CN113667683A - Polynucleotide for expressing HPV39L1, expression vector, host cell and application thereof - Google Patents

Abstract

The invention provides a polynucleotide for expressing HPV39L1, an expression vector, a host cell and application thereof. The HPV39L1 protein produced by the polynucleotide has high yield. The HPV39L1 protein prepared by the method can be used for preparing vaccines for preventing HPV39 infection.

Description

Polynucleotide for expressing HPV39L1, expression vector, host cell and application thereof

Technical Field

The invention relates to the technical field of biology, relates to a method for producing HPV39L1 protein, and particularly relates to an expression HPV39L1 polynucleotide, an expression vector, a host cell and application thereof.

Background

Human Papillomaviruses (HPV) are non-enveloped small double-stranded circular DNA viruses belonging to the papovaviridae family, members of the genus papillomavirus a. To date, the HPV viruses have identified over 200 genotypes, of which 13 human papillomaviruses may induce carcinogenesis after persistent infection, and are considered high-risk HPV (hrHPV). According to data published by the International Agency for Research on Cancer (IARC), HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59 has been shown to convert infected cells into malignant cells and thereby initiate cervical Cancer. Biological agents a review of human cardio genetics. IARC simple Eval cardio circlis Hum 2012; 100B ] HPV viruses appear spherical with a diameter of about 60nm when observed under an electron microscope, and are virus particles composed of a nucleic acid of about 8000 base pairs surrounded by a capsid with regular icosahedral symmetry. [ Knipe, 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 is used as a transcription template, and comprises ten open reading frames, an early region (early region, E) encoding three genomic regions of the coding strand, i.e., 6 viral regulatory proteins (E1, E2, E4, E5, E6 and E7), a late region (late region, L) encoding two viral capsid proteins L1 and L2, and a Long Control Region (LCR) regulating replication, transcription and translation of the viral genome.

The antigenic component of the preventive HPV vaccines currently on the market is mainly Virus-like particles (VLPs) consisting of capsid protein (L1). VLP is recombinant protein expressed by genetic engineering means, namely, viral capsid protein is produced by a heterologous recombinant expression system, and the expression product is purified to obtain virus-like particles which do not contain viral nucleic acid and have a spatial structure similar to that of natural viruses. The VLP vaccine has no capability of infecting host because of lack of virus genetic material, but the characteristic close to natural virus structure can stimulate the organism to generate effective humoral immunity and cellular immunity, thus playing the role of preventing infection and disease. The vaccine produced by the strategy method has single and stable component, strong immunogenicity and higher safety. The Global Vaccine council (Global Advisory Committee on Vaccine Safety, GACVS) WHO collaborated with the World Health Organization (WHO) regularly organized review of Safety data relating to HPV vaccines, summarized data over 2.7 billion doses post vaccination in the last review of 20 months, 7, 2017, with the conclusion that: HPV vaccines are very safe and there is currently no clear evidence that HPV vaccines are associated with any serious side effects or major medical conditions. (GACVS. safety update of HPV vaccines. https:// www.who.int/vaccine _ safety/committee/topics/HPV/June _ 2017/en/; 2017.]

A large number of studies indicate that the HPV major capsid protein L1 can be expressed in a variety of expression systems and assembled into virus-like particles with morphological structures similar to native HPV without the aid of the minor capsid protein L2. Currently, three companies' prophylactic HPV vaccines are on the market: bivalent vaccine of Kurarin Schke

(HPV16, 18), tetravalent vaccine from Moshadong

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

(HPV 6, 11, 16, 18, 31, 33, 45, 52, 58), and Xiamengtai sea Biotechnology Limited bivalent vaccine

(HPV16, 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 protein, and the purified antigen adsorbs an adjuvant to prepare the VLP vaccine for preventing HPV infection.

However, as a high-risk HPV capable of inducing malignant tumors such as cervical cancer, the HPV39 has not been reported to express the VLP assembled by HPV39L1 protein by using Hansenula polymorpha.

Disclosure of Invention

The invention aims to provide a polynucleotide for expressing HPV39L1, an expression vector, a host cell and application thereof.

In one aspect, the present invention provides a polynucleotide for encoding HPV39L1 protein, the sequence of the polynucleotide is as shown in SEQ ID NO: 3, respectively.

Further, the amino acid sequence of the HPV39L1 protein is shown as SEQ ID NO: 1 is shown.

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

Further, the recombinant expression vector is a vector obtained by converting a nucleotide sequence shown as SEQ ID NO: 3 into a plasmid. The plasmid may be one 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 TT.

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

Further, the host cell is a yeast.

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

In a fourth aspect, the present invention provides a method for producing HPV39L1 protein, the method comprising: constructing a polypeptide comprising a nucleotide sequence integrated with or comprising a nucleotide sequence set forth in SEQ ID NO: 3, culturing the recombinant hansenula polymorpha of the polynucleotide shown in the formula 3, collecting thalli, crushing the thalli to obtain lysate, and separating and purifying the lysate to obtain the HPV39L1 protein.

Further, the polynucleotide is integrated into a plasmid, which is contained in the Hansenula.

Further, the conditions of the culture include: the pH value is 5.0-7.0, 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%.

Further, culturing the recombinant Hansenula polymorpha strain in a culture medium containing glycerol; in the culture process, when the glycerol in the culture medium is completely consumed and the wet weight of the thalli is more than 100g/L, adding the glycerol at a feed rate of 200-600 g/h; and when the wet weight of the thalli is more than 200g/L, starting to add methanol to 0.5% (w/v) at one time, entering a methanol induction period, starting to add methanol in a flowing manner when the methanol is completely consumed and the dissolved oxygen rises to 80%, gradually adjusting the methanol flowing speed along with the acceleration of the thalli by using the methanol, controlling the dissolved oxygen to be more than 20% in the induction process, and finishing fermentation after the wet weight of the thalli reaches 300-400 g/L after induction is carried out for 30-50 hours.

Further, the separation and purification means that the lysate of the cells is passed through a cation chromatographic column and then a chromatographic column CHT.

Further, the cation exchange chromatography packing is POROS HS or Nanogel SP or the like.

In a fifth aspect, the invention provides an HPV39L1 protein, obtained by the method for producing the HPV39L1 protein.

The sixth aspect of the present invention provides the use of the aforementioned polynucleotide for encoding HPV39L1 protein, or recombinant expression vector, or host cell, or HPV39L1 protein, in the preparation of an HPV vaccine.

The seventh aspect of the invention provides a method for preparing an anti-HPV vaccine, which comprises the following steps: the HPV39L1 protein is prepared by the method for producing the HPV39L1 protein, and a pharmaceutically acceptable vaccine adjuvant is added.

The eighth aspect of the invention provides an anti-HPV vaccine, which is obtained by adopting the preparation method of the anti-HPV vaccine.

The beneficial technical effects are as follows: the present invention provides SEQ ID NO: 3, the yield of the encoded HPV39L1 protein is far higher than that of other polynucleotide sequences. The hansenula polymorpha serving as a eukaryotic unicellular organism has the advantages of low culture cost, rapid growth, clear molecular biological background and the like, and compared with a prokaryotic expression system, the hansenula polymorpha has a more complete 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), the hansenula polymorpha has the advantages of stable genetic character, high yield and more reasonable glycosylation of products, and can avoid the problems of low integrated copy number of exogenous genes of pichia pastoris and the like.

Drawings

FIG. 1: the pMTZ vector of one embodiment of the present invention is a structural diagram.

FIG. 2: the structure of the 39L1-1-pMTZ vector of one embodiment of the invention.

FIG. 3: the structure of the 39L1-2-pMTZ vector of one embodiment of the invention.

FIG. 4: the structure of the 39L1-3-pMTZ vector of one embodiment of the invention.

FIG. 5: the structure of the 39L1-4-pMTZ vector of one embodiment of the invention.

FIG. 6: enzyme-linked immunosorbent assay is used for detecting the 39L1 protein expression condition of the recombinant hansenula polymorpha engineering strain containing different nucleotide coding sequences of 39L1-1, 39L1-2, 39L1-3 and 39L 1-4;

FIG. 7: SDS-PAGE detection of HPV39L1 protein expression during fermentation. M: a molecular weight standard; 1: before induction; 2: inducing for 10 hours; 3: inducing for 20 hours; 4: inducing for 30 hours; 5: and (5) putting the strain into a tank.

FIG. 8: western Blot detection of HPV39L1 protein expression during fermentation. M: a molecular weight standard; 1: before induction; 2: inducing for 10 hours; 3: inducing for 20 hours; 4: inducing for 30 hours; 5: and (5) putting the strain into a tank.

FIG. 9: POROS HS and CHT purified SDS-PAGE detection of HPV39L1 protein. M: a molecular weight standard; 1: loading the column sample; 2: POROS HS eluent; 3: CHT eluent.

FIG. 10: and (3) observing the result of the purified HPV39L1 protein by using a transmission electron microscope.

Detailed Description

In order to realize the high-efficiency expression of the HPV39L1 protein in Hansenula polymorpha, the invention discloses a nucleotide sequence for coding the HPV39L1 protein, a preparation method of a recombinant Hansenula polymorpha strain for expressing the HPV39L1 protein, and a fermentation process for ensuring the high-efficiency expression of the HPV39L1 VLP. The expressed HPV39L1 protein is purified sequentially through a cation chromatography column POROS HS and a chromatography column CHT to obtain a high-purity target protein solution which can be used as an antigen component of a univalent recombinant HPV39L1 vaccine or a multivalent recombinant HPV vaccine, so that HPV39 infection is prevented, and 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, prostatic cancer, ovarian cancer, colorectal adenoma and other cancers and precancerous lesions thereof) of cervical cancer and the like caused by HPV39 infection are further prevented.

The invention synthesizes 4 different DNA coding sequences according to the amino acid sequence of HPV39L1 protein. The synthesized DNA sequences are respectively constructed on hansenula polymorpha expression vectors to obtain 4 recombinant expression plasmids carrying HPV39L1 protein coding genes, and the 4 recombinant hansenula polymorpha expression plasmids belong to intracellular expression plasmids. The recombinant plasmid is integrated into a hansenula polymorpha genome by a genetic engineering method, and expression screening shows that the recombinant plasmid contains SEQ ID NO: the HPV39L1 protein expression of the strain with the 3 genes is better than that of other DNA coding sequences. Mixing a mixture containing SEQ ID NO: 3, fermenting and culturing the high-expression strain of the gene in a fermentation tank, purifying and chromatographing to obtain high-purity HPV39L1 protein, and adsorbing by an aluminum adjuvant to obtain the HPV39L1 vaccine.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

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

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various methods set forth herein will be apparent to those skilled in the art without departing from the scope and spirit of the present 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 above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

EXAMPLE 1 HPV39L1 protein engineering Strain construction

Selection of HPV39L1 amino acid sequence

The full-length HPV39L1 protein consists of 505 amino acids, and after NCBI GenBank retrieval and alignment analysis, the most representative conserved sequence (GenBank: AAA47056.1) is selected as the amino acid sequence of HPV39L1, and the sequence information is shown as SEQ ID NO: 1 is shown.

SEQ ID NO：1

MAMWRSSDSMVYLPPPSVAKVVNTDDYVTRTGIYYYAGSSRLLTVGHPYFKVGMNGGRKQDIPKVSAYQYRVFRVTLPDPNKFSIPDASLYNPETQRLVWACVGVEVGRGQPLGVGISGHPLYNRQDDTENSPFSSTTNKDSRDNVSVDYKQTQLCIIGCVPAIGEHWGKGKACKPNNVSTGDCPPLELVNTPIEDGDMIDTGYGAMDFGALQETKSEVPLDICQSICKYPDYLQMSADVYGDSMFFCLRREQLFARHFWNRGGMVGDAIPAQLYIKGTDIRANPGSSVYCPSPSGSMVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTSIESSIPSTYDPSKFKEYTRHVEEYDLQFIFQLCTVTLTTDVMSYIHTMNSSILDNWNFAVAPPPSASLVDTYRYLQSAAITCQKDAPAPEKKDPYDGLKFWNVDLREKFSLELDQFPLGRKFLLQARVRRRPTIGPRKRPAASTSSSSATKHKRKRVSK

Design and Synthesis of HPV39L1 encoding Gene

In order to efficiently express HPV39L1 protein in Hansenula polymorpha, the invention optimizes the nucleotide coding sequence of HPV39L1 by adopting Hansenula polymorpha codon optimization strategy based on the nucleotide sequence of wild type strain of HPV39L1 with GenBank ID of AAA47056.1 to respectively obtain 4 different codon-optimized nucleotide sequences, such as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, respectively. According to the optimized nucleotide coding sequence, the Suzhou Jinzhi biotechnology limited company is entrusted to synthesize a full-length gene, and the synthesized gene sequence is subjected to sequencing verification.

SEQ ID NO：2

atggctatgtggagatcctctgactcgatggtttacctgcctccaccttctgttgctaaggtcgttaacaccgacgactacgttactagaacgggcatctactattacgcaggttcttccagactcctgaccgttggacacccatacttcaaggtgggcatgaacggtggcagaaagcaggacatccctaaggtctccgcctaccaatacagagtgttcagagtcaccctgccagaccctaacaagttctcgatcccagacgcttccttgtacaaccctgagacccagagactcgtctgggcatgtgttggtgtggaagttggcagaggacaaccactgggtgttggcatctctggacaccctctttacaacagacaggacgatactgagaactcgccattctcctctaccacgaacaaggactccagagacaacgtttcggtcgactacaagcagacccagctgtgcattatcggctgtgttcctgccattggagaacactggggaaagggcaaggcctgcaagccaaataacgtgtctaccggtgactgtccacctttggagcttgtcaacacccctatcgaagacggagatatgatcgacactggctacggagctatggactttggtgccctccaagagaccaagtccgaggttccactggacatttgtcagtcgatctgtaagtacccagactatttgcagatgtctgctgacgtgtacggtgactccatgttcttctgcctgagaagagagcagctctttgccagacacttctggaacagaggtggaatggttggtgacgccattcctgcacagttgtacatcaagggcaccgacatcagagccaacccaggttcgtctgtctactgtcctagtccatccggatctatggtcacctctgactcccagctcttcaacaagccttactggctgcacaaggcccagggccacaacaatggtatctgctggcacaaccagttgttccttacggtcgttgacactaccagatccaccaacttcaccctctcgacttctatcgaatcctcgattccatctacctatgacccttccaagttcaaggagtacaccagacacgtggaggaatacgacttgcagttcatctttcagctgtgcacggtgacacttaccactgacgtcatgtcttacatccacaccatgaactcgtccattctggacaactggaacttcgccgttgctccacctccatctgcatccttggtcgacacctacagatacctgcaatcggcagccattacctgccagaaggacgctcctgcacctgagaagaaagacccatacgacggcttgaagttctggaacgtggacctcagagagaagttctccttggagctggatcagttccctctcggcagaaagttcctgttgcaggccagagtcagacgcagaccaacgatcggacctagaaagagaccagcagcctccacttcttcgtccagcgctaccaagcacaagaggaagagagtgtcgaagtaatag

SEQ ID NO：3

atggcaatgtggagatcttccgactctatggtctacttgccacctccatccgtcgcaaaggttgtgaacactgacgattacgtcaccagaaccggtatctactactatgccggctcctctagactgttgacggttggccacccttacttcaaggtcggtatgaacggcggaagaaagcaggacattcccaaggtttctgcttaccagtatagagtcttcagagtgaccctccctgacccaaacaagttttccattcctgacgcctctctgtataacccagaaactcagagattggtttgggcctgcgtgggcgttgaggtcggaagaggtcagcctcttggcgtgggtatctccggtcacccactgtacaacagacaggatgacaccgaaaactcccctttctcgtccacgaccaacaaggattcgagagacaacgtgtctgtcgactacaagcagacccaactctgtatcattggttgcgtgccagccatcggagagcactggggcaagggtaaagcttgtaagcctaacaatgtctccacgggcgactgccctccactggagttggttaacaccccaattgaggacggcgacatgatcgacaccggatacggtgctatggacttcggcgctttgcaggaaactaagtctgaggtccctcttgacatctgccaatccatctgcaagtacccagactacctccagatgtccgccgatgtctacggcgactctatgttcttctgcctgagaagagagcagttgttcgcaagacacttctggaacagaggcggtatggtgggagacgcaatcccagcccagctctatatcaagggtacggacattagagctaaccctggttcctcggtttactgcccatccccttctggctcgatggttacttccgactctcagctgtttaacaagccatactggttgcacaaggcacaaggacacaataacggcatctgttggcacaaccagctcttcttgaccgttgtggacaccacgagaagcactaacttcaccctgtccacctctatcgagagttccatcccttcgacctacgacccatctaagttcaaggaatacaccagacacgtcgaagagtacgacctgcaattcattttccagctctgtaccgttactcttacgaccgacgttatgtcctacatccacaccatgaactcctcgatccttgacaactggaactttgctgtggcccctccaccttccgcttctctggttgacacttacagatacttgcagtccgccgctatcacctgtcagaaggacgccccagctccagaaaagaaggacccttacgacggtctgaagttctggaacgttgacctgagagagaagttctcgctcgaattggaccagtttccacttggtagaaagttcttgctgcaggcaagagttagaagaagacctaccattggcccaaggaagagacctgccgcatctacctcctcatcgtccgcaactaagcacaagagaaagagagtctcgaagtaatag

SEQ ID NO：4

atggctatgtggagatcgtccgactctatggtctacctgccacctccatctgttgctaaggttgtcaacaccgacgattacgttacgagaactggcatctattactatgcaggttcctctagactcctgaccgttggccacccttacttcaaggtgggcatgaacggcggtagaaagcaagacatccctaaggtctccgcctaccagtacagagtcttcagagtgaccctgccagaccctaacaagttctccatccctgacgcttcgctgtacaaccctgaaacccagagactggtttgggcatgtgtcggtgtggaggttggcagaggtcaaccactgggcgttggtatctccggacaccctctttacaacagacaggacgatactgagaactccccattctcgtctaccactaacaaggattcgagagacaacgtttccgtcgactacaagcagacccagctgtgcatcattggctgcgttcctgccattggagaacactggggaaagggcaaggcctgcaagccaaataacgtgtctaccggtgactgtcctccactggagctcgtcaacacccctatcgaggacggcgacatgatcgacactggctacggtgccatggactttggtgccctccaagagaccaagtccgaggttccactggacatttgtcagtcgatctgtaagtaccctgactacctgcaaatgtcggccgatgtgtacggtgactcgatgttcttctgcctgagaagagagcagctcttcgccagacacttctggaacagaggcggtatggttggcgacgcaattcctgcccagttgtacatcaagggcaccgacatcagagccaacccaggttcgtctgtctactgtcctagtccatccggatctatggtcacctctgactcccagctcttcaacaagccttactggctgcacaaggcccagggccacaacaatggtatctgctggcacaaccagttgttccttacggtcgttgacactaccagatccaccaacttcaccctctcgacttcgatcgaatcttccattccatccacctatgacccttccaagttcaaggagtacaccagacacgtggaagagtacgacttgcaattcatcttccagctgtgcacagtgacgcttactaccgacgtcatgtcttacatccacaccatgaactcctcgattctggacaactggaacttcgccgttgctcctccaccatccgcatctctggtcgacacctacagatatctgcaatccgccgcaattacctgccagaaggacgcccctgctcctgagaagaaagacccatacgatggcctgaagttctggaacgtggacctgagagagaagttctccctggagctggaccagttccctctgggcagaaagttcctcctgcaggccagagtcagaagaagaccaacgatcggacctagaaagagaccagccgcatctacttcctcttcgtccgctaccaagcacaagaggaagagagtttccaagtaatag

SEQ ID NO：5

atggctatgtggagatcttctgactctatggtttacttgccaccaccatctgttgctaaggttgttaacactgacgactacgttactagaactggtatttactactacgctggttcttctagattgttgactgttggtcacccatacttcaaggttggtatgaacggtggtagaaagcaagacattccaaaggtttctgcttaccaatacagagttttcagagttactttgccagacccaaacaagttctctattccagacgcttctttgtacaacccagaaactcaaagattggtttgggcttgtgttggtgttgaagttggtagaggtcaaccattgggtgttggtatttctggtcacccattgtacaacagacaagacgacactgaaaactctccattctcttctactactaacaaggactctagagacaacgtttctgttgactacaagcaaactcaattgtgtattattggttgtgttccagctattggtgaacactggggtaagggtaaggcttgtaagccaaacaacgtttctactggtgactgtccaccattggaattggttaacactccaattgaagacggtgacatgattgacactggttacggtgctatggacttcggtgctttgcaagaaactaagtctgaagttccattggacatttgtcaatctatttgtaagtacccagactacttgcaaatgtctgctgacgtttacggtgactctatgttcttctgtttgagaagagaacaattgttcgctagacacttctggaacagaggtggtatggttggtgacgctattccagctcaattgtacattaagggtactgacattagagctaacccaggttcttctgtttactgtccatctccatctggttctatggttacttctgactctcaattgttcaacaagccatactggttgcacaaggctcaaggtcacaacaacggtatttgttggcacaaccaattgttcttgactgttgttgacactactagatctactaacttcactttgtctacttctattgaatcttctattccatctacttacgacccatctaagttcaaggaatacactagacacgttgaagaatacgacttgcaattcattttccaattgtgtactgttactttgactactgacgttatgtcttacattcacactatgaactcttctattttggacaactggaacttcgctgttgctccaccaccatctgcttctttggttgacacttacagatacttgcaatctgctgctattacttgtcaaaaggacgctccagctccagaaaagaaggacccatacgacggtttgaagttctggaacgttgacttgagagaaaagttctctttggaattggaccaattcccattgggtagaaagttcttgttgcaagctagagttagaagaagaccaactattggtccaagaaagagaccagctgcttctacttcttcttcttctgctactaagcacaagagaaagagagtttctaagtaatag

Construction of HPV39L1 protein recombinant expression vector

The hansenula polymorpha expression vector applied by the invention is self-modified by the company (the vector is modified from a commercial vector pPICZ B, and the original promoter and transcription terminator of the pPICZ B are replaced by the promoter and transcription terminator of the hansenula polymorpha). The pMTZ vector of (SEQ ID NO: 6, FIG. 1). The optimized 4 HPV39L1 coding sequences were cloned into pMTZ vectors via BstBI cleavage sites at the 5 'end and KpnI cleavage sites at the 3' end, respectively, to obtain expression vectors 39L1-1-pMTZ (SEQ ID NO: 7, FIG. 2), 39L1-2-pMTZ (SEQ ID NO: 8, FIG. 3), 39L1-3-pMTZ (SEQ ID NO: 9, FIG. 4) and 39L1-4-pMTZ (SEQ ID NO: 10, FIG. 5). Transcription of the HPV39L1 coding sequence is regulated by the Hansenula polymorpha methanol oxidase promoter pMOX and the MOX transcriptional termination region.

pMTZ vector sequence (SEQ ID NO: 6):

agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgaggaattcacgtggcccagccggccgtctcggatcggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc

39L1-1-pMTZ(SEQ ID NO：7)：

agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctatgtggagatcctctgactcgatggtttacctgcctccaccttctgttgctaaggtcgttaacaccgacgactacgttactagaacgggcatctactattacgcaggttcttccagactcctgaccgttggacacccatacttcaaggtgggcatgaacggtggcagaaagcaggacatccctaaggtctccgcctaccaatacagagtgttcagagtcaccctgccagaccctaacaagttctcgatcccagacgcttccttgtacaaccctgagacccagagactcgtctgggcatgtgttggtgtggaagttggcagaggacaaccactgggtgttggcatctctggacaccctctttacaacagacaggacgatactgagaactcgccattctcctctaccacgaacaaggactccagagacaacgtttcggtcgactacaagcagacccagctgtgcattatcggctgtgttcctgccattggagaacactggggaaagggcaaggcctgcaagccaaataacgtgtctaccggtgactgtccacctttggagcttgtcaacacccctatcgaagacggagatatgatcgacactggctacggagctatggactttggtgccctccaagagaccaagtccgaggttccactggacatttgtcagtcgatctgtaagtacccagactatttgcagatgtctgctgacgtgtacggtgactccatgttcttctgcctgagaagagagcagctctttgccagacacttctggaacagaggtggaatggttggtgacgccattcctgcacagttgtacatcaagggcaccgacatcagagccaacccaggttcgtctgtctactgtcctagtccatccggatctatggtcacctctgactcccagctcttcaacaagccttactggctgcacaaggcccagggccacaacaatggtatctgctggcacaaccagttgttccttacggtcgttgacactaccagatccaccaacttcaccctctcgacttctatcgaatcctcgattccatctacctatgacccttccaagttcaaggagtacaccagacacgtggaggaatacgacttgcagttcatctttcagctgtgcacggtgacacttaccactgacgtcatgtcttacatccacaccatgaactcgtccattctggacaactggaacttcgccgttgctccacctccatctgcatccttggtcgacacctacagatacctgcaatcggcagccattacctgccagaaggacgctcctgcacctgagaagaaagacccatacgacggcttgaagttctggaacgtggacctcagagagaagttctccttggagctggatcagttccctctcggcagaaagttcctgttgcaggccagagtcagacgcagaccaacgatcggacctagaaagagaccagcagcctccacttcttcgtccagcgctaccaagcacaagaggaagagagtgtcgaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc

39L1-2-pMTZ(SEQ ID NO：8)：

agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggcaatgtggagatcttccgactctatggtctacttgccacctccatccgtcgcaaaggttgtgaacactgacgattacgtcaccagaaccggtatctactactatgccggctcctctagactgttgacggttggccacccttacttcaaggtcggtatgaacggcggaagaaagcaggacattcccaaggtttctgcttaccagtatagagtcttcagagtgaccctccctgacccaaacaagttttccattcctgacgcctctctgtataacccagaaactcagagattggtttgggcctgcgtgggcgttgaggtcggaagaggtcagcctcttggcgtgggtatctccggtcacccactgtacaacagacaggatgacaccgaaaactcccctttctcgtccacgaccaacaaggattcgagagacaacgtgtctgtcgactacaagcagacccaactctgtatcattggttgcgtgccagccatcggagagcactggggcaagggtaaagcttgtaagcctaacaatgtctccacgggcgactgccctccactggagttggttaacaccccaattgaggacggcgacatgatcgacaccggatacggtgctatggacttcggcgctttgcaggaaactaagtctgaggtccctcttgacatctgccaatccatctgcaagtacccagactacctccagatgtccgccgatgtctacggcgactctatgttcttctgcctgagaagagagcagttgttcgcaagacacttctggaacagaggcggtatggtgggagacgcaatcccagcccagctctatatcaagggtacggacattagagctaaccctggttcctcggtttactgcccatccccttctggctcgatggttacttccgactctcagctgtttaacaagccatactggttgcacaaggcacaaggacacaataacggcatctgttggcacaaccagctcttcttgaccgttgtggacaccacgagaagcactaacttcaccctgtccacctctatcgagagttccatcccttcgacctacgacccatctaagttcaaggaatacaccagacacgtcgaagagtacgacctgcaattcattttccagctctgtaccgttactcttacgaccgacgttatgtcctacatccacaccatgaactcctcgatccttgacaactggaactttgctgtggcccctccaccttccgcttctctggttgacacttacagatacttgcagtccgccgctatcacctgtcagaaggacgccccagctccagaaaagaaggacccttacgacggtctgaagttctggaacgttgacctgagagagaagttctcgctcgaattggaccagtttccacttggtagaaagttcttgctgcaggcaagagttagaagaagacctaccattggcccaaggaagagacctgccgcatctacctcctcatcgtccgcaactaagcacaagagaaagagagtctcgaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc

39L1-3-pMTZ(SEQ ID NO：9)：

agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctatgtggagatcgtccgactctatggtctacctgccacctccatctgttgctaaggttgtcaacaccgacgattacgttacgagaactggcatctattactatgcaggttcctctagactcctgaccgttggccacccttacttcaaggtgggcatgaacggcggtagaaagcaagacatccctaaggtctccgcctaccagtacagagtcttcagagtgaccctgccagaccctaacaagttctccatccctgacgcttcgctgtacaaccctgaaacccagagactggtttgggcatgtgtcggtgtggaggttggcagaggtcaaccactgggcgttggtatctccggacaccctctttacaacagacaggacgatactgagaactccccattctcgtctaccactaacaaggattcgagagacaacgtttccgtcgactacaagcagacccagctgtgcatcattggctgcgttcctgccattggagaacactggggaaagggcaaggcctgcaagccaaataacgtgtctaccggtgactgtcctccactggagctcgtcaacacccctatcgaggacggcgacatgatcgacactggctacggtgccatggactttggtgccctccaagagaccaagtccgaggttccactggacatttgtcagtcgatctgtaagtaccctgactacctgcaaatgtcggccgatgtgtacggtgactcgatgttcttctgcctgagaagagagcagctcttcgccagacacttctggaacagaggcggtatggttggcgacgcaattcctgcccagttgtacatcaagggcaccgacatcagagccaacccaggttcgtctgtctactgtcctagtccatccggatctatggtcacctctgactcccagctcttcaacaagccttactggctgcacaaggcccagggccacaacaatggtatctgctggcacaaccagttgttccttacggtcgttgacactaccagatccaccaacttcaccctctcgacttcgatcgaatcttccattccatccacctatgacccttccaagttcaaggagtacaccagacacgtggaagagtacgacttgcaattcatcttccagctgtgcacagtgacgcttactaccgacgtcatgtcttacatccacaccatgaactcctcgattctggacaactggaacttcgccgttgctcctccaccatccgcatctctggtcgacacctacagatatctgcaatccgccgcaattacctgccagaaggacgcccctgctcctgagaagaaagacccatacgatggcctgaagttctggaacgtggacctgagagagaagttctccctggagctggaccagttccctctgggcagaaagttcctcctgcaggccagagtcagaagaagaccaacgatcggacctagaaagagaccagccgcatctacttcctcttcgtccgctaccaagcacaagaggaagagagtttccaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc

39L1-4-pMTZ(SEQ ID NO：10)：

agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctatgtggagatcttctgactctatggtttacttgccaccaccatctgttgctaaggttgttaacactgacgactacgttactagaactggtatttactactacgctggttcttctagattgttgactgttggtcacccatacttcaaggttggtatgaacggtggtagaaagcaagacattccaaaggtttctgcttaccaatacagagttttcagagttactttgccagacccaaacaagttctctattccagacgcttctttgtacaacccagaaactcaaagattggtttgggcttgtgttggtgttgaagttggtagaggtcaaccattgggtgttggtatttctggtcacccattgtacaacagacaagacgacactgaaaactctccattctcttctactactaacaaggactctagagacaacgtttctgttgactacaagcaaactcaattgtgtattattggttgtgttccagctattggtgaacactggggtaagggtaaggcttgtaagccaaacaacgtttctactggtgactgtccaccattggaattggttaacactccaattgaagacggtgacatgattgacactggttacggtgctatggacttcggtgctttgcaagaaactaagtctgaagttccattggacatttgtcaatctatttgtaagtacccagactacttgcaaatgtctgctgacgtttacggtgactctatgttcttctgtttgagaagagaacaattgttcgctagacacttctggaacagaggtggtatggttggtgacgctattccagctcaattgtacattaagggtactgacattagagctaacccaggttcttctgtttactgtccatctccatctggttctatggttacttctgactctcaattgttcaacaagccatactggttgcacaaggctcaaggtcacaacaacggtatttgttggcacaaccaattgttcttgactgttgttgacactactagatctactaacttcactttgtctacttctattgaatcttctattccatctacttacgacccatctaagttcaaggaatacactagacacgttgaagaatacgacttgcaattcattttccaattgtgtactgttactttgactactgacgttatgtcttacattcacactatgaactcttctattttggacaactggaacttcgctgttgctccaccaccatctgcttctttggttgacacttacagatacttgcaatctgctgctattacttgtcaaaaggacgctccagctccagaaaagaaggacccatacgacggtttgaagttctggaacgttgacttgagagaaaagttctctttggaattggaccaattcccattgggtagaaagttcttgttgcaagctagagttagaagaagaccaactattggtccaagaaagagaccagctgcttctacttcttcttcttctgctactaagcacaagagaaagagagtttctaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc

construction of HPV39L1 protein recombinant expression strain

The Hansenula polymorpha host bacteria used in the present invention were derived from the wild-type Hansenula polymorpha CBS4732 strain (ATCC 34438) and purchased from American Type Culture Collection (ATCC). 39L1-1-pMTZ, 39L1-2-pMTZ, 39L1-3-pMTZ and 39L1-4-pMTZ recombinant expression plasmids were linearized with ScaI enzyme, respectively, and Hansenula polymorpha was electrotransferred under conditions of 1500V, 120. omega., 50. mu.F. After the electrotransformation, the bacterial solution is coated on YPD plates (200 mu g/mL Zeocin), and inverted culture is carried out at 37 ℃ for 1-2 days.

Example 2 expression screening of HPV39L1 recombinant engineered Strain

1. Glass test tube expression screening

Single colonies of 6 recombinant Hansenula polymorpha were randomly picked from electrically transformed YPD plates of 39L1-1-pMTZ, 39L1-2-pMTZ, 39L1-3-pMTZ and 39L1-4-pMTZ, respectively, inoculated into YPD liquid medium, and cultured overnight at 37 ℃. Centrifuging part of the bacterial liquid, removing YPD culture medium, adding induction culture medium BMMY, inducing at 37 deg.C for 48 hr, and collecting thallus. The acid-treated glass beads are shaken violently to break the thalli, the broken thalli are collected after centrifugation, the expression condition of HPV39L1 protein in the broken thalli supernatant is quantitatively detected by an enzyme-linked immunosorbent assay (ELISA), and the result is shown in figure 6: the recombinant engineering strains containing different HPV39L1 coding sequences have definite expression, but the expression conditions of HPV39L1 proteins of different coding sequences have certain difference. In contrast, the expression level of the recombinant engineering bacteria containing the 39L1-1 and 39L1-2 coding sequences is significantly higher than that of the recombinant engineering bacteria containing the 39L1-3 and 39L1-4 coding sequences, and meanwhile, the expression level of the recombinant engineering bacteria containing the 39L1-2 coding sequences is significantly higher than that of the recombinant engineering bacteria containing the 35L1-1 coding sequences, and the results have statistical significance (p is less than 0.05, and p is less than 0.001).

2. Fermenter expression screening

To further compare the expression advantage of the 39L1-1 and 39L1-2 coding sequences, 1 strain each was picked from the engineered strains containing the 39L1-1 and 39L1-2 coding sequences for fermenter expression validation, and the 39L1 protein expression of both strains was compared.

The main fermentation parameters are as follows: fermentation volume of 30L; the culture temperature of the thalli is 37 ℃; culturing at pH 5.00, and proliferating with 3 times of glycerol. Induction pH 6.50, 30 hours.

Cell disruption parameters: adding a bacterium breaking buffer solution (containing 0.4mol/L sodium chloride and 0.1mol/L MOPS) into wet bacteria in a fermentation tank according to the proportion of 1:4, carrying out heavy suspension and uniform stirring on the bacteria, filtering the bacterial suspension by using a screen, carrying out ice bath cooling on the filtered bacterial suspension to 4 ℃, and breaking the ice-bath bacterial suspension for 5 times under the pressure of 1500 bar. Centrifuging the crushed solution at 4 deg.C and 8500 for 20min, collecting supernatant, and detecting antigen content. The results showed that the antigen expression level of the strain containing 39L1-2 was significantly higher than that of the strain containing 39L 1-1.

Surface ELISA detects the antigen content of 39L1 protein in the broken bacteria supernatant of different strains

Type of Strain	Antigen content (μ g/ml)
		Strain comprising 39L1-1	967.195
Strain comprising 39L1-2	1739.481

Example 3 fermentation Process of HPV39L1 recombinant Hansenula polymorpha expression Strain

Preparing a seed solution: the strain of example 2, comprising 39L1-2, was inoculated into 1000mL of sterilized shake flask YPG medium in a clean bench (under sterile operating conditions). The shake flask is placed in a constant temperature oscillator for culture, the culture temperature is 37 ℃, the rotation speed of a shaking table is 190rpm, and the culture time is 24 h. When the seed liquid OD₆₀₀When the value reaches 2.0, stopping shake culture, and storing at 4 ℃ after the verification is qualified to be used as fermentation seed liquid.

Fermentation in a fermentation tank: according to the formula of BSM1 (the formula of a BSM1 culture medium: 26.7ml/L of 85% phosphoric acid, 0.93g/L of calcium sulfate dihydrate, 18.2g/L of potassium sulfate, 14.9g/L of magnesium sulfate dihydrate, 4.13g/L of potassium hydroxide, 40g/L of glycerol and 14 g/L of PTM 14 ml) and 20L of basal culture medium, and the basal culture medium is sterilized for 30min at 121 ℃. Inoculating the cultured qualified fermented seed liquid into a 30L fermentation tank according to a proportion of 5% under the protection of flame. In the fermentation culture process, the pH is controlled to be 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 basic culture medium is consumed and the wet weight of the thalli is about 100g/L, feeding the glycerol at a feed rate of 200-600 g/h. When the wet weight of the thalli is more than 200g/L, beginning to add methanol in a methanol induction period, gradually adjusting the methanol adding speed as the speed of the thalli using the methanol is increased, controlling the dissolved oxygen by more than 20 percent in the induction process, and finishing the fermentation after inducing the thalli for 30 hours. The thalli is stored at the temperature of minus 20 ℃ for purification after high-speed centrifugation. The fermentation supernatants at different times were subjected to SDS-PAGE (FIG. 7) and Western Blot identification (FIG. 8). The result shows that the expression of the HPV39L1 protein is continuously increased along with the prolonging of the induction time, and the fermentation expression quantity meets the requirement of large-scale production.

Example 4 purification Process of HPV39L1 recombinant protein

And (3) crushing thalli: taking HPV39L1 stored at the temperature of minus 20 ℃ for fermentation, putting wet thalli into a tank, adding a bacterium breaking buffer solution (containing 0.4mol/L sodium chloride and 0.1mol/L MOPS) according to the proportion of 1:4, carrying out heavy suspension and uniform stirring on the thalli, filtering the bacterial suspension by using a screen, carrying out ice-bath cooling on the filtered bacterial suspension to 4 ℃, breaking the bacterial suspension in the ice-bath for 5 times under the pressure of 1500bar, and examining the bacterium breaking rate by a microscope to be more than or equal to 80%. The disruption solution was centrifuged at 8500 for 20min at 4 ℃ and the supernatant was collected.

Column chromatography: loading the clarified liquid to a cation chromatographic column POROS HS for primary purification, eluting by using 1.5mol/L sodium chloride solution, and collecting the eluate of the primary purification; the primarily purified protein solution is loaded to a chromatographic column CHT for refining and purification, 200mol/L phosphate buffer solution is used for elution, and the eluted HPV39L1 protein is collected (shown in figure 9).

Example 5 Transmission Electron microscopy of HPV39L1 recombinant protein

The purified HPV39L1 protein was added dropwise to a clean plastic plate to form droplets. And inserting the copper mesh into the middle of the liquid drop by using tweezers to ensure that the upper surface and the lower surface of the copper mesh are immersed by the liquid, standing at room temperature for 20 minutes, taking out the copper mesh by using the tweezers, and sucking the liquid from the edge of the copper mesh by using filter paper. And placing the copper mesh adsorbed with the sample on the surface of the dye solution, dyeing for 10 seconds at room temperature, taking out the copper mesh, sucking the excess liquid by using filter paper, and drying. The virus-like particle morphology was observed by transmission electron microscope observation (JEM-2100, Japan Electron Co., Ltd.). The transmission electron microscopy observation of HPV39L1 protein is shown in FIG. 10.

Example 6 preparation of a vaccine containing HPV39L1 protein

Diluting the HPV39L1 protein stock solution prepared according to the embodiment 1-4 to 250 mu g/mL by using stock solution dilution buffer solution, adding 250 mu g/mL aluminum phosphate adjuvant into 1mL diluted protein solution for mixing, adsorbing for 1-3 h to obtain the HPV39L1 protein vaccine, and storing at 4 ℃ in a dark place.

Example 7 immunogenicity of HPV39L1 protein vaccine

Different doses of HPV39L1 vaccine were administered to mice separately, the positive conversion rate of specific antibodies in serum was determined by enzyme-linked immunosorbent assay (ELISA), the percentage of positive serum was calculated for each dose group, and ED was calculated using SPSS software₅₀(half effective dose) value, thereby evaluating the immunogenicity of the vaccine.

1. Immunization of animals

60 Balb/c female mice, 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, the blank aluminum adjuvant diluent is used for diluting according to the following table, and the sample is required to be completely mixed when diluted and used for immunizing animals. Injecting 0.5 mL/injection at five subcutaneous points, immunizing 1 needle at 0 day, collecting blood in orbit after 28 days, and separating serum for detecting the positive conversion rate of the neutralizing antibody.

The animals were grouped as follows:

group of	Test article	Dosage (μ g/0.5mL)	Immunization procedure	Mouse
						1	HPV39L1 vaccine	0.01000	0 day injection	10
2	HPV39L1 vaccine	0.00250	0 day injection	10
					3	HPV39L1 vaccine	0.00063	0 day injection	10
4	HPV39L1 vaccine	0.00016	0 day injection	10
					5	Physiological saline	/	0 day injection	10

ELISA method for detecting antibody positive conversion rate in serum

The test procedure was as follows: 1) coating: stock HPV39L1 was diluted to 5. mu.g/mL with phosphate buffer (0.01mol/mL, pH7.4), 100. mu.L/well was added to the microplate, and left overnight at 4 ℃ or incubated at 37 ℃ for 2 hours. 2) And (3) sealing: the plate was washed 6 times with 300. mu.L/well wash solution, 200. mu.L of blocking solution was added to each well, and blocked at 37 ℃ for 2 hours. 3) Using PBST diluent containing 2.0% skimmed milk powder according to the weight ratio of 1: serum was diluted 1000 times, an ELISA plate was added at 100. mu.L/well, double well assay, incubation at 37 ℃ for 1 hour, and positive and blank controls were set. 4) Adding an enzyme-labeled secondary antibody: wash plate 6 times with 300 μ L/well wash, dilute 1: 10000 diluted goat anti-mouse-HRP, 100 mu L/hole added enzyme label plate, 37 degrees C were incubated for 1 hours. 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 to develop color at 37 ℃ for 10 minutes. 6) End reading: adding the stop solution into the plate at a concentration of 50 μ L/well, slightly oscillating, mixing, reading with an enzyme-linked immunosorbent assay, and measuring at 450nm and 620nm as reference wavelength.

3. In vivo efficacy ED₅₀Is calculated by

In vivo efficacy ED of HPV39L1 vaccine calculated from antibody positive conversion results of mouse sera at different dose levels₅₀The value of (A) is 0.0004. mu.g, showing that the HPV39L1 vaccine has good immunogenicity.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, 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 above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Sequence listing

<110> Shanghai Bowei Biotechnology Ltd

Chongqing Bowei Baitai biopharmaceutical Co.,Ltd.

<120> polynucleotide for expressing HPV39L1, and expression vector, host cell and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 505

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ala Met Trp Arg Ser Ser Asp Ser Met Val Tyr Leu Pro Pro Pro

1 5 10 15

Ser Val Ala Lys Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Gly

20 25 30

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

35 40 45

Tyr Phe Lys Val Gly Met Asn Gly Gly Arg Lys Gln Asp Ile Pro Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Gly Val Gly Ile Ser Gly His Pro Leu Tyr Asn Arg Gln Asp Asp

115 120 125

Thr Glu Asn Ser Pro Phe Ser Ser Thr Thr Asn Lys Asp Ser Arg Asp

130 135 140

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

145 150 155 160

Val Pro Ala Ile Gly Glu His Trp Gly Lys Gly Lys Ala Cys Lys Pro

165 170 175

Asn Asn Val Ser Thr Gly Asp Cys Pro Pro Leu Glu Leu Val Asn Thr

180 185 190

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

195 200 205

Phe Gly Ala Leu Gln Glu Thr Lys Ser Glu Val Pro Leu Asp Ile Cys

210 215 220

Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp Val

225 230 235 240

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

245 250 255

Arg His Phe Trp Asn Arg Gly Gly Met Val Gly Asp Ala Ile Pro Ala

260 265 270

Gln Leu Tyr Ile Lys Gly Thr Asp Ile Arg Ala Asn Pro Gly Ser Ser

275 280 285

Val Tyr Cys Pro Ser Pro Ser Gly Ser Met Val Thr Ser Asp Ser Gln

290 295 300

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

305 310 315 320

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

325 330 335

Arg Ser Thr Asn Phe Thr Leu Ser Thr Ser Ile Glu Ser Ser Ile Pro

340 345 350

Ser Thr Tyr Asp Pro Ser Lys Phe Lys Glu Tyr Thr Arg His Val Glu

355 360 365

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

370 375 380

Thr Asp Val Met Ser Tyr Ile His Thr Met Asn Ser Ser Ile Leu Asp

385 390 395 400

Asn Trp Asn Phe Ala Val Ala Pro Pro Pro Ser Ala Ser Leu Val Asp

405 410 415

Thr Tyr Arg Tyr Leu Gln Ser Ala Ala Ile Thr Cys Gln Lys Asp Ala

420 425 430

Pro Ala Pro Glu Lys Lys Asp Pro Tyr Asp Gly Leu Lys Phe Trp Asn

435 440 445

Val Asp Leu Arg Glu Lys Phe Ser Leu Glu Leu Asp Gln Phe Pro Leu

450 455 460

Gly Arg Lys Phe Leu Leu Gln Ala Arg Val Arg Arg Arg Pro Thr Ile

465 470 475 480

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

485 490 495

Lys His Lys Arg Lys Arg Val Ser Lys

500 505

<210> 2

<211> 1521

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggctatgt ggagatcctc tgactcgatg gtttacctgc ctccaccttc tgttgctaag 60

gtcgttaaca ccgacgacta cgttactaga acgggcatct actattacgc aggttcttcc 120

agactcctga ccgttggaca cccatacttc aaggtgggca tgaacggtgg cagaaagcag 180

gacatcccta aggtctccgc ctaccaatac agagtgttca gagtcaccct gccagaccct 240

aacaagttct cgatcccaga cgcttccttg tacaaccctg agacccagag actcgtctgg 300

gcatgtgttg gtgtggaagt tggcagagga caaccactgg gtgttggcat ctctggacac 360

cctctttaca acagacagga cgatactgag aactcgccat tctcctctac cacgaacaag 420

gactccagag acaacgtttc ggtcgactac aagcagaccc agctgtgcat tatcggctgt 480

gttcctgcca ttggagaaca ctggggaaag ggcaaggcct gcaagccaaa taacgtgtct 540

accggtgact gtccaccttt ggagcttgtc aacaccccta tcgaagacgg agatatgatc 600

gacactggct acggagctat ggactttggt gccctccaag agaccaagtc cgaggttcca 660

ctggacattt gtcagtcgat ctgtaagtac ccagactatt tgcagatgtc tgctgacgtg 720

tacggtgact ccatgttctt ctgcctgaga agagagcagc tctttgccag acacttctgg 780

aacagaggtg gaatggttgg tgacgccatt cctgcacagt tgtacatcaa gggcaccgac 840

atcagagcca acccaggttc gtctgtctac tgtcctagtc catccggatc tatggtcacc 900

tctgactccc agctcttcaa caagccttac tggctgcaca aggcccaggg ccacaacaat 960

ggtatctgct ggcacaacca gttgttcctt acggtcgttg acactaccag atccaccaac 1020

ttcaccctct cgacttctat cgaatcctcg attccatcta cctatgaccc ttccaagttc 1080

aaggagtaca ccagacacgt ggaggaatac gacttgcagt tcatctttca gctgtgcacg 1140

gtgacactta ccactgacgt catgtcttac atccacacca tgaactcgtc cattctggac 1200

aactggaact tcgccgttgc tccacctcca tctgcatcct tggtcgacac ctacagatac 1260

ctgcaatcgg cagccattac ctgccagaag gacgctcctg cacctgagaa gaaagaccca 1320

tacgacggct tgaagttctg gaacgtggac ctcagagaga agttctcctt ggagctggat 1380

cagttccctc tcggcagaaa gttcctgttg caggccagag tcagacgcag accaacgatc 1440

ggacctagaa agagaccagc agcctccact tcttcgtcca gcgctaccaa gcacaagagg 1500

aagagagtgt cgaagtaata g 1521

<210> 3

<211> 1521

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggcaatgt ggagatcttc cgactctatg gtctacttgc cacctccatc cgtcgcaaag 60

gttgtgaaca ctgacgatta cgtcaccaga accggtatct actactatgc cggctcctct 120

agactgttga cggttggcca cccttacttc aaggtcggta tgaacggcgg aagaaagcag 180

gacattccca aggtttctgc ttaccagtat agagtcttca gagtgaccct ccctgaccca 240

aacaagtttt ccattcctga cgcctctctg tataacccag aaactcagag attggtttgg 300

gcctgcgtgg gcgttgaggt cggaagaggt cagcctcttg gcgtgggtat ctccggtcac 360

ccactgtaca acagacagga tgacaccgaa aactcccctt tctcgtccac gaccaacaag 420

gattcgagag acaacgtgtc tgtcgactac aagcagaccc aactctgtat cattggttgc 480

gtgccagcca tcggagagca ctggggcaag ggtaaagctt gtaagcctaa caatgtctcc 540

acgggcgact gccctccact ggagttggtt aacaccccaa ttgaggacgg cgacatgatc 600

gacaccggat acggtgctat ggacttcggc gctttgcagg aaactaagtc tgaggtccct 660

cttgacatct gccaatccat ctgcaagtac ccagactacc tccagatgtc cgccgatgtc 720

tacggcgact ctatgttctt ctgcctgaga agagagcagt tgttcgcaag acacttctgg 780

aacagaggcg gtatggtggg agacgcaatc ccagcccagc tctatatcaa gggtacggac 840

attagagcta accctggttc ctcggtttac tgcccatccc cttctggctc gatggttact 900

tccgactctc agctgtttaa caagccatac tggttgcaca aggcacaagg acacaataac 960

ggcatctgtt ggcacaacca gctcttcttg accgttgtgg acaccacgag aagcactaac 1020

ttcaccctgt ccacctctat cgagagttcc atcccttcga cctacgaccc atctaagttc 1080

aaggaataca ccagacacgt cgaagagtac gacctgcaat tcattttcca gctctgtacc 1140

gttactctta cgaccgacgt tatgtcctac atccacacca tgaactcctc gatccttgac 1200

aactggaact ttgctgtggc ccctccacct tccgcttctc tggttgacac ttacagatac 1260

ttgcagtccg ccgctatcac ctgtcagaag gacgccccag ctccagaaaa gaaggaccct 1320

tacgacggtc tgaagttctg gaacgttgac ctgagagaga agttctcgct cgaattggac 1380

cagtttccac ttggtagaaa gttcttgctg caggcaagag ttagaagaag acctaccatt 1440

ggcccaagga agagacctgc cgcatctacc tcctcatcgt ccgcaactaa gcacaagaga 1500

aagagagtct cgaagtaata g 1521

<210> 4

<211> 1521

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggctatgt ggagatcgtc cgactctatg gtctacctgc cacctccatc tgttgctaag 60

gttgtcaaca ccgacgatta cgttacgaga actggcatct attactatgc aggttcctct 120

agactcctga ccgttggcca cccttacttc aaggtgggca tgaacggcgg tagaaagcaa 180

gacatcccta aggtctccgc ctaccagtac agagtcttca gagtgaccct gccagaccct 240

aacaagttct ccatccctga cgcttcgctg tacaaccctg aaacccagag actggtttgg 300

gcatgtgtcg gtgtggaggt tggcagaggt caaccactgg gcgttggtat ctccggacac 360

cctctttaca acagacagga cgatactgag aactccccat tctcgtctac cactaacaag 420

gattcgagag acaacgtttc cgtcgactac aagcagaccc agctgtgcat cattggctgc 480

gttcctgcca ttggagaaca ctggggaaag ggcaaggcct gcaagccaaa taacgtgtct 540

accggtgact gtcctccact ggagctcgtc aacaccccta tcgaggacgg cgacatgatc 600

gacactggct acggtgccat ggactttggt gccctccaag agaccaagtc cgaggttcca 660

ctggacattt gtcagtcgat ctgtaagtac cctgactacc tgcaaatgtc ggccgatgtg 720

tacggtgact cgatgttctt ctgcctgaga agagagcagc tcttcgccag acacttctgg 780

aacagaggcg gtatggttgg cgacgcaatt cctgcccagt tgtacatcaa gggcaccgac 840

atcagagcca acccaggttc gtctgtctac tgtcctagtc catccggatc tatggtcacc 900

tctgactccc agctcttcaa caagccttac tggctgcaca aggcccaggg ccacaacaat 960

ggtatctgct ggcacaacca gttgttcctt acggtcgttg acactaccag atccaccaac 1020

ttcaccctct cgacttcgat cgaatcttcc attccatcca cctatgaccc ttccaagttc 1080

aaggagtaca ccagacacgt ggaagagtac gacttgcaat tcatcttcca gctgtgcaca 1140

gtgacgctta ctaccgacgt catgtcttac atccacacca tgaactcctc gattctggac 1200

aactggaact tcgccgttgc tcctccacca tccgcatctc tggtcgacac ctacagatat 1260

ctgcaatccg ccgcaattac ctgccagaag gacgcccctg ctcctgagaa gaaagaccca 1320

tacgatggcc tgaagttctg gaacgtggac ctgagagaga agttctccct ggagctggac 1380

cagttccctc tgggcagaaa gttcctcctg caggccagag tcagaagaag accaacgatc 1440

ggacctagaa agagaccagc cgcatctact tcctcttcgt ccgctaccaa gcacaagagg 1500

aagagagttt ccaagtaata g 1521

<210> 5

<211> 1521

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggctatgt ggagatcttc tgactctatg gtttacttgc caccaccatc tgttgctaag 60

gttgttaaca ctgacgacta cgttactaga actggtattt actactacgc tggttcttct 120

agattgttga ctgttggtca cccatacttc aaggttggta tgaacggtgg tagaaagcaa 180

gacattccaa aggtttctgc ttaccaatac agagttttca gagttacttt gccagaccca 240

aacaagttct ctattccaga cgcttctttg tacaacccag aaactcaaag attggtttgg 300

gcttgtgttg gtgttgaagt tggtagaggt caaccattgg gtgttggtat ttctggtcac 360

ccattgtaca acagacaaga cgacactgaa aactctccat tctcttctac tactaacaag 420

gactctagag acaacgtttc tgttgactac aagcaaactc aattgtgtat tattggttgt 480

gttccagcta ttggtgaaca ctggggtaag ggtaaggctt gtaagccaaa caacgtttct 540

actggtgact gtccaccatt ggaattggtt aacactccaa ttgaagacgg tgacatgatt 600

gacactggtt acggtgctat ggacttcggt gctttgcaag aaactaagtc tgaagttcca 660

ttggacattt gtcaatctat ttgtaagtac ccagactact tgcaaatgtc tgctgacgtt 720

tacggtgact ctatgttctt ctgtttgaga agagaacaat tgttcgctag acacttctgg 780

aacagaggtg gtatggttgg tgacgctatt ccagctcaat tgtacattaa gggtactgac 840

attagagcta acccaggttc ttctgtttac tgtccatctc catctggttc tatggttact 900

tctgactctc aattgttcaa caagccatac tggttgcaca aggctcaagg tcacaacaac 960

ggtatttgtt ggcacaacca attgttcttg actgttgttg acactactag atctactaac 1020

ttcactttgt ctacttctat tgaatcttct attccatcta cttacgaccc atctaagttc 1080

aaggaataca ctagacacgt tgaagaatac gacttgcaat tcattttcca attgtgtact 1140

gttactttga ctactgacgt tatgtcttac attcacacta tgaactcttc tattttggac 1200

aactggaact tcgctgttgc tccaccacca tctgcttctt tggttgacac ttacagatac 1260

ttgcaatctg ctgctattac ttgtcaaaag gacgctccag ctccagaaaa gaaggaccca 1320

tacgacggtt tgaagttctg gaacgttgac ttgagagaaa agttctcttt ggaattggac 1380

caattcccat tgggtagaaa gttcttgttg caagctagag ttagaagaag accaactatt 1440

ggtccaagaa agagaccagc tgcttctact tcttcttctt ctgctactaa gcacaagaga 1500

aagagagttt ctaagtaata g 1521

<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> 6238

<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 ctatgtggag atcctctgac tcgatggttt 1560

acctgcctcc accttctgtt gctaaggtcg ttaacaccga cgactacgtt actagaacgg 1620

gcatctacta ttacgcaggt tcttccagac tcctgaccgt tggacaccca tacttcaagg 1680

tgggcatgaa cggtggcaga aagcaggaca tccctaaggt ctccgcctac caatacagag 1740

tgttcagagt caccctgcca gaccctaaca agttctcgat cccagacgct tccttgtaca 1800

accctgagac ccagagactc gtctgggcat gtgttggtgt ggaagttggc agaggacaac 1860

cactgggtgt tggcatctct ggacaccctc tttacaacag acaggacgat actgagaact 1920

cgccattctc ctctaccacg aacaaggact ccagagacaa cgtttcggtc gactacaagc 1980

agacccagct gtgcattatc ggctgtgttc ctgccattgg agaacactgg ggaaagggca 2040

aggcctgcaa gccaaataac gtgtctaccg gtgactgtcc acctttggag cttgtcaaca 2100

cccctatcga agacggagat atgatcgaca ctggctacgg agctatggac tttggtgccc 2160

tccaagagac caagtccgag gttccactgg acatttgtca gtcgatctgt aagtacccag 2220

actatttgca gatgtctgct gacgtgtacg gtgactccat gttcttctgc ctgagaagag 2280

agcagctctt tgccagacac ttctggaaca gaggtggaat ggttggtgac gccattcctg 2340

cacagttgta catcaagggc accgacatca gagccaaccc aggttcgtct gtctactgtc 2400

ctagtccatc cggatctatg gtcacctctg actcccagct cttcaacaag ccttactggc 2460

tgcacaaggc ccagggccac aacaatggta tctgctggca caaccagttg ttccttacgg 2520

tcgttgacac taccagatcc accaacttca ccctctcgac ttctatcgaa tcctcgattc 2580

catctaccta tgacccttcc aagttcaagg agtacaccag acacgtggag gaatacgact 2640

tgcagttcat ctttcagctg tgcacggtga cacttaccac tgacgtcatg tcttacatcc 2700

acaccatgaa ctcgtccatt ctggacaact ggaacttcgc cgttgctcca cctccatctg 2760

catccttggt cgacacctac agatacctgc aatcggcagc cattacctgc cagaaggacg 2820

ctcctgcacc tgagaagaaa gacccatacg acggcttgaa gttctggaac gtggacctca 2880

gagagaagtt ctccttggag ctggatcagt tccctctcgg cagaaagttc ctgttgcagg 2940

ccagagtcag acgcagacca acgatcggac ctagaaagag accagcagcc tccacttctt 3000

cgtccagcgc taccaagcac aagaggaaga gagtgtcgaa gtaataggta ccggagacgt 3060

ggaaggacat accgcttttg agaagcgtgt ttgaaaatag ttctttttct ggtttatatc 3120

gtttatgaag tgatgagatg aaaagctgaa atagcgagta taggaaaatt taatgaaaat 3180

taaattaaat attttcttag gctattagtc accttcaaaa tgccggccgc ttctaagaac 3240

gttgtcatga tcgacaacta cgactcgttt acctggaacc tgtacgagta cctgtgtcag 3300

gagggagcca atgtcgaggt tttcaggaac gatcagatca ccattccgga gattgagcag 3360

ctcaagccgg acgttgtggt gatatcccct ggtcctggcc atccaagaac agactcggga 3420

atatctcgcg acgtgatcag ccattttaaa ggcaagattc ctgtctttgg tgtctgtatg 3480

ggccagcagt gtatcttcga ggagtttggc ggagacgtcg agtatgcggg cgagattgtc 3540

catggaaaaa cgtccactgt taagcacgac aacaagggaa tgttcaaaaa cgttccgcaa 3600

gatgttgctg tcaccagata ccactcgctg gccggaacgc tcaagtcgct tccggactgt 3660

ctagagatca ctgctcgcac agacaacggg atcattatgg gtgtgagaca caagaagtac 3720

accatcgagg gcgtccagtt tcatccagag agcattctga ccgaggaggg ccatctgatg 3780

atccagaata tcctcaacgt ttccggtggt tactgggagg aaaatgccaa cggcgcggct 3840

cagagaaagg aaagcatatt ggagaaaata tacgcgcaga gacgaaaaga ctacgagttt 3900

gagatgaaca gaccggggcg cagatttgct gatctagaac tgtacttgtc catgggactg 3960

caccgccgct aatcaatttt tacgacagat tggagcagaa catcagcgcc ggcaaggttg 4020

caattctcag cgaaatcaag agagcgtcgc cttctaaagg cgtcatcgac ggagacgcta 4080

acgctgccaa acaggccctc aactacgcca aggctggagt tgccacaatt tctgttttga 4140

ccgagccaac ctggtttaaa ggaaatatcc aggacctgga ggtggccaga aaagccattg 4200

actctgtggc caatagaccg tgtattttgc ggaaggagtt tatcttcaac aagtaccaaa 4260

ttctagaggc ccgactggcg ggagcagaca cggttctgct gattgtcaag atgctgagct 4320

cggatccccc acacaccata gcttcaaaat gtttctactc cttttttact cttccagatt 4380

ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc atactaaatt 4440

ttccctcttt cttcctctag ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa 4500

aaagagaccg cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt 4560

ttctttttct tgaaattttt ttttttagtt tttttctctt tcagtgacct ccattgatat 4620

ttaagttaat aaacggtctt caatttctca agtttcagtt tcatttttct tgttctatta 4680

caactttttt tacttcttgt tcattagaaa gaaagcatag caatctaatc taaggggcgg 4740

tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa 4800

ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg 4860

agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt 4920

cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt 4980

gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg 5040

gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg 5100

cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt 5160

cgtggccgag gagcaggact gacacgtccg acggcggccc acgggtccca ggcctcggag 5220

atccgtcccc cttttccttt gtcgatatca tgtaattagt tatgtcacgc ttacattcac 5280

gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg 5340

tccctattta tttttttata gttatgttag tattaagaac gttatttata tttcaaattt 5400

ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg 5460

agaaggtttt gggacgctcg aaggctttaa tttgcaagct ggagaccaac atgtgagcaa 5520

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 5580

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 5640

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 5700

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 5760

ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 5820

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 5880

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 5940

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6000

acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6060

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6120

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6180

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagatc 6238

<210> 8

<211> 6238

<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 caatgtggag atcttccgac tctatggtct 1560

acttgccacc tccatccgtc gcaaaggttg tgaacactga cgattacgtc accagaaccg 1620

gtatctacta ctatgccggc tcctctagac tgttgacggt tggccaccct tacttcaagg 1680

tcggtatgaa cggcggaaga aagcaggaca ttcccaaggt ttctgcttac cagtatagag 1740

tcttcagagt gaccctccct gacccaaaca agttttccat tcctgacgcc tctctgtata 1800

acccagaaac tcagagattg gtttgggcct gcgtgggcgt tgaggtcgga agaggtcagc 1860

ctcttggcgt gggtatctcc ggtcacccac tgtacaacag acaggatgac accgaaaact 1920

cccctttctc gtccacgacc aacaaggatt cgagagacaa cgtgtctgtc gactacaagc 1980

agacccaact ctgtatcatt ggttgcgtgc cagccatcgg agagcactgg ggcaagggta 2040

aagcttgtaa gcctaacaat gtctccacgg gcgactgccc tccactggag ttggttaaca 2100

ccccaattga ggacggcgac atgatcgaca ccggatacgg tgctatggac ttcggcgctt 2160

tgcaggaaac taagtctgag gtccctcttg acatctgcca atccatctgc aagtacccag 2220

actacctcca gatgtccgcc gatgtctacg gcgactctat gttcttctgc ctgagaagag 2280

agcagttgtt cgcaagacac ttctggaaca gaggcggtat ggtgggagac gcaatcccag 2340

cccagctcta tatcaagggt acggacatta gagctaaccc tggttcctcg gtttactgcc 2400

catccccttc tggctcgatg gttacttccg actctcagct gtttaacaag ccatactggt 2460

tgcacaaggc acaaggacac aataacggca tctgttggca caaccagctc ttcttgaccg 2520

ttgtggacac cacgagaagc actaacttca ccctgtccac ctctatcgag agttccatcc 2580

cttcgaccta cgacccatct aagttcaagg aatacaccag acacgtcgaa gagtacgacc 2640

tgcaattcat tttccagctc tgtaccgtta ctcttacgac cgacgttatg tcctacatcc 2700

acaccatgaa ctcctcgatc cttgacaact ggaactttgc tgtggcccct ccaccttccg 2760

cttctctggt tgacacttac agatacttgc agtccgccgc tatcacctgt cagaaggacg 2820

ccccagctcc agaaaagaag gacccttacg acggtctgaa gttctggaac gttgacctga 2880

gagagaagtt ctcgctcgaa ttggaccagt ttccacttgg tagaaagttc ttgctgcagg 2940

caagagttag aagaagacct accattggcc caaggaagag acctgccgca tctacctcct 3000

catcgtccgc aactaagcac aagagaaaga gagtctcgaa gtaataggta ccggagacgt 3060

ggaaggacat accgcttttg agaagcgtgt ttgaaaatag ttctttttct ggtttatatc 3120

gtttatgaag tgatgagatg aaaagctgaa atagcgagta taggaaaatt taatgaaaat 3180

taaattaaat attttcttag gctattagtc accttcaaaa tgccggccgc ttctaagaac 3240

gttgtcatga tcgacaacta cgactcgttt acctggaacc tgtacgagta cctgtgtcag 3300

gagggagcca atgtcgaggt tttcaggaac gatcagatca ccattccgga gattgagcag 3360

ctcaagccgg acgttgtggt gatatcccct ggtcctggcc atccaagaac agactcggga 3420

atatctcgcg acgtgatcag ccattttaaa ggcaagattc ctgtctttgg tgtctgtatg 3480

ggccagcagt gtatcttcga ggagtttggc ggagacgtcg agtatgcggg cgagattgtc 3540

catggaaaaa cgtccactgt taagcacgac aacaagggaa tgttcaaaaa cgttccgcaa 3600

gatgttgctg tcaccagata ccactcgctg gccggaacgc tcaagtcgct tccggactgt 3660

ctagagatca ctgctcgcac agacaacggg atcattatgg gtgtgagaca caagaagtac 3720

accatcgagg gcgtccagtt tcatccagag agcattctga ccgaggaggg ccatctgatg 3780

atccagaata tcctcaacgt ttccggtggt tactgggagg aaaatgccaa cggcgcggct 3840

cagagaaagg aaagcatatt ggagaaaata tacgcgcaga gacgaaaaga ctacgagttt 3900

gagatgaaca gaccggggcg cagatttgct gatctagaac tgtacttgtc catgggactg 3960

caccgccgct aatcaatttt tacgacagat tggagcagaa catcagcgcc ggcaaggttg 4020

caattctcag cgaaatcaag agagcgtcgc cttctaaagg cgtcatcgac ggagacgcta 4080

acgctgccaa acaggccctc aactacgcca aggctggagt tgccacaatt tctgttttga 4140

ccgagccaac ctggtttaaa ggaaatatcc aggacctgga ggtggccaga aaagccattg 4200

actctgtggc caatagaccg tgtattttgc ggaaggagtt tatcttcaac aagtaccaaa 4260

ttctagaggc ccgactggcg ggagcagaca cggttctgct gattgtcaag atgctgagct 4320

cggatccccc acacaccata gcttcaaaat gtttctactc cttttttact cttccagatt 4380

ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc atactaaatt 4440

ttccctcttt cttcctctag ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa 4500

aaagagaccg cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt 4560

ttctttttct tgaaattttt ttttttagtt tttttctctt tcagtgacct ccattgatat 4620

ttaagttaat aaacggtctt caatttctca agtttcagtt tcatttttct tgttctatta 4680

caactttttt tacttcttgt tcattagaaa gaaagcatag caatctaatc taaggggcgg 4740

tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa 4800

ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg 4860

agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt 4920

cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt 4980

gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg 5040

gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg 5100

cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt 5160

cgtggccgag gagcaggact gacacgtccg acggcggccc acgggtccca ggcctcggag 5220

atccgtcccc cttttccttt gtcgatatca tgtaattagt tatgtcacgc ttacattcac 5280

gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg 5340

tccctattta tttttttata gttatgttag tattaagaac gttatttata tttcaaattt 5400

ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg 5460

agaaggtttt gggacgctcg aaggctttaa tttgcaagct ggagaccaac atgtgagcaa 5520

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 5580

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 5640

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 5700

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 5760

ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 5820

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 5880

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 5940

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6000

acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6060

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6120

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6180

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagatc 6238

<210> 9

<211> 6238

<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 ctatgtggag atcgtccgac tctatggtct 1560

acctgccacc tccatctgtt gctaaggttg tcaacaccga cgattacgtt acgagaactg 1620

gcatctatta ctatgcaggt tcctctagac tcctgaccgt tggccaccct tacttcaagg 1680

tgggcatgaa cggcggtaga aagcaagaca tccctaaggt ctccgcctac cagtacagag 1740

tcttcagagt gaccctgcca gaccctaaca agttctccat ccctgacgct tcgctgtaca 1800

accctgaaac ccagagactg gtttgggcat gtgtcggtgt ggaggttggc agaggtcaac 1860

cactgggcgt tggtatctcc ggacaccctc tttacaacag acaggacgat actgagaact 1920

ccccattctc gtctaccact aacaaggatt cgagagacaa cgtttccgtc gactacaagc 1980

agacccagct gtgcatcatt ggctgcgttc ctgccattgg agaacactgg ggaaagggca 2040

aggcctgcaa gccaaataac gtgtctaccg gtgactgtcc tccactggag ctcgtcaaca 2100

cccctatcga ggacggcgac atgatcgaca ctggctacgg tgccatggac tttggtgccc 2160

tccaagagac caagtccgag gttccactgg acatttgtca gtcgatctgt aagtaccctg 2220

actacctgca aatgtcggcc gatgtgtacg gtgactcgat gttcttctgc ctgagaagag 2280

agcagctctt cgccagacac ttctggaaca gaggcggtat ggttggcgac gcaattcctg 2340

cccagttgta catcaagggc accgacatca gagccaaccc aggttcgtct gtctactgtc 2400

ctagtccatc cggatctatg gtcacctctg actcccagct cttcaacaag ccttactggc 2460

tgcacaaggc ccagggccac aacaatggta tctgctggca caaccagttg ttccttacgg 2520

tcgttgacac taccagatcc accaacttca ccctctcgac ttcgatcgaa tcttccattc 2580

catccaccta tgacccttcc aagttcaagg agtacaccag acacgtggaa gagtacgact 2640

tgcaattcat cttccagctg tgcacagtga cgcttactac cgacgtcatg tcttacatcc 2700

acaccatgaa ctcctcgatt ctggacaact ggaacttcgc cgttgctcct ccaccatccg 2760

catctctggt cgacacctac agatatctgc aatccgccgc aattacctgc cagaaggacg 2820

cccctgctcc tgagaagaaa gacccatacg atggcctgaa gttctggaac gtggacctga 2880

gagagaagtt ctccctggag ctggaccagt tccctctggg cagaaagttc ctcctgcagg 2940

ccagagtcag aagaagacca acgatcggac ctagaaagag accagccgca tctacttcct 3000

cttcgtccgc taccaagcac aagaggaaga gagtttccaa gtaataggta ccggagacgt 3060

ggaaggacat accgcttttg agaagcgtgt ttgaaaatag ttctttttct ggtttatatc 3120

gtttatgaag tgatgagatg aaaagctgaa atagcgagta taggaaaatt taatgaaaat 3180

taaattaaat attttcttag gctattagtc accttcaaaa tgccggccgc ttctaagaac 3240

gttgtcatga tcgacaacta cgactcgttt acctggaacc tgtacgagta cctgtgtcag 3300

gagggagcca atgtcgaggt tttcaggaac gatcagatca ccattccgga gattgagcag 3360

ctcaagccgg acgttgtggt gatatcccct ggtcctggcc atccaagaac agactcggga 3420

atatctcgcg acgtgatcag ccattttaaa ggcaagattc ctgtctttgg tgtctgtatg 3480

ggccagcagt gtatcttcga ggagtttggc ggagacgtcg agtatgcggg cgagattgtc 3540

catggaaaaa cgtccactgt taagcacgac aacaagggaa tgttcaaaaa cgttccgcaa 3600

gatgttgctg tcaccagata ccactcgctg gccggaacgc tcaagtcgct tccggactgt 3660

ctagagatca ctgctcgcac agacaacggg atcattatgg gtgtgagaca caagaagtac 3720

accatcgagg gcgtccagtt tcatccagag agcattctga ccgaggaggg ccatctgatg 3780

atccagaata tcctcaacgt ttccggtggt tactgggagg aaaatgccaa cggcgcggct 3840

cagagaaagg aaagcatatt ggagaaaata tacgcgcaga gacgaaaaga ctacgagttt 3900

gagatgaaca gaccggggcg cagatttgct gatctagaac tgtacttgtc catgggactg 3960

caccgccgct aatcaatttt tacgacagat tggagcagaa catcagcgcc ggcaaggttg 4020

caattctcag cgaaatcaag agagcgtcgc cttctaaagg cgtcatcgac ggagacgcta 4080

acgctgccaa acaggccctc aactacgcca aggctggagt tgccacaatt tctgttttga 4140

ccgagccaac ctggtttaaa ggaaatatcc aggacctgga ggtggccaga aaagccattg 4200

actctgtggc caatagaccg tgtattttgc ggaaggagtt tatcttcaac aagtaccaaa 4260

ttctagaggc ccgactggcg ggagcagaca cggttctgct gattgtcaag atgctgagct 4320

cggatccccc acacaccata gcttcaaaat gtttctactc cttttttact cttccagatt 4380

ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc atactaaatt 4440

ttccctcttt cttcctctag ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa 4500

aaagagaccg cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt 4560

ttctttttct tgaaattttt ttttttagtt tttttctctt tcagtgacct ccattgatat 4620

ttaagttaat aaacggtctt caatttctca agtttcagtt tcatttttct tgttctatta 4680

caactttttt tacttcttgt tcattagaaa gaaagcatag caatctaatc taaggggcgg 4740

tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa 4800

ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg 4860

agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt 4920

cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt 4980

gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg 5040

gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg 5100

cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt 5160

cgtggccgag gagcaggact gacacgtccg acggcggccc acgggtccca ggcctcggag 5220

atccgtcccc cttttccttt gtcgatatca tgtaattagt tatgtcacgc ttacattcac 5280

gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg 5340

tccctattta tttttttata gttatgttag tattaagaac gttatttata tttcaaattt 5400

ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg 5460

agaaggtttt gggacgctcg aaggctttaa tttgcaagct ggagaccaac atgtgagcaa 5520

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 5580

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 5640

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 5700

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 5760

ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 5820

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 5880

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 5940

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6000

acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6060

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6120

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6180

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagatc 6238

<210> 10

<211> 6238

<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 ctatgtggag atcttctgac tctatggttt 1560

acttgccacc accatctgtt gctaaggttg ttaacactga cgactacgtt actagaactg 1620

gtatttacta ctacgctggt tcttctagat tgttgactgt tggtcaccca tacttcaagg 1680

ttggtatgaa cggtggtaga aagcaagaca ttccaaaggt ttctgcttac caatacagag 1740

ttttcagagt tactttgcca gacccaaaca agttctctat tccagacgct tctttgtaca 1800

acccagaaac tcaaagattg gtttgggctt gtgttggtgt tgaagttggt agaggtcaac 1860

cattgggtgt tggtatttct ggtcacccat tgtacaacag acaagacgac actgaaaact 1920

ctccattctc ttctactact aacaaggact ctagagacaa cgtttctgtt gactacaagc 1980

aaactcaatt gtgtattatt ggttgtgttc cagctattgg tgaacactgg ggtaagggta 2040

aggcttgtaa gccaaacaac gtttctactg gtgactgtcc accattggaa ttggttaaca 2100

ctccaattga agacggtgac atgattgaca ctggttacgg tgctatggac ttcggtgctt 2160

tgcaagaaac taagtctgaa gttccattgg acatttgtca atctatttgt aagtacccag 2220

actacttgca aatgtctgct gacgtttacg gtgactctat gttcttctgt ttgagaagag 2280

aacaattgtt cgctagacac ttctggaaca gaggtggtat ggttggtgac gctattccag 2340

ctcaattgta cattaagggt actgacatta gagctaaccc aggttcttct gtttactgtc 2400

catctccatc tggttctatg gttacttctg actctcaatt gttcaacaag ccatactggt 2460

tgcacaaggc tcaaggtcac aacaacggta tttgttggca caaccaattg ttcttgactg 2520

ttgttgacac tactagatct actaacttca ctttgtctac ttctattgaa tcttctattc 2580

catctactta cgacccatct aagttcaagg aatacactag acacgttgaa gaatacgact 2640

tgcaattcat tttccaattg tgtactgtta ctttgactac tgacgttatg tcttacattc 2700

acactatgaa ctcttctatt ttggacaact ggaacttcgc tgttgctcca ccaccatctg 2760

cttctttggt tgacacttac agatacttgc aatctgctgc tattacttgt caaaaggacg 2820

ctccagctcc agaaaagaag gacccatacg acggtttgaa gttctggaac gttgacttga 2880

gagaaaagtt ctctttggaa ttggaccaat tcccattggg tagaaagttc ttgttgcaag 2940

ctagagttag aagaagacca actattggtc caagaaagag accagctgct tctacttctt 3000

cttcttctgc tactaagcac aagagaaaga gagtttctaa gtaataggta ccggagacgt 3060

ggaaggacat accgcttttg agaagcgtgt ttgaaaatag ttctttttct ggtttatatc 3120

gtttatgaag tgatgagatg aaaagctgaa atagcgagta taggaaaatt taatgaaaat 3180

taaattaaat attttcttag gctattagtc accttcaaaa tgccggccgc ttctaagaac 3240

gttgtcatga tcgacaacta cgactcgttt acctggaacc tgtacgagta cctgtgtcag 3300

gagggagcca atgtcgaggt tttcaggaac gatcagatca ccattccgga gattgagcag 3360

ctcaagccgg acgttgtggt gatatcccct ggtcctggcc atccaagaac agactcggga 3420

atatctcgcg acgtgatcag ccattttaaa ggcaagattc ctgtctttgg tgtctgtatg 3480

ggccagcagt gtatcttcga ggagtttggc ggagacgtcg agtatgcggg cgagattgtc 3540

catggaaaaa cgtccactgt taagcacgac aacaagggaa tgttcaaaaa cgttccgcaa 3600

gatgttgctg tcaccagata ccactcgctg gccggaacgc tcaagtcgct tccggactgt 3660

ctagagatca ctgctcgcac agacaacggg atcattatgg gtgtgagaca caagaagtac 3720

accatcgagg gcgtccagtt tcatccagag agcattctga ccgaggaggg ccatctgatg 3780

atccagaata tcctcaacgt ttccggtggt tactgggagg aaaatgccaa cggcgcggct 3840

cagagaaagg aaagcatatt ggagaaaata tacgcgcaga gacgaaaaga ctacgagttt 3900

gagatgaaca gaccggggcg cagatttgct gatctagaac tgtacttgtc catgggactg 3960

caccgccgct aatcaatttt tacgacagat tggagcagaa catcagcgcc ggcaaggttg 4020

caattctcag cgaaatcaag agagcgtcgc cttctaaagg cgtcatcgac ggagacgcta 4080

acgctgccaa acaggccctc aactacgcca aggctggagt tgccacaatt tctgttttga 4140

ccgagccaac ctggtttaaa ggaaatatcc aggacctgga ggtggccaga aaagccattg 4200

actctgtggc caatagaccg tgtattttgc ggaaggagtt tatcttcaac aagtaccaaa 4260

ttctagaggc ccgactggcg ggagcagaca cggttctgct gattgtcaag atgctgagct 4320

cggatccccc acacaccata gcttcaaaat gtttctactc cttttttact cttccagatt 4380

ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc atactaaatt 4440

ttccctcttt cttcctctag ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa 4500

aaagagaccg cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt 4560

ttctttttct tgaaattttt ttttttagtt tttttctctt tcagtgacct ccattgatat 4620

ttaagttaat aaacggtctt caatttctca agtttcagtt tcatttttct tgttctatta 4680

caactttttt tacttcttgt tcattagaaa gaaagcatag caatctaatc taaggggcgg 4740

tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa 4800

ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg 4860

agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt 4920

cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt 4980

gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg 5040

gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg 5100

cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt 5160

cgtggccgag gagcaggact gacacgtccg acggcggccc acgggtccca ggcctcggag 5220

atccgtcccc cttttccttt gtcgatatca tgtaattagt tatgtcacgc ttacattcac 5280

gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg 5340

tccctattta tttttttata gttatgttag tattaagaac gttatttata tttcaaattt 5400

ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg 5460

agaaggtttt gggacgctcg aaggctttaa tttgcaagct ggagaccaac atgtgagcaa 5520

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 5580

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 5640

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 5700

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 5760

ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 5820

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 5880

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 5940

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6000

acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6060

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6120

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6180

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagatc 6238

Claims (10)

1. A polynucleotide encoding HPV39L1 protein, wherein the sequence of the polynucleotide is as set forth in SEQ ID NO: 3, respectively.

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

3. A host cell comprising or incorporating the recombinant expression vector of claim 2.

4. The host cell of claim 3, wherein the host cell is a yeast; preferably, Hansenula polymorpha; more preferably, it is Hansenula polymorpha.

5. A method of producing HPV39L1 protein, comprising the steps of: constructing a polypeptide integrated with or containing a nucleotide sequence shown as SEQ ID NO: 3, culturing, collecting thalli, crushing the thalli to obtain lysate, separating and purifying the lysate to obtain the HPV39L1 protein.

6. The method of producing HPV39L1 protein according to claim 5, further comprising one or more of the following features:

1) the polynucleotide is integrated into a plasmid, and the recombinant hansenula polymorpha strain contains the plasmid;

2) the conditions of the culture include: the pH value 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%;

3) culturing the recombinant hansenula polymorpha strain in a culture medium containing glycerol; in the culture process, when the glycerol in the culture medium is completely consumed and the wet weight of the thalli is more than 100g/L, adding the glycerol at a feed rate of 200-600 g/h; when the wet weight of the thalli is more than 200g/L, starting to add methanol to 0.5% (w/v) at one time, entering a methanol induction period, starting to add methanol in a flowing manner when methanol is completely consumed and dissolved oxygen rises to 80%, gradually adjusting the methanol flowing speed along with the acceleration of the thalli by using the methanol, controlling the dissolved oxygen to be more than 20% in the induction process, and finishing fermentation after the wet weight of the thalli reaches 300-400 g/L after induction is carried out for 30-50 hours;

4) the separation and purification means that the thalli lysate is subjected to cation exchange chromatography and then CHT chromatography.

7. An HPV39L1 protein obtainable by a method of producing an HPV39L1 protein according to any one of claims 5 to 6.

8. Use of the polynucleotide of claim 1 encoding an HPV39L1 protein, or the recombinant expression vector of claim 2, or the host cell of claim 3, or the HPV39L1 protein of claim 7, for the preparation of an HPV vaccine.

9. A preparation method of an anti-HPV vaccine comprises the following steps: the method for producing HPV39L1 protein according to any one of claims 5-6 is used to prepare HPV39L1 protein and add pharmaceutically acceptable vaccine adjuvant.

10. An anti-HPV vaccine obtained by the method of claim 9 for the preparation of an anti-HPV vaccine.

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