CN113604482A - Polynucleotide for expressing HPV68L1, expression vector, host cell and application thereof - Google Patents

Polynucleotide for expressing HPV68L1, expression vector, host cell and application thereof Download PDF

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CN113604482A
CN113604482A CN202110981111.5A CN202110981111A CN113604482A CN 113604482 A CN113604482 A CN 113604482A CN 202110981111 A CN202110981111 A CN 202110981111A CN 113604482 A CN113604482 A CN 113604482A
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hpv68l1
protein
thalli
polynucleotide
methanol
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袁靖宇
沈琼
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Shanghai Bowei Biotechnology Co ltd
Chongqing Bloomer Bio Pharmaceutical Co ltd
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Chongqing Bloomer Bio Pharmaceutical Co ltd
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Abstract

The invention provides a polynucleotide for expressing HPV68L1, an expression vector, a host cell and application thereof. The HPV68L1 protein produced by the polynucleotide has high yield. The HPV68L1 protein prepared by the method can be used for preparing vaccines for preventing HPV68 infection.

Description

Polynucleotide for expressing HPV68L1, expression vector, host cell and application thereof
Technical Field
The invention relates to the technical field of biology, relates to a method for producing HPV68L1 protein, and particularly relates to an expression HPV68L1 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
Figure BDA0003229149290000021
(HPV16, 18), tetravalent vaccine from Moshadong
Figure BDA0003229149290000022
(HPV 6, 11, 16, 18) and nine-valent vaccines
Figure BDA0003229149290000023
9(HPV 6, 11, 16, 18, 31, 33, 45, 52, 58), and Xiamengtai sea Biotechnology Limited bivalent vaccine
Figure BDA0003229149290000024
(HPV16, 18). The three companies adopt insect cell-baculovirus expression systems and brewage respectivelyPreparing HPV L1 protein by using a wine yeast expression system and an escherichia coli expression system, and preparing the VLP vaccine for preventing HPV infection after adsorbing an adjuvant by using the purified antigen.
Disclosure of Invention
The invention aims to provide a polynucleotide for expressing HPV68L1, an expression vector, a host cell and application thereof.
In one aspect, the present invention provides a polynucleotide for encoding HPV68L1 protein, the sequence of the polynucleotide is as shown in SEQ ID NO: 2, respectively.
Further, the amino acid sequence of the HPV68L1 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: 2 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, Hansenula polymorpha (Hansenula polymorpha) is used.
In a fourth aspect, the present invention provides a method for producing HPV68L1 protein, comprising the steps of: constructing a polypeptide integrated with or containing a nucleotide sequence shown as SEQ ID NO: 2, culturing, collecting thalli, crushing the thalli to obtain lysate, and separating and purifying the lysate to obtain the HPV68L1 protein.
Further, the polynucleotide is integrated into a plasmid contained in the recombinant Hansenula polymorpha strain.
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; 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;
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 exchange chromatography packing of the cation chromatography column is POROS HS, Nanogel SP or the like.
In the fifth aspect, the invention provides HPV68L1 protein, which is obtained by adopting the method for producing the HPV68L1 protein.
The sixth aspect of the invention provides the use of the aforementioned polynucleotide for encoding HPV68L1 protein, or recombinant expression vector, or host cell, or HPV68L1 protein in the preparation of HPV vaccine.
The seventh aspect of the invention provides a method for preparing an anti-HPV vaccine, which comprises the following steps: the HPV68L1 protein is prepared by the method for producing the HPV68L1 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 invention has the beneficial technical effects that: the present invention provides SEQ ID NO: 2, the yield of the encoded HPV68L1 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.
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FIG. 1: the pMTZ vector of one embodiment of the present invention is a structural diagram.
FIG. 2: the structure of 68L1-1-pMTZ vector of one embodiment of the present invention.
FIG. 3: the structure of 68L1-2-pMTZ vector in one embodiment of the invention.
FIG. 4: the structure of 68L1-3-pMTZ vector in one embodiment of the invention.
FIG. 5: the structure of 68L1-4-pMTZ vector in one embodiment of the invention.
FIG. 6: detecting the expression condition of 68L1 protein of the recombinant hansenula polymorpha engineering strain containing different nucleotide coding sequences of 68L1-1, 68L1-2, 68L1-3 and 68L1-4 by an enzyme-linked immunosorbent assay;
FIG. 7: SDS-PAGE detection of HPV68L1 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 HPV68L1 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: SDS-PAGE detection of purified HPV68L1 protein. M: a molecular weight standard; 1: purified HPV68L1 protein.
FIG. 10: and (3) observing the result of the purified HPV68L1 protein by using a transmission electron microscope.
Detailed Description
In order to realize the high-efficiency expression of the HPV68L1 protein in Hansenula polymorpha, the invention discloses a nucleotide sequence for coding the HPV68L1 protein, a preparation method of a recombinant Hansenula polymorpha strain for expressing the HPV68L1 protein, and a fermentation process for ensuring the high-efficiency expression of the HPV68L1 VLP. The expressed HPV68L1 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 HPV68L1 vaccine or a multivalent recombinant HPV vaccine, so that HPV68 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 HPV68 infection are further prevented.
The invention synthesizes 4 different DNA coding sequences according to the amino acid sequence of HPV68L1 protein. The synthesized DNA sequences are respectively constructed on hansenula polymorpha expression vectors to obtain 4 recombinant expression plasmids carrying HPV68L1 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 HPV68L1 protein expression of the 2 gene strain is better than that of other DNA coding sequences. Mixing a mixture containing SEQ ID NO: 2, fermenting and culturing the high-expression strain of the gene in a fermentation tank, purifying and carrying out chromatography to obtain high-purity HPV68L1 protein, and adsorbing by an aluminum adjuvant to obtain the HPV68L1 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 construction of HPV68L1 protein engineered Strain
Selection of HPV68L1 amino acid sequence
The full-length HPV68L1 protein consists of 505 amino acids, and after NCBI GenBank retrieval and alignment analysis, the most representative conserved sequence (GenBank: ACL12341.1) is selected as the amino acid sequence of HPV68L1, and the sequence information is shown in SEQ ID NO: 1 is shown.
