gccggacgtt gtggtgatat cccctggtcc tggccatcca 1920

agaacagact cgggaatatc tcgcgacgtg atcagccatt ttaaaggcaa gattcctgtc 1980

tttggtgtct gtatgggcca gcagtgtatc ttcgaggagt ttggcggaga cgtcgagtat 2040

gcgggcgaga ttgtccatgg aaaaacgtcc actgttaagc acgacaacaa gggaatgttc 2100

aaaaacgttc cgcaagatgt tgctgtcacc agataccact cgctggccgg aacgctcaag 2160

tcgcttccgg actgtctaga gatcactgct cgcacagaca acgggatcat tatgggtgtg 2220

agacacaaga agtacaccat cgagggcgtc cagtttcatc cagagagcat tctgaccgag 2280

gagggccatc tgatgatcca gaatatcctc aacgtttccg gtggttactg ggaggaaaat 2340

gccaacggcg cggctcagag aaaggaaagc atattggaga aaatatacgc gcagagacga 2400

aaagactacg agtttgagat gaacagaccg gggcgcagat ttgctgatct agaactgtac 2460

ttgtccatgg gactgcaccg ccgctaatca atttttacga cagattggag cagaacatca 2520

gcgccggcaa ggttgcaatt ctcagcgaaa tcaagagagc gtcgccttct aaaggcgtca 2580

tcgacggaga cgctaacgct gccaaacagg ccctcaacta cgccaaggct ggagttgcca 2640

caatttctgt tttgaccgag ccaacctggt ttaaaggaaa tatccaggac ctggaggtgg 2700

ccagaaaagc cattgactct gtggccaata gaccgtgtat tttgcggaag gagtttatct 2760

tcaacaagta ccaaattcta gaggcccgac tggcgggagc agacacggtt ctgctgattg 2820

tcaagatgct gagctcggat cccccacaca ccatagcttc aaaatgtttc tactcctttt 2880

ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc 2940

acagcatact aaattttccc tctttcttcc tctagggtgt cgttaattac ccgtactaaa 3000

ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa 3060

aaatttttat cacgtttctt tttcttgaaa tttttttttt tagttttttt ctctttcagt 3120

gacctccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt 3180

tttcttgttc tattacaact ttttttactt cttgttcatt agaaagaaag catagcaatc 3240

taatctaagg ggcggtgttg acaattaatc atcggcatag tatatcggca tagtataata 3300

cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc 3360

gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt 3420

cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt 3480

ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga 3540

gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc 3600

catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg 3660

caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtccgacggc ggcccacggg 3720

tcccaggcct cggagatccg tccccctttt cctttgtcga tatcatgtaa ttagttatgt 3780

cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac 3840

aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat 3900

ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac 3960

tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc aagctggaga 4020

ccaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 4080

cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 4140

ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 4200

tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 4260

gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc 4320

gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 4380

gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 4440

ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 4500

ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 4560

ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 4620

gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 4680

ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 4740

tggtcatgag atc 4753

<210> 7

<211> 6220

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60

taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120

gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180

ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240

ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300

tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360

ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420

ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480

tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540

gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600

acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660

atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720

tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780

aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840

aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900

agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960

actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020

gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080

aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140

aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200

cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260

agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320

caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380

tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440

accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500

taaagtacaa aaacaaattc gaaacgatgg ctacttggag accttccgag aacaaggttt 1560

acctgccacc taccccagtg tctaaggtcg ttgccacgga ctcctacgtc aagagaacct 1620

cgatcttcta ccacgcaggc tcgtccagat tgctggccgt tggacaccct tactactctg 1680

tgaccaagga caacaccaag acgaacatcc caaaggtttc cgcctaccag tacagagtct 1740

tcagagttag actgccagac cctaacaagt ttggtctccc tgacaccaac atctacaacc 1800

cagaccagga gagacttgtg tgggcatgtg ttggtctgga ggttggcaga ggacagcctt 1860

tgggtgctgg actctctggc caccctctgt ttaacagact cgacgatact gagtcgtcca 1920

acttggccaa caataacgtc attgaggact ccagagacaa catctctgtt gacggcaagc 1980

agacccagtt gtgcattgtc ggatgtactc ctgctatggg tgaacactgg acgaagggtg 2040

cagtgtgcaa gtccacccag gttaccacag gagactgccc acctcttgcc ttgatcaaca 2100

cgccaattga ggacggtgac atgatcgaca ccggattcgg tgccatggac ttcaaggtgc 2160

tgcaggagtc gaaggctgag gtccctctgg acatcgttca gtccacctgt aagtacccag 2220

actacctgaa gatgtcggca gacgcctatg gtgactccat gtggttctac ttgagaagag 2280

agcagctctt tgccagacac tacttcaaca gagccggcaa ggtcggagag accattcctg 2340

cagagctgta cctcaagggc tcgaacggta gagaaccacc tccatcttcc gtctacgttg 2400

ctactccttc cggatctatg atcacgtcgg aggcccagtt gttcaacaag ccatattggc 2460

tgcagcgtgc tcaaggccac aataacggca tctgctgggg taaccaactg ttcgtgactg 2520

ttgtcgacac cactagatcc accaacatga cgatttccac agctaccgaa cagctgtcga 2580

agtacgacgc cagaaagatc aaccagtacc tcagacacgt ggaggaatac gagttgcagt 2640

tcgtcttcca gctctgcaag atcacccttt ctgccgaggt tatggcatac ctgcacaaca 2700

tgaatgccaa cttgctggag gactggaaca ttggattgtc ccctccagtt gccaccagcc 2760

tcgaagacaa gtacagatac gtgagatcga ctgcaatcac ctgtcagaga gagcagccac 2820

ctacggagaa gcaggaccca cttgctaagt acaagttctg ggacgtcaac ctgcaagact 2880

ccttctcgac cgacctggac cagtttcctt tgggcagaaa gttcctcatg caactgggaa 2940

ccagatctaa gccagctgtc gccacctcca agaagagatc agcacctact tccacctcga 3000

cgccagccaa gagaaagcgc agataatagg taccggagac gtggaaggac ataccgcttt 3060

tgagaagcgt gtttgaaaat agttcttttt ctggtttata tcgtttatga agtgatgaga 3120

tgaaaagctg aaatagcgag tataggaaaa tttaatgaaa attaaattaa atattttctt 3180

aggctattag tcaccttcaa aatgccggcc gcttctaaga acgttgtcat gatcgacaac 3240

tacgactcgt ttacctggaa cctgtacgag tacctgtgtc aggagggagc caatgtcgag 3300

gttttcagga acgatcagat caccattccg gagattgagc agctcaagcc ggacgttgtg 3360

gtgatatccc ctggtcctgg ccatccaaga acagactcgg gaatatctcg cgacgtgatc 3420

agccatttta aaggcaagat tcctgtcttt ggtgtctgta tgggccagca gtgtatcttc 3480

gaggagtttg gcggagacgt cgagtatgcg ggcgagattg tccatggaaa aacgtccact 3540

gttaagcacg acaacaaggg aatgttcaaa aacgttccgc aagatgttgc tgtcaccaga 3600

taccactcgc tggccggaac gctcaagtcg cttccggact gtctagagat cactgctcgc 3660

acagacaacg ggatcattat gggtgtgaga cacaagaagt acaccatcga gggcgtccag 3720

tttcatccag agagcattct gaccgaggag ggccatctga tgatccagaa tatcctcaac 3780

gtttccggtg gttactggga ggaaaatgcc aacggcgcgg ctcagagaaa ggaaagcata 3840

ttggagaaaa tatacgcgca gagacgaaaa gactacgagt ttgagatgaa cagaccgggg 3900

cgcagatttg ctgatctaga actgtacttg tccatgggac tgcaccgccg ctaatcaatt 3960

tttacgacag attggagcag aacatcagcg ccggcaaggt tgcaattctc agcgaaatca 4020

agagagcgtc gccttctaaa ggcgtcatcg acggagacgc taacgctgcc aaacaggccc 4080

tcaactacgc caaggctgga gttgccacaa tttctgtttt gaccgagcca acctggttta 4140

aaggaaatat ccaggacctg gaggtggcca gaaaagccat tgactctgtg gccaatagac 4200

cgtgtatttt gcggaaggag tttatcttca acaagtacca aattctagag gcccgactgg 4260

cgggagcaga cacggttctg ctgattgtca agatgctgag ctcggatccc ccacacacca 4320

tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat 4380

cgccgtacca cttcaaaaca cccaagcaca gcatactaaa ttttccctct ttcttcctct 4440

agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt 4500

ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaattt 4560

ttttttttag tttttttctc tttcagtgac ctccattgat atttaagtta ataaacggtc 4620

ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt 4680

gttcattaga aagaaagcat agcaatctaa tctaaggggc ggtgttgaca attaatcatc 4740

ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 4800

gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 4860

cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 4920

cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 4980

ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 5040

gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 5100

ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 5160

ctgacacgtc cgacggcggc ccacgggtcc caggcctcgg agatccgtcc cccttttcct 5220

ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg 5280

ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta 5340

tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 5400

gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct 5460

cgaaggcttt aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca 5520

ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5580

atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5640

aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5700

gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5760

ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5820

ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5880

acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5940

gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 6000

ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6060

ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 6120

gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6180

ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc 6220

<210> 8

<211> 6220

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60

taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120

gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180

ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240

ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300

tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360

ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420

ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480

tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540

gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600

acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660

atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720

tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780

aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840

aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900

agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960

actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020

gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080

aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140

aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200

cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260

agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320

caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380

tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440

accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500

taaagtacaa aaacaaattc gaaacgatgg caacctggag accatctgag aacaaggtct 1560

acttgcctcc aactcctgtt tccaaggttg tcgcaaccga ctcgtacgtg aagagaacgt 1620

ccattttcta ccacgccggt tcctctagac tgctcgctgt cggtcaccca tattactccg 1680

ttacgaagga caacacgaag accaacatcc ctaaggtgtc tgcataccaa tacagagttt 1740

tcagagtcag actccctgat ccaaacaagt tcggcctgcc agacacgaac atctacaacc 1800

ctgaccaaga gagactggtc tgggcttgcg tgggattgga ggtcggtaga ggccagccac 1860

tgggagccgg tctttccgga cacccattgt tcaacagact ggatgacacc gaatcctcga 1920

acctggccaa taacaacgtg atcgaggaca gtagagacaa catttcggtc gatggtaaac 1980

agacgcagct gtgtatcgtt ggctgcaccc cagccatggg agagcactgg actaagggag 2040

ccgtttgtaa gtctactcag gtcacaaccg gcgactgtcc tccattggct ctgattaaca 2100

cccctatcga ggacggagat atgatcgaca ccggttttgg agctatggac ttcaaggttc 2160

tccaggagtc caaggccgag gtgccattgg acatcgtgca gtctacatgc aagtaccccg 2220

actatttgaa gatgtccgcc gatgcttacg gagactctat gtggttttac cttagacgtg 2280

agcagttgtt cgcaagacat tacttcaaca gagccggaaa ggttggtgag accatcccag 2340

ctgagttgta cctgaaggga tccaacggca gagagcctcc accttcgtct gtgtacgtcg 2400

caaccccaag cggttccatg attacctctg aagcacagct gtttaacaag ccttactggc 2460

ttcagagagc ccagggtcac aacaatggaa tttgttgggg caaccagctc ttcgttaccg 2520

tcgtggacac taccagatct actaacatga ccatctcgac ggccactgag cagctttcta 2580

agtacgacgc tagaaagatc aaccaatacc tgagacacgt tgaagagtac gagctgcagt 2640

ttgtgttcca gctgtgtaag attaccctct ccgctgaggt tatggcctac ttgcacaaca 2700

tgaacgcaaa cctgttggag gactggaaca tcggcttgtc tccacctgtc gcaacctccc 2760

tggaggacaa gtacagatac gttagatcta ccgccatcac ttgccagaga gagcagcctc 2820

caaccgagaa gcaggaccct ctggccaagt acaagttctg ggacgttaac ttgcaggact 2880

cgttctccac cgacctcgac cagttcccac tgggtagaaa gtttttgatg caacttggca 2940

ccagatccaa gcctgccgtt gctacttcta agaagagatc cgccccaacg tcgacttcca 3000

cccctgctaa aagaaagaga agataatagg taccggagac gtggaaggac ataccgcttt 3060

tgagaagcgt gtttgaaaat agttcttttt ctggtttata tcgtttatga agtgatgaga 3120

tgaaaagctg aaatagcgag tataggaaaa tttaatgaaa attaaattaa atattttctt 3180

aggctattag tcaccttcaa aatgccggcc gcttctaaga acgttgtcat gatcgacaac 3240

tacgactcgt ttacctggaa cctgtacgag tacctgtgtc aggagggagc caatgtcgag 3300

gttttcagga acgatcagat caccattccg gagattgagc agctcaagcc ggacgttgtg 3360

gtgatatccc ctggtcctgg ccatccaaga acagactcgg gaatatctcg cgacgtgatc 3420

agccatttta aaggcaagat tcctgtcttt ggtgtctgta tgggccagca gtgtatcttc 3480

gaggagtttg gcggagacgt cgagtatgcg ggcgagattg tccatggaaa aacgtccact 3540

gttaagcacg acaacaaggg aatgttcaaa aacgttccgc aagatgttgc tgtcaccaga 3600

taccactcgc tggccggaac gctcaagtcg cttccggact gtctagagat cactgctcgc 3660

acagacaacg ggatcattat gggtgtgaga cacaagaagt acaccatcga gggcgtccag 3720

tttcatccag agagcattct gaccgaggag ggccatctga tgatccagaa tatcctcaac 3780

gtttccggtg gttactggga ggaaaatgcc aacggcgcgg ctcagagaaa ggaaagcata 3840

ttggagaaaa tatacgcgca gagacgaaaa gactacgagt ttgagatgaa cagaccgggg 3900

cgcagatttg ctgatctaga actgtacttg tccatgggac tgcaccgccg ctaatcaatt 3960

tttacgacag attggagcag aacatcagcg ccggcaaggt tgcaattctc agcgaaatca 4020

agagagcgtc gccttctaaa ggcgtcatcg acggagacgc taacgctgcc aaacaggccc 4080

tcaactacgc caaggctgga gttgccacaa tttctgtttt gaccgagcca acctggttta 4140

aaggaaatat ccaggacctg gaggtggcca gaaaagccat tgactctgtg gccaatagac 4200

cgtgtatttt gcggaaggag tttatcttca acaagtacca aattctagag gcccgactgg 4260

cgggagcaga cacggttctg ctgattgtca agatgctgag ctcggatccc ccacacacca 4320

tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat 4380

cgccgtacca cttcaaaaca cccaagcaca gcatactaaa ttttccctct ttcttcctct 4440

agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt 4500

ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaattt 4560

ttttttttag tttttttctc tttcagtgac ctccattgat atttaagtta ataaacggtc 4620

ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt 4680

gttcattaga aagaaagcat agcaatctaa tctaaggggc ggtgttgaca attaatcatc 4740

ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 4800

gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 4860

cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 4920

cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 4980

ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 5040

gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 5100

ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 5160

ctgacacgtc cgacggcggc ccacgggtcc caggcctcgg agatccgtcc cccttttcct 5220

ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg 5280

ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta 5340

tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 5400

gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct 5460

cgaaggcttt aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca 5520

ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5580

atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5640

aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5700

gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5760

ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5820

ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5880

acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5940

gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 6000

ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6060

ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 6120

gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6180

ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc 6220

<210> 9

<211> 6220

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60

taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120

gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180

ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240

ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300

tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360

ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420

ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480

tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540

gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600

acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660

atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720

tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780

aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840

aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900

agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960

actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020

gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080

aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140

aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200

cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260

agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320

caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380

tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440

accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500

taaagtacaa aaacaaattc gaaacgatgg ccacttggag accatctgaa aacaaggttt 1560

acttgccacc tactccagtc tccaaggttg tcgctactga ctcttacgtt aagagaactt 1620

ctatcttcta ccacgctgga tcttccagac ttttggcagt gggccaccca tactattcgg 1680

tcactaagga caacactaag acaaacatcc ctaaagtgtc tgcttaccag tacagagtct 1740

tcagagttcg tttgcctgac ccaaacaagt tcggattgcc agacactaac atctacaacc 1800

cagaccagga aagattagtt tgggcctgtg tcggcctcga agttggaaga ggtcagcctc 1860

ttggtgctgg cttgtctgga cacccactct tcaacagatt ggacgatact gagtcatcca 1920

acctggctaa caacaatgtt atcgaagact ctagagataa catttccgtt gacggaaagc 1980

agactcagtt gtgtattgtt ggttgtactc cagcaatggg cgagcattgg accaagggtg 2040

ctgtttgtaa gagcactcaa gttaccactg gtgactgccc tccactggca ctcatcaaca 2100

ctccaatcga ggatggtgac atgatcgaca ccggctttgg tgctatggac ttcaaggtct 2160

tgcaggagtc taaagccgaa gttcctttag acattgttca atccacctgc aagtaccccg 2220

actacttgaa gatgtctgct gatgcctacg gtgactctat gtggttctac ttgcgtagag 2280

agcagctgtt tgctagacac tacttcaaca gagctggtaa agtcggagaa acgattccag 2340

ccgagttgta cttgaagggt tctaacggaa gagaacctcc tccatcctct gtctacgttg 2400

ccactccttc tggttccatg attacctctg aggctcagct ctttaataag ccttactggt 2460

tgcagcgtgc ccaaggtcac aacaatggaa tctgctgggg taaccagttg ttcgttactg 2520

tcgttgacac cactagatcc accaacatga cgatttctac agctaccgaa cagttgtcca 2580

agtacgatgc cagaaagatc aaccaatact tgagacacgt tgaggaatac gagcttcagt 2640

tcgtctttca attgtgcaag atcactttgt ctgccgaagt tatggcttac ttgcacaaca 2700

tgaatgccaa ccttttggag gactggaaca ttggattgtc tcctccagtt gctaccagtt 2760

tggaggacaa gtacagatat gtcagatcca ctgctatcac ctgtcaaaga gagcagccac 2820

ctactgaaaa gcaggaccca ctggctaaat acaagttctg ggatgtcaac ttgcaagact 2880

ccttctctac cgaccttgat cagttcccat tgggtagaaa gttccttatg cagttgggaa 2940

ccagatccaa acctgctgtt gccacctcca agaagagatc ggctccaact tctacctcca 3000

ctcctgccaa gagaaagcgt agataatagg taccggagac gtggaaggac ataccgcttt 3060

tgagaagcgt gtttgaaaat agttcttttt ctggtttata tcgtttatga agtgatgaga 3120

tgaaaagctg aaatagcgag tataggaaaa tttaatgaaa attaaattaa atattttctt 3180

aggctattag tcaccttcaa aatgccggcc gcttctaaga acgttgtcat gatcgacaac 3240

tacgactcgt ttacctggaa cctgtacgag tacctgtgtc aggagggagc caatgtcgag 3300

gttttcagga acgatcagat caccattccg gagattgagc agctcaagcc ggacgttgtg 3360

gtgatatccc ctggtcctgg ccatccaaga acagactcgg gaatatctcg cgacgtgatc 3420

agccatttta aaggcaagat tcctgtcttt ggtgtctgta tgggccagca gtgtatcttc 3480

gaggagtttg gcggagacgt cgagtatgcg ggcgagattg tccatggaaa aacgtccact 3540

gttaagcacg acaacaaggg aatgttcaaa aacgttccgc aagatgttgc tgtcaccaga 3600

taccactcgc tggccggaac gctcaagtcg cttccggact gtctagagat cactgctcgc 3660

acagacaacg ggatcattat gggtgtgaga cacaagaagt acaccatcga gggcgtccag 3720

tttcatccag agagcattct gaccgaggag ggccatctga tgatccagaa tatcctcaac 3780

gtttccggtg gttactggga ggaaaatgcc aacggcgcgg ctcagagaaa ggaaagcata 3840

ttggagaaaa tatacgcgca gagacgaaaa gactacgagt ttgagatgaa cagaccgggg 3900

cgcagatttg ctgatctaga actgtacttg tccatgggac tgcaccgccg ctaatcaatt 3960

tttacgacag attggagcag aacatcagcg ccggcaaggt tgcaattctc agcgaaatca 4020

agagagcgtc gccttctaaa ggcgtcatcg acggagacgc taacgctgcc aaacaggccc 4080

tcaactacgc caaggctgga gttgccacaa tttctgtttt gaccgagcca acctggttta 4140

aaggaaatat ccaggacctg gaggtggcca gaaaagccat tgactctgtg gccaatagac 4200

cgtgtatttt gcggaaggag tttatcttca acaagtacca aattctagag gcccgactgg 4260

cgggagcaga cacggttctg ctgattgtca agatgctgag ctcggatccc ccacacacca 4320

tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat 4380

cgccgtacca cttcaaaaca cccaagcaca gcatactaaa ttttccctct ttcttcctct 4440

agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt 4500

ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaattt 4560

ttttttttag tttttttctc tttcagtgac ctccattgat atttaagtta ataaacggtc 4620

ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt 4680

gttcattaga aagaaagcat agcaatctaa tctaaggggc ggtgttgaca attaatcatc 4740

ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 4800

gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 4860

cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 4920

cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 4980

ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 5040

gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 5100

ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 5160

ctgacacgtc cgacggcggc ccacgggtcc caggcctcgg agatccgtcc cccttttcct 5220

ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg 5280

ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta 5340

tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 5400

gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct 5460

cgaaggcttt aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca 5520

ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5580

atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5640

aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5700

gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5760

ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5820

ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5880

acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5940

gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 6000

ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6060

ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 6120

gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6180

ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc 6220

<210> 10

<211> 6220

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60

taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120

gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180

ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240

ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300

tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360

ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420

ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480

tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540

gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600

acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660

atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720

tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780

aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840

aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900

agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960

actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020

gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080

aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140

aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200

cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260

agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320

caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380

tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440

accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500

taaagtacaa aaacaaattc gaaacgatgg ctacctggag accttccgag aacaaggtgt 1560

acctccctcc aacccctgtg tcgaaggtcg ttgctaccga ctcctacgtc aagagaacct 1620

ccattttcta ccacgcaggc tcctctagat tgctggccgt tggacaccct tattactccg 1680

ttaccaagga caacaccaag actaacatcc caaaggtttc cgcctaccaa tacagagtgt 1740

ttagagtcag acttccagac cctaacaagt tcggcttgcc tgacacgaac atctacaacc 1800

ctgaccagga gcgtctagtc tgggcttgcg ttggtctgga ggtcggcaga ggacagccat 1860

tgggtgcagg attatccggt caccctctgt ttaacagact cgatgacact gaatcttcca 1920

acttggccaa caataacgtg attgaggact ccagagacaa catctctgtc gacggtaaac 1980

aaacccagct ctgcatcgtt ggatgcactc ctgctatggg tgaacactgg actaagggag 2040

ccgtttgtaa gtctacccag gttactaccg gcgactgtcc acctttggcc ttgattaaca 2100

cccctatcga ggacggagac atgatcgata ctggtttcgg agcaatggac ttcaaggttc 2160

ttcaagagag taaggctgag gttcctttgg acatcgtcca gtctacttgt aagtatccag 2220

actacctgaa gatgtccgcc gacgcttacg gcgactccat gtggttctac ctgagaagag 2280

agcagttgtt cgccagacac tacttcaaca gagccggaaa ggttggtgag actatccctg 2340

ctgaactgta ccttaagggc tccaacggta gagagccacc accttcttca gtttacgtcg 2400

ctaccccatc cggttctatg atcacttccg aagcccaact gttcaacaag ccatactggc 2460

tccagagagc acagggccac aataacggta tttgttgggg aaaccagttg ttcgtcactg 2520

ttgtggacac tacgagatct actaacatga cgatctccac cgcaacagag cagctttcta 2580

agtacgacgc tagaaagatc aaccagtacc tgagacacgt ggaagagtac gagttgcaat 2640

tcgttttcca gctgtgtaag atcaccttgt ccgctgaggt catggcctac ctgcacaaca 2700

tgaacgctaa cttgctggaa gactggaaca tcggcttgtc cccacctgtc gcaacctctc 2760

tggaggacaa gtacagatac gttagatcta ccgcaattac ttgccagaga gagcaacctc 2820

caaccgagaa gcaagacccc cttgccaagt acaagttctg ggacgttaac ctgcaggact 2880

ctttcagcac tgacctggac caattccctt tgggaagaaa gttcttgatg cagttaggca 2940

ctagatctaa gccagccgtc gctacttcta agaagagatc tgcccctacc tcgacttcta 3000

ccccagctaa gagaaagaga cgctaatagg taccggagac gtggaaggac ataccgcttt 3060

tgagaagcgt gtttgaaaat agttcttttt ctggtttata tcgtttatga agtgatgaga 3120

tgaaaagctg aaatagcgag tataggaaaa tttaatgaaa attaaattaa atattttctt 3180

aggctattag tcaccttcaa aatgccggcc gcttctaaga acgttgtcat gatcgacaac 3240

tacgactcgt ttacctggaa cctgtacgag tacctgtgtc aggagggagc caatgtcgag 3300

gttttcagga acgatcagat caccattccg gagattgagc agctcaagcc ggacgttgtg 3360

gtgatatccc ctggtcctgg ccatccaaga acagactcgg gaatatctcg cgacgtgatc 3420

agccatttta aaggcaagat tcctgtcttt ggtgtctgta tgggccagca gtgtatcttc 3480

gaggagtttg gcggagacgt cgagtatgcg ggcgagattg tccatggaaa aacgtccact 3540

gttaagcacg acaacaaggg aatgttcaaa aacgttccgc aagatgttgc tgtcaccaga 3600

taccactcgc tggccggaac gctcaagtcg cttccggact gtctagagat cactgctcgc 3660

acagacaacg ggatcattat gggtgtgaga cacaagaagt acaccatcga gggcgtccag 3720

tttcatccag agagcattct gaccgaggag ggccatctga tgatccagaa tatcctcaac 3780

gtttccggtg gttactggga ggaaaatgcc aacggcgcgg ctcagagaaa ggaaagcata 3840

ttggagaaaa tatacgcgca gagacgaaaa gactacgagt ttgagatgaa cagaccgggg 3900

cgcagatttg ctgatctaga actgtacttg tccatgggac tgcaccgccg ctaatcaatt 3960

tttacgacag attggagcag aacatcagcg ccggcaaggt tgcaattctc agcgaaatca 4020

agagagcgtc gccttctaaa ggcgtcatcg acggagacgc taacgctgcc aaacaggccc 4080

tcaactacgc caaggctgga gttgccacaa tttctgtttt gaccgagcca acctggttta 4140

aaggaaatat ccaggacctg gaggtggcca gaaaagccat tgactctgtg gccaatagac 4200

cgtgtatttt gcggaaggag tttatcttca acaagtacca aattctagag gcccgactgg 4260

cgggagcaga cacggttctg ctgattgtca agatgctgag ctcggatccc ccacacacca 4320

tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat 4380

cgccgtacca cttcaaaaca cccaagcaca gcatactaaa ttttccctct ttcttcctct 4440

agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt 4500

ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaattt 4560

ttttttttag tttttttctc tttcagtgac ctccattgat atttaagtta ataaacggtc 4620

ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt 4680

gttcattaga aagaaagcat agcaatctaa tctaaggggc ggtgttgaca attaatcatc 4740

ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 4800

gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 4860

cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 4920

cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 4980

ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 5040

gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 5100

ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 5160

ctgacacgtc cgacggcggc ccacgggtcc caggcctcgg agatccgtcc cccttttcct 5220

ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg 5280

ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta 5340

tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 5400

gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct 5460

cgaaggcttt aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca 5520

ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5580

atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5640

aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5700

gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5760

ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5820

ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5880

acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5940

gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 6000

ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6060

ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 6120

gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6180

ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc 6220

Claims (10)

1. A polynucleotide for encoding HPV56L1 protein, wherein the polynucleotide is set forth in SEQ ID NO: 4.

2. A recombinant expression vector comprising the polynucleotide of claim 1.

3. A host cell comprising or having integrated therein the recombinant expression vector of claim 2.

4. The host cell of claim 3, wherein the host cell is Hansenula polymorpha.

5. A method for preparing a recombinant hansenula species expressing HPV56L1 protein, the method comprising the steps of:

1) The nucleotide sequence is shown as SEQ ID NO:4, inserting the polynucleotide shown in the figure into an expression vector to construct a recombinant expression vector;

2) Transforming the recombinant expression vector obtained in the step 1) into Hansenula species;

3) Culturing the hansenula polymorpha strain obtained in the step 2) to obtain a recombinant hansenula polymorpha strain containing exogenous polynucleotides.

6. A method of producing HPV56L1 protein, the method comprising: construction of a nucleotide sequence such as SEQ ID NO:4, culturing recombinant hansenula polymorpha of the polynucleotide shown in the formula 4, collecting thalli, crushing the thalli to obtain lysate, and separating and purifying the lysate to obtain HPV56L1 protein.

7. The method of claim 6, wherein the polynucleotide sequence is integrated into a plasmid, and the hansenula contains the plasmid.

8. The method according to claim 6, wherein the pH is 5.0-7.0, the fermentation temperature is 30-37 ℃, the stirring speed is less than or equal to 950rpm, the air flow is less than or equal to 2.0VVM, the tank pressure is less than or equal to 0.10MPa, and the dissolved oxygen is more than 10%.

9. The method according to claim 6, wherein the glycerol feed rate is 200-600 g/h when the glycerol in the basal medium is consumed during the culture process and the cell wet weight is greater than 100 g/L; when the thallus wet weight is greater than 200g/L, methanol is added to 0.5% (w/v) at a time, the methanol is introduced into a methanol induction period, after the dissolved oxygen is increased to 80% after the methanol is completely consumed, the methanol is fed in, the methanol flow acceleration is gradually regulated along with the acceleration of the thallus by using the methanol, the dissolved oxygen is controlled to be more than 20% in the induction process, the thallus wet weight is induced to be 300-400 g/L for 30-50 h, and then fermentation is finished.

10. The method according to claim 6, wherein the separation and purification are performed by subjecting the cell lysate to cation exchange chromatography followed by CHT chromatography.

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