SEQ ID NO：1
MALWRSSDNMVYLPPPSVAKVVNTDDYVTRTGIYYYAGTSRLLTVGHPYFKVPMSGGRKQ
DIPKVSAYQYRVFRISLPDPNKFSLPESTLYNPDTQRLVWACVGVEIGRGQPLGVGLSGHPLY
NRLDDTENSPFSSNKNPKDSRDNVSVDYKQTQLCIIGCVPAIGEHWAKGKSCKPSNVQPGD
CPPLELVNTPIQDGDMIDTGYGAMDFSTLQETKSEVPLDICQSVCKYPDYLQMSADVYGDS
MFFCLRREQLFARHFWNRGGMVGDTIPTELYIKGTDIRDSPSSYVYAPSPSGSMVSSDSQLF
NKPYWLHKAQGHNNGICWHNQLFLTVVDTTRSTNFTLSTTTESAVPNIYDPNKFKEYIRHV
EEYDLQFIFQLCTITLSTDVMSYIHTMNPAILDDWNFGVAPPPSASLVDTYRYLQSAAITCQK
DAPAPTKKDPYDGLNFWNVNLKEKFSSELDQFPLGRKFLLQAGVRRRPTIGPRKRPATATTA
STSKHKRKRVSK
Design and Synthesis of HPV68L1 encoding Gene
In order to efficiently express HPV68L1 protein in Hansenula polymorpha, the invention optimizes the nucleotide coding sequence of HPV68L1 by adopting a Hansenula polymorpha codon optimization strategy based on the nucleotide sequence of a wild-type strain of HPV68L1 with GenBank ID of ACL12341.1 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
atggctctgtggagatcctctgacaacatggtttacttgcctccaccttccgtcgccaaggttgtcaacaccgacgacta
cgtgaccagaacgggcatctactattacgccggtacttcgagattgctcactgttggacacccatacttcaaggtcccta
tgtctggtggcagaaagcaggacatccctaaggtgtccgcttaccagtacagagttttcagaatctccctgcctgaccca
aacaagttctcgctcccagagtccaccttgtacaaccctgacacgcagagactggtctgggcctgtgttggtgtggaaat
tggcagaggacagccacttggtgttggcctgtctggtcacccattgtacaacagattggacgataccgagaactctccat
tctcctcgaacaagaatcctaaggactccagagacaacgtctctgtggactacaagcagacccaactgtgcattatcggc
tgtgttccagctattggagagcactgggccaagggcaagtcgtgcaagccttccaacgtccagccaggtgactgtcctcc
attggagttggtgaacacccctatccaggatggcgatatgatcgacactggatacggtgctatggacttttcgacccttc
aggagaccaagtccgaggttcctctcgacatttgccagtctgtttgtaagtaccctgattacttgcagatgtcggcagac
gtgtacggagactccatgttcttttgcctgagaagagagcagctcttcgccagacacttctggaacagaggtggcatggt
tggagacactatcccaaccgagttgtacattaagggcactgacatcagagactcgccttcttcctatgtctacgctccat
ctccttcaggttctatggttagctccgactcgcagctgttcaacaagccatactggcttcacaaggcccagggacacaac
aatggcatctgctggcacaaccagctcttcctgacggttgtcgacaccactagatccacaaacttcaccttgtcgactac
gaccgagtctgcagttccaaacatctacgaccctaacaagttcaaggagtatattagacacgttgaggaatatgacctgc
agttcatcttccagctctgcaccattacattgtccactgacgtgatgtcctacatccatacgatgaacccagccattctg
gatgactggaactttggtgttgcaccacctccatctgcttccttggtcgacacctacagatacctgcaatcggccgctat
cacgtgtcagaaggacgccccagcacctaccaagaaggacccttacgacggattgaacttctggaatgtcaacctcaagg
agaagttctcgtctgaactggaccagtttccattgggcagaaagttcctgttgcaggccggtgtcagacgcagacctacc
atcggtccaagaaagagacctgccacagcaactacggcttctacctccaagcacaagcgtaagagagtgtccaagtaata
g
SEQ ID NO：3
atggccttgtggagatcgtctgacaacatggtctacctgccacctccatctgttgctaaggtcgttaacactgacgatta
cgtcacgagaaccggtatctactactatgctggaacctccagactgttgaccgtcggtcacccttacttcaaggtgccaa
tgtccggaggtagaaagcaggacatcccaaaggtttctgcctaccagtacagagtcttcagaatctctttgccagaccct
aacaagttctccctgcctgagtcgaccctgtacaacccagacactcagagattggtttgggcttgcgtcggcgttgagat
cggtagaggccaacctctgggagtgggtctctccggccaccctctgtacaacagactcgatgacaccgagaactcccctt
tctcttccaataagaacccaaaggactccagagataacgtttccgtcgactacaagcagactcagctttgtatcattggt
tgcgtgcctgccatcggtgaacactgggcaaagggaaagtcctgtaagccatcgaacgttcagccaggagactgcccacc
tctggagcttgttaacactccaatccaagacggtgacatgattgacaccggctacggagctatggacttctctaccttgc
aggagacgaagtctgaggtcccattggacatctgccagtccgtgtgcaagtacccagactaccttcaaatgtccgctgac
gtttacggcgactctatgttcttctgtttgagaagagagcagctgttcgctagacacttttggaacagaggcggtatggt
tggtgacaccattcctacggaactctacatcaagggtacagacatcagagactctccatccagctacgtttatgcccctt
ccccatcgggctcaatggtctcttccgactcgcagttgttcaacaagccttactggctgcacaaggctcaaggccacaat
aacggtatttgttggcacaaccagctgttcctcaccgtggttgacacaaccagatccaccaactttacgctttccaccac
taccgaatctgccgtccctaacatctacgacccaaacaagttcaaggaatacatcagacacgtggaagagtacgacctcc
aattcatcttccagttgtgcactatcaccctgtctaccgacgttatgtcttacattcacacgatgaaccctgcaatcttg
gacgattggaacttcggcgtcgcccctccaccttccgcatctcttgttgacacttacagatacttgcagtccgcagccat
cacttgccaaaaggacgctcctgccccaactaagaaggacccatacgacggcctgaacttctggaacgttaacttgaagg
agaagttctcctcggaactcgaccagttccctctgggtagaaagttcctcctgcaggcaggagtgagacgcagaccaacg
atcggcccaagaaagagaccagctaccgccaccactgcatccacgtctaagcacaagagaaagagagtctcgaagtaata
g
SEQ ID NO：4
atggctctgtggagatcctctgacaacatggtttacttgcctccaccttccgtcgccaaggttgtcaacaccgacgactacgtgaccagaacggg
catctactattacgccggtacttcgagattgctcactgttggacacccatacttcaaggtccctatgtctggtggcagaaagcaggacatccctaag
gtgtccgcttaccagtacagagttttcagaatctccctgcctgacccaaacaagttctcgctcccagagtccaccttgtacaaccctgacacgcag
agactggtctgggcctgtgttggtgtggaaattggcagaggacagccacttggtgttggcctgtctggtcacccattgtacaacagattggacgat
accgagaactctccattctcctcgaacaagaatcctaaggactccagagacaacgtctctgtggactacaagcagacccaactgtgcattatcgg
ctgtgttccagctattggagagcactgggccaagggcaagtcgtgcaagccttccaacgtccagccaggtgactgtcctccattggagttggtga
acacccctatccaggatggcgatatgatcgacactggatacggtgctatggacttttcgacccttcaggagaccaagtccgaggttcctctcgac
atttgccagtctgtttgtaagtaccctgattacttgcagatgtcggcagacgtgtacggagactccatgttcttttgcctgagaagagagcagctctt
cgccagacacttctggaacagaggtggcatggttggagacactatcccaaccgagttgtacattaagggcactgacatcagagactcgccttctt
cctatgtctacgctccatctccttcaggttctatggttagctccgactcgcagctgttcaacaagccatactggcttcacaaggcccagggacacaa
caatggcatctgctggcacaaccagctcttcctgacggttgtcgacaccactagatccacaaacttcaccttgtcgactacgaccgagtctgcagt
tccaaacatctacgaccctaacaagttcaaggagtatattagacacgttgaggaatatgacctgcagttcatcttccagctctgcaccattacattgt
ccactgacgtgatgtcctacatccatacgatgaacccagccattctggatgactggaactttggtgttgcaccacctccatctgcttccttggtcgac
acctacagatacctgcaatcggccgctatcacgtgtcagaaggacgccccagcacctaccaagaaggacccttacgacggattgaacttctgg
aatgtcaacctcaaggagaagttctcgtctgaactggaccagtttccattgggcagaaagttcctgttgcaggccggtgtcagacgcagacctac
catcggtccaagaaagagacctgccacagcaactacggcttctacctccaagcacaagcgtaagagagtgtccaagtaatag
SEQ ID NO：5
atggccttgtggagatcgtctgacaacatggtctacctgccacctccatctgttgctaaggtcgttaacactgacgattacgtcacgagaaccggt
atctactactatgctggaacctccagactgttgaccgtcggtcacccttacttcaaggtgccaatgtccggaggtagaaagcaggacatcccaaa
ggtttctgcctaccagtacagagtcttcagaatctctttgccagaccctaacaagttctccctgcctgagtcgaccctgtacaacccagacactcag
agattggtttgggcttgcgtcggcgttgagatcggtagaggccaacctctgggagtgggtctctccggccaccctctgtacaacagactcgatga
caccgagaactcccctttctcttccaataagaacccaaaggactccagagataacgtttccgtcgactacaagcagactcagctttgtatcattggt
tgcgtgcctgccatcggtgaacactgggcaaagggaaagtcctgtaagccatcgaacgttcagccaggagactgcccacctctggagcttgtta
acactccaatccaagacggtgacatgattgacaccggctacggagctatggacttctctaccttgcaggagacgaagtctgaggtcccattgga
catctgccagtccgtgtgcaagtacccagactaccttcaaatgtccgctgacgtttacggcgactctatgttcttctgtttgagaagagagcagctgt
tcgctagacacttttggaacagaggcggtatggttggtgacaccattcctacggaactctacatcaagggtacagacatcagagactctccatcc
agctacgtttatgccccttccccatcgggctcaatggtctcttccgactcgcagttgttcaacaagccttactggctgcacaaggctcaaggccac
aataacggtatttgttggcacaaccagctgttcctcaccgtggttgacacaaccagatccaccaactttacgctttccaccactaccgaatctgccg
tccctaacatctacgacccaaacaagttcaaggaatacatcagacacgtggaagagtacgacctccaattcatcttccagttgtgcactatcaccc
tgtctaccgacgttatgtcttacattcacacgatgaaccctgcaatcttggacgattggaacttcggcgtcgcccctccaccttccgcatctcttgttg
acacttacagatacttgcagtccgcagccatcacttgccaaaaggacgctcctgccccaactaagaaggacccatacgacggcctgaacttctg
gaacgttaacttgaaggagaagttctcctcggaactcgaccagttccctctgggtagaaagttcctcctgcaggcaggagtgagacgcagacca
acgatcggcccaagaaagagaccagctaccgccaccactgcatccacgtctaagcacaagagaaagagagtctcgaagtaatag
Construction of HPV68L1 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 HPV68L1 coding sequences were cloned into pMTZ vector by 5 'BstBI cleavage site and 3' KpnI cleavage site to obtain expression vectors 68L1-1-pMTZ (SEQ ID NO: 7, FIG. 2), 68L1-2-pMTZ (SEQ ID NO: 8, FIG. 3), 68L1-3-pMTZ (SEQ ID NO: 9, FIG. 4) and 68L1-4-pMTZ (SEQ ID NO: 10, FIG. 5). Transcription of the HPV68L1 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
68L1-1-pMTZ(SEQ ID NO：7)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcg
gaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaa
ttcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccat
aatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtc
gctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaattt
ttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctcc
aacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgat
gtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggcttt
ggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccact
gcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttcctt
gagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccg
caagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcga
ggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctg
tgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgg
gtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgc
cgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattc
gaaacgatggctctgtggagatcctctgacaacatggtttacttgcctccaccttccgtcgccaaggttgtcaacaccgacgactacgtgaccag
aacgggcatctactattacgccggtacttcgagattgctcactgttggacacccatacttcaaggtccctatgtctggtggcagaaagcaggacat
ccctaaggtgtccgcttaccagtacagagttttcagaatctccctgcctgacccaaacaagttctcgctcccagagtccaccttgtacaaccctgac
acgcagagactggtctgggcctgtgttggtgtggaaattggcagaggacagccacttggtgttggcctgtctggtcacccattgtacaacagatt
ggacgataccgagaactctccattctcctcgaacaagaatcctaaggactccagagacaacgtctctgtggactacaagcagacccaactgtgc
attatcggctgtgttccagctattggagagcactgggccaagggcaagtcgtgcaagccttccaacgtccagccaggtgactgtcctccattgga
gttggtgaacacccctatccaggatggcgatatgatcgacactggatacggtgctatggacttttcgacccttcaggagaccaagtccgaggttc
ctctcgacatttgccagtctgtttgtaagtaccctgattacttgcagatgtcggcagacgtgtacggagactccatgttcttttgcctgagaagagag
cagctcttcgccagacacttctggaacagaggtggcatggttggagacactatcccaaccgagttgtacattaagggcactgacatcagagact
cgccttcttcctatgtctacgctccatctccttcaggttctatggttagctccgactcgcagctgttcaacaagccatactggcttcacaaggcccag
ggacacaacaatggcatctgctggcacaaccagctcttcctgacggttgtcgacaccactagatccacaaacttcaccttgtcgactacgaccga
gtctgcagttccaaacatctacgaccctaacaagttcaaggagtatattagacacgttgaggaatatgacctgcagttcatcttccagctctgcacc
attacattgtccactgacgtgatgtcctacatccatacgatgaacccagccattctggatgactggaactttggtgttgcaccacctccatctgcttcc
ttggtcgacacctacagatacctgcaatcggccgctatcacgtgtcagaaggacgccccagcacctaccaagaaggacccttacgacggattg
aacttctggaatgtcaacctcaaggagaagttctcgtctgaactggaccagtttccattgggcagaaagttcctgttgcaggccggtgtcagacgc
agacctaccatcggtccaagaaagagacctgccacagcaactacggcttctacctccaagcacaagcgtaagagagtgtccaagtaataggta
ccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctg
aaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcat
gatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcacca
ttccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgat
cagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcga
gattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgc
tggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtaca
ccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgg
gaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatga
acagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacat
cagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacagg
ccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccaga
aaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagc
agacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcg
gactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaa
aggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttag
tttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttctt
gttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggt
gaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccgg
ctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtg
gtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccggg
acgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcac
ttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatg
taattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctattt
atttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttg
cttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaa
aaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgaca
ggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcc
cttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccc
ccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggt
aacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtat
ctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgc
aagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgtta
agggattttggtcatgagatc
68L1-2-pMTZ(SEQ ID NO：8)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccttgtggagatcgtctgacaacatggtctacctgccacctccatctgttgctaaggtcgttaacactgacgattacgtcacgagaaccggtatctactactatgctggaacctccagactgttgaccgtcggtcacccttacttcaaggtgccaatgtccggaggtagaaagcaggacatcccaaaggtttctgcctaccagtacagagtcttcagaatctctttgccagaccctaacaagttctccctgcctgagtcgaccctgtacaacccagacactcagagattggtttgggcttgcgtcggcgttgagatcggtagaggccaacctctgggagtgggtctctccggccaccctctgtacaacagactcgatgacaccgagaactcccctttctcttccaataagaacccaaaggactccagagataacgtttccgtcgactacaagcagactcagctttgtatcattggttgcgtgcctgccatcggtgaacactgggcaaagggaaagtcctgtaagccatcgaacgttcagccaggagactgcccacctctggagcttgttaacactccaatccaagacggtgacatgattgacaccggctacggagctatggacttctctaccttgcaggagacgaagtctgaggtcccattggacatctgccagtccgtgtgcaagtacccagactaccttcaaatgtccgctgacgtttacggcgactctatgttcttctgtttgagaagagagcagctgttcgctagacacttttggaacagaggcggtatggttggtgacaccattcctacggaactctacatcaagggtacagacatcagagactctccatccagctacgtttatgccccttccccatcgggctcaatggtctcttccgactcgcagttgttcaacaagccttactggctgcacaaggctcaaggccacaataacggtatttgttggcacaaccagctgttcctcaccgtggttgacacaaccagatccaccaactttacgctttccaccactaccgaatctgccgtccctaacatctacgacccaaacaagttcaaggaatacatcagacacgtggaagagtacgacctccaattcatcttccagttgtgcactatcaccctgtctaccgacgttatgtcttacattcacacgatgaaccctgcaatcttggacgattggaacttcggcgtcgcccctccaccttccgcatctcttgttgacacttacagatacttgcagtccgcagccatcacttgccaaaaggacgctcctgccccaactaagaaggacccatacgacggcctgaacttctggaacgttaacttgaaggagaagttctcctcggaactcgaccagttccctctgggtagaaagttcctcctgcaggcaggagtgagacgcagaccaacgatcggcccaagaaagagaccagctaccgccaccactgcatccacgtctaagcacaagagaaagagagtctcgaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
68L1-3-pMTZ(SEQ ID NO：9)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctctgtggagatcctctgacaacatggtttacttgcctccaccttccgtcgccaaggttgtcaacaccgacgactacgtgaccagaacgggcatctactattacgccggtacttcgagattgctcactgttggacacccatacttcaaggtccctatgtctggtggcagaaagcaggacatccctaaggtgtccgcttaccagtacagagttttcagaatctccctgcctgacccaaacaagttctcgctcccagagtccaccttgtacaaccctgacacgcagagactggtctgggcctgtgttggtgtggaaattggcagaggacagccacttggtgttggcctgtctggtcacccattgtacaacagattggacgataccgagaactctccattctcctcgaacaagaatcctaaggactccagagacaacgtctctgtggactacaagcagacccaactgtgcattatcggctgtgttccagctattggagagcactgggccaagggcaagtcgtgcaagccttccaacgtccagccaggtgactgtcctccattggagttggtgaacacccctatccaggatggcgatatgatcgacactggatacggtgctatggacttttcgacccttcaggagaccaagtccgaggttcctctcgacatttgccagtctgtttgtaagtaccctgattacttgcagatgtcggcagacgtgtacggagactccatgttcttttgcctgagaagagagcagctcttcgccagacacttctggaacagaggtggcatggttggagacactatcccaaccgagttgtacattaagggcactgacatcagagactcgccttcttcctatgtctacgctccatctccttcaggttctatggttagctccgactcgcagctgttcaacaagccatactggcttcacaaggcccagggacacaacaatggcatctgctggcacaaccagctcttcctgacggttgtcgacaccactagatccacaaacttcaccttgtcgactacgaccgagtctgcagttccaaacatctacgaccctaacaagttcaaggagtatattagacacgttgaggaatatgacctgcagttcatcttccagctctgcaccattacattgtccactgacgtgatgtcctacatccatacgatgaacccagccattctggatgactggaactttggtgttgcaccacctccatctgcttccttggtcgacacctacagatacctgcaatcggccgctatcacgtgtcagaaggacgccccagcacctaccaagaaggacccttacgacggattgaacttctggaatgtcaacctcaaggagaagttctcgtctgaactggaccagtttccattgggcagaaagttcctgttgcaggccggtgtcagacgcagacctaccatcggtccaagaaagagacctgccacagcaactacggcttctacctccaagcacaagcgtaagagagtgtccaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
68L1-4-pMTZ(SEQ ID NO：10)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccttgtggagatcgtctgacaacatggtctacctgccacctccatctgttgctaaggtcgttaacactgacgattacgtcacgagaaccggtatctactactatgctggaacctccagactgttgaccgtcggtcacccttacttcaaggtgccaatgtccggaggtagaaagcaggacatcccaaaggtttctgcctaccagtacagagtcttcagaatctctttgccagaccctaacaagttctccctgcctgagtcgaccctgtacaacccagacactcagagattggtttgggcttgcgtcggcgttgagatcggtagaggccaacctctgggagtgggtctctccggccaccctctgtacaacagactcgatgacaccgagaactcccctttctcttccaataagaacccaaaggactccagagataacgtttccgtcgactacaagcagactcagctttgtatcattggttgcgtgcctgccatcggtgaacactgggcaaagggaaagtcctgtaagccatcgaacgttcagccaggagactgcccacctctggagcttgttaacactccaatccaagacggtgacatgattgacaccggctacggagctatggacttctctaccttgcaggagacgaagtctgaggtcccattggacatctgccagtccgtgtgcaagtacccagactaccttcaaatgtccgctgacgtttacggcgactctatgttcttctgtttgagaagagagcagctgttcgctagacacttttggaacagaggcggtatggttggtgacaccattcctacggaactctacatcaagggtacagacatcagagactctccatccagctacgtttatgccccttccccatcgggctcaatggtctcttccgactcgcagttgttcaacaagccttactggctgcacaaggctcaaggccacaataacggtatttgttggcacaaccagctgttcctcaccgtggttgacacaaccagatccaccaactttacgctttccaccactaccgaatctgccgtccctaacatctacgacccaaacaagttcaaggaatacatcagacacgtggaagagtacgacctccaattcatcttccagttgtgcactatcaccctgtctaccgacgttatgtcttacattcacacgatgaaccctgcaatcttggacgattggaacttcggcgtcgcccctccaccttccgcatctcttgttgacacttacagatacttgcagtccgcagccatcacttgccaaaaggacgctcctgccccaactaagaaggacccatacgacggcctgaacttctggaacgttaacttgaaggagaagttctcctcggaactcgaccagttccctctgggtagaaagttcctcctgcaggcaggagtgagacgcagaccaacgatcggcccaagaaagagaccagctaccgccaccactgcatccacgtctaagcacaagagaaagagagtctcgaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
construction of HPV68L1 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). 68L1-1-pMTZ, 68L1-2-pMTZ, 68L1-3-pMTZ and 68L1-4-pMTZ recombinant expression plasmids were linearized with ScaI enzyme, respectively, and transferred to Hansenula polymorpha under the conditions of 1500V, 120. omega. and 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 HPV68L1 recombinant engineered Strain
1. Glass test tube expression screening
Single colonies of 6 recombinant Hansenula polymorpha were randomly picked from YPD plates electroporated with 68L1-1-pMTZ, 68L1-2-pMTZ, 68L1-3-pMTZ and 68L1-4-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 HPV68L1 protein in the broken thalli is quantitatively detected by an enzyme-linked immunosorbent assay (ELISA), and the result is shown in figure 6: the recombinant engineering strains containing different HPV68L1 coding sequences have clear expression, but the expression conditions of HPV68L1 proteins of different coding sequences have certain difference. In contrast, the expression effect of the recombinant engineering bacteria containing the coding sequences of 68L1-1 and 68L1-2 is better than that of the recombinant engineering bacteria containing the coding sequences of 68L1-3 and 68L1-4, and meanwhile, the expression level of the recombinant engineering bacteria containing the coding sequences of 68L1-1 is obviously higher than that of the recombinant engineering bacteria containing the coding sequences of 68L1-2, and the result has statistical significance (p is less than 0.05 and p is less than 0.01).
2. Fermenter expression screening
In order to further compare the expression advantages of the coding sequences of 68L1-1 and 68L1-2, from the engineering strains containing the coding sequences of 68L1-1 and 68L1-2, 1 strain was selected for fermentation tank expression verification, and the expression of 68L1 protein of the two 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 68L1-1 was significantly higher than that of the strain containing 68L 1-2.
Surface ELISA detects the antigen content of 68L1 protein in the broken bacteria supernatant of different strains
Type of Strain Antigen content (μ g/ml)
Strain comprising 68L1-1 359.482
Strain comprising 68L1-2 108.469
EXAMPLE 3 fermentation Process of HPV68L1 recombinant Hansenula polymorpha expression Strain
Preparing a seed solution: the strain of example 2, comprising 68L1-1, 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: the basic culture medium is prepared according to the formula of BSM1, 20L, and sterilized for 30min at 121 ℃ for standby. 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 HPV68L1 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 HPV68L1 recombinant protein
And (3) crushing thalli: taking HPV68L1 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 HPV68L1 protein is collected (shown in figure 9).
Example 5 Transmission Electron microscopy of HPV68L1 recombinant protein
The purified HPV68L1 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 HPV68L1 protein is shown in FIG. 10.
Example 6 preparation of HPV68L1 containing protein vaccine
Diluting the HPV68L1 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 HPV68L1 protein vaccine, and storing at 4 ℃ in a dark place.
Example 7 immunogenicity of HPV68L1 protein vaccine
Different doses of HPV68L1 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:
Figure BDA0003229149290000201
Figure BDA0003229149290000211
ELISA method for detecting antibody positive conversion rate in serum
The test procedure was as follows: 1) coating: HPV68L1 stock 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 HPV68L1 vaccine calculated from antibody positive conversion rate results of mouse sera at different dose levels50The value of (A) is 0.00007. mu.g, showing that the HPV68L1 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> Chongqing Bowei Baitai biopharmaceutical Co., Ltd
SHANGHAI BOWEI BIOTECHNOLOGY Co.,Ltd.
<120> polynucleotide for expressing HPV68L1, 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 Leu Trp Arg Ser Ser Asp Asn 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 Thr Ser Arg Leu Leu Thr Val Gly His Pro
35 40 45
Tyr Phe Lys Val Pro Met Ser Gly Gly Arg Lys Gln Asp Ile Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Ile Ser Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Ser Leu Pro Glu Ser Thr Leu Tyr Asn Pro Asp Thr Gln
85 90 95
Arg Leu Val Trp Ala Cys Val Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Leu Tyr Asn Arg Leu Asp Asp
115 120 125
Thr Glu Asn Ser Pro Phe Ser Ser Asn Lys Asn Pro Lys Asp Ser Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Ile Gly
145 150 155 160
Cys Val Pro Ala Ile Gly Glu His Trp Ala Lys Gly Lys Ser Cys Lys
165 170 175
Pro Ser Asn Val Gln Pro Gly Asp Cys Pro Pro Leu Glu Leu Val Asn
180 185 190
Thr Pro Ile Gln Asp Gly Asp Met Ile Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Ser Thr Leu Gln Glu Thr Lys Ser Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Val Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Val Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Gly Gly Met Val Gly Asp Thr Ile Pro
260 265 270
Thr Glu Leu Tyr Ile Lys Gly Thr Asp Ile Arg Asp Ser Pro Ser Ser
275 280 285
Tyr Val Tyr Ala Pro Ser Pro Ser Gly Ser Met Val Ser Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn
305 310 315 320
Asn Gly Ile Cys Trp His Asn Gln Leu Phe Leu Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Phe Thr Leu Ser Thr Thr Thr Glu Ser Ala Val
340 345 350
Pro Asn Ile Tyr Asp Pro Asn Lys Phe Lys Glu Tyr Ile Arg His Val
355 360 365
Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu
370 375 380
Ser Thr Asp Val Met Ser Tyr Ile His Thr Met Asn Pro Ala Ile Leu
385 390 395 400
Asp Asp Trp Asn Phe Gly Val Ala Pro Pro Pro Ser Ala Ser Leu Val
405 410 415
Asp Thr Tyr Arg Tyr Leu Gln Ser Ala Ala Ile Thr Cys Gln Lys Asp
420 425 430
Ala Pro Ala Pro Thr Lys Lys Asp Pro Tyr Asp Gly Leu Asn Phe Trp
435 440 445
Asn Val Asn Leu Lys Glu Lys Phe Ser Ser Glu Leu Asp Gln Phe Pro
450 455 460
Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly Val Arg Arg Arg Pro Thr
465 470 475 480
Ile Gly Pro Arg Lys Arg Pro Ala Thr Ala Thr Thr Ala Ser Thr Ser
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
atggctctgt ggagatcctc tgacaacatg gtttacttgc ctccaccttc cgtcgccaag 60
gttgtcaaca ccgacgacta cgtgaccaga acgggcatct actattacgc cggtacttcg 120
agattgctca ctgttggaca cccatacttc aaggtcccta tgtctggtgg cagaaagcag 180
gacatcccta aggtgtccgc ttaccagtac agagttttca gaatctccct gcctgaccca 240
aacaagttct cgctcccaga gtccaccttg tacaaccctg acacgcagag actggtctgg 300
gcctgtgttg gtgtggaaat tggcagagga cagccacttg gtgttggcct gtctggtcac 360
ccattgtaca acagattgga cgataccgag aactctccat tctcctcgaa caagaatcct 420
aaggactcca gagacaacgt ctctgtggac tacaagcaga cccaactgtg cattatcggc 480
tgtgttccag ctattggaga gcactgggcc aagggcaagt cgtgcaagcc ttccaacgtc 540
cagccaggtg actgtcctcc attggagttg gtgaacaccc ctatccagga tggcgatatg 600
atcgacactg gatacggtgc tatggacttt tcgacccttc aggagaccaa gtccgaggtt 660
cctctcgaca tttgccagtc tgtttgtaag taccctgatt acttgcagat gtcggcagac 720
gtgtacggag actccatgtt cttttgcctg agaagagagc agctcttcgc cagacacttc 780
tggaacagag gtggcatggt tggagacact atcccaaccg agttgtacat taagggcact 840
gacatcagag actcgccttc ttcctatgtc tacgctccat ctccttcagg ttctatggtt 900
agctccgact cgcagctgtt caacaagcca tactggcttc acaaggccca gggacacaac 960
aatggcatct gctggcacaa ccagctcttc ctgacggttg tcgacaccac tagatccaca 1020
aacttcacct tgtcgactac gaccgagtct gcagttccaa acatctacga ccctaacaag 1080
ttcaaggagt atattagaca cgttgaggaa tatgacctgc agttcatctt ccagctctgc 1140
accattacat tgtccactga cgtgatgtcc tacatccata cgatgaaccc agccattctg 1200
gatgactgga actttggtgt tgcaccacct ccatctgctt ccttggtcga cacctacaga 1260
tacctgcaat cggccgctat cacgtgtcag aaggacgccc cagcacctac caagaaggac 1320
ccttacgacg gattgaactt ctggaatgtc aacctcaagg agaagttctc gtctgaactg 1380
gaccagtttc cattgggcag aaagttcctg ttgcaggccg gtgtcagacg cagacctacc 1440
atcggtccaa gaaagagacc tgccacagca actacggctt ctacctccaa gcacaagcgt 1500
aagagagtgt ccaagtaata g 1521
<210> 3
<211> 1521
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atggccttgt ggagatcgtc tgacaacatg gtctacctgc cacctccatc tgttgctaag 60
gtcgttaaca ctgacgatta cgtcacgaga accggtatct actactatgc tggaacctcc 120
agactgttga ccgtcggtca cccttacttc aaggtgccaa tgtccggagg tagaaagcag 180
gacatcccaa aggtttctgc ctaccagtac agagtcttca gaatctcttt gccagaccct 240
aacaagttct ccctgcctga gtcgaccctg tacaacccag acactcagag attggtttgg 300
gcttgcgtcg gcgttgagat cggtagaggc caacctctgg gagtgggtct ctccggccac 360
cctctgtaca acagactcga tgacaccgag aactcccctt tctcttccaa taagaaccca 420
aaggactcca gagataacgt ttccgtcgac tacaagcaga ctcagctttg tatcattggt 480
tgcgtgcctg ccatcggtga acactgggca aagggaaagt cctgtaagcc atcgaacgtt 540
cagccaggag actgcccacc tctggagctt gttaacactc caatccaaga cggtgacatg 600
attgacaccg gctacggagc tatggacttc tctaccttgc aggagacgaa gtctgaggtc 660
ccattggaca tctgccagtc cgtgtgcaag tacccagact accttcaaat gtccgctgac 720
gtttacggcg actctatgtt cttctgtttg agaagagagc agctgttcgc tagacacttt 780
tggaacagag gcggtatggt tggtgacacc attcctacgg aactctacat caagggtaca 840
gacatcagag actctccatc cagctacgtt tatgcccctt ccccatcggg ctcaatggtc 900
tcttccgact cgcagttgtt caacaagcct tactggctgc acaaggctca aggccacaat 960
aacggtattt gttggcacaa ccagctgttc ctcaccgtgg ttgacacaac cagatccacc 1020
aactttacgc tttccaccac taccgaatct gccgtcccta acatctacga cccaaacaag 1080
ttcaaggaat acatcagaca cgtggaagag tacgacctcc aattcatctt ccagttgtgc 1140
actatcaccc tgtctaccga cgttatgtct tacattcaca cgatgaaccc tgcaatcttg 1200
gacgattgga acttcggcgt cgcccctcca ccttccgcat ctcttgttga cacttacaga 1260
tacttgcagt ccgcagccat cacttgccaa aaggacgctc ctgccccaac taagaaggac 1320
ccatacgacg gcctgaactt ctggaacgtt aacttgaagg agaagttctc ctcggaactc 1380
gaccagttcc ctctgggtag aaagttcctc ctgcaggcag gagtgagacg cagaccaacg 1440
atcggcccaa gaaagagacc agctaccgcc accactgcat ccacgtctaa gcacaagaga 1500
aagagagtct cgaagtaata g 1521
<210> 4
<211> 1521
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atggctctgt ggagatcctc tgacaacatg gtttacttgc ctccaccttc cgtcgccaag 60
gttgtcaaca ccgacgacta cgtgaccaga acgggcatct actattacgc cggtacttcg 120
agattgctca ctgttggaca cccatacttc aaggtcccta tgtctggtgg cagaaagcag 180
gacatcccta aggtgtccgc ttaccagtac agagttttca gaatctccct gcctgaccca 240
aacaagttct cgctcccaga gtccaccttg tacaaccctg acacgcagag actggtctgg 300
gcctgtgttg gtgtggaaat tggcagagga cagccacttg gtgttggcct gtctggtcac 360
ccattgtaca acagattgga cgataccgag aactctccat tctcctcgaa caagaatcct 420
aaggactcca gagacaacgt ctctgtggac tacaagcaga cccaactgtg cattatcggc 480
tgtgttccag ctattggaga gcactgggcc aagggcaagt cgtgcaagcc ttccaacgtc 540
cagccaggtg actgtcctcc attggagttg gtgaacaccc ctatccagga tggcgatatg 600
atcgacactg gatacggtgc tatggacttt tcgacccttc aggagaccaa gtccgaggtt 660
cctctcgaca tttgccagtc tgtttgtaag taccctgatt acttgcagat gtcggcagac 720
gtgtacggag actccatgtt cttttgcctg agaagagagc agctcttcgc cagacacttc 780
tggaacagag gtggcatggt tggagacact atcccaaccg agttgtacat taagggcact 840
gacatcagag actcgccttc ttcctatgtc tacgctccat ctccttcagg ttctatggtt 900
agctccgact cgcagctgtt caacaagcca tactggcttc acaaggccca gggacacaac 960
aatggcatct gctggcacaa ccagctcttc ctgacggttg tcgacaccac tagatccaca 1020
aacttcacct tgtcgactac gaccgagtct gcagttccaa acatctacga ccctaacaag 1080
ttcaaggagt atattagaca cgttgaggaa tatgacctgc agttcatctt ccagctctgc 1140
accattacat tgtccactga cgtgatgtcc tacatccata cgatgaaccc agccattctg 1200
gatgactgga actttggtgt tgcaccacct ccatctgctt ccttggtcga cacctacaga 1260
tacctgcaat cggccgctat cacgtgtcag aaggacgccc cagcacctac caagaaggac 1320
ccttacgacg gattgaactt ctggaatgtc aacctcaagg agaagttctc gtctgaactg 1380
gaccagtttc cattgggcag aaagttcctg ttgcaggccg gtgtcagacg cagacctacc 1440
atcggtccaa gaaagagacc tgccacagca actacggctt ctacctccaa gcacaagcgt 1500
aagagagtgt ccaagtaata g 1521
<210> 5
<211> 1521
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atggccttgt ggagatcgtc tgacaacatg gtctacctgc cacctccatc tgttgctaag 60
gtcgttaaca ctgacgatta cgtcacgaga accggtatct actactatgc tggaacctcc 120
agactgttga ccgtcggtca cccttacttc aaggtgccaa tgtccggagg tagaaagcag 180
gacatcccaa aggtttctgc ctaccagtac agagtcttca gaatctcttt gccagaccct 240
aacaagttct ccctgcctga gtcgaccctg tacaacccag acactcagag attggtttgg 300
gcttgcgtcg gcgttgagat cggtagaggc caacctctgg gagtgggtct ctccggccac 360
cctctgtaca acagactcga tgacaccgag aactcccctt tctcttccaa taagaaccca 420
aaggactcca gagataacgt ttccgtcgac tacaagcaga ctcagctttg tatcattggt 480
tgcgtgcctg ccatcggtga acactgggca aagggaaagt cctgtaagcc atcgaacgtt 540
cagccaggag actgcccacc tctggagctt gttaacactc caatccaaga cggtgacatg 600
attgacaccg gctacggagc tatggacttc tctaccttgc aggagacgaa gtctgaggtc 660
ccattggaca tctgccagtc cgtgtgcaag tacccagact accttcaaat gtccgctgac 720
gtttacggcg actctatgtt cttctgtttg agaagagagc agctgttcgc tagacacttt 780
tggaacagag gcggtatggt tggtgacacc attcctacgg aactctacat caagggtaca 840
gacatcagag actctccatc cagctacgtt tatgcccctt ccccatcggg ctcaatggtc 900
tcttccgact cgcagttgtt caacaagcct tactggctgc acaaggctca aggccacaat 960
aacggtattt gttggcacaa ccagctgttc ctcaccgtgg ttgacacaac cagatccacc 1020
aactttacgc tttccaccac taccgaatct gccgtcccta acatctacga cccaaacaag 1080
ttcaaggaat acatcagaca cgtggaagag tacgacctcc aattcatctt ccagttgtgc 1140
actatcaccc tgtctaccga cgttatgtct tacattcaca cgatgaaccc tgcaatcttg 1200
gacgattgga acttcggcgt cgcccctcca ccttccgcat ctcttgttga cacttacaga 1260
tacttgcagt ccgcagccat cacttgccaa aaggacgctc ctgccccaac taagaaggac 1320
ccatacgacg gcctgaactt ctggaacgtt aacttgaagg agaagttctc ctcggaactc 1380
gaccagttcc ctctgggtag aaagttcctc ctgcaggcag gagtgagacg cagaccaacg 1440
atcggcccaa gaaagagacc agctaccgcc accactgcat ccacgtctaa gcacaagaga 1500
aagagagtct cgaagtaata 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 ctctgtggag atcctctgac aacatggttt 1560
acttgcctcc accttccgtc gccaaggttg tcaacaccga cgactacgtg accagaacgg 1620
gcatctacta ttacgccggt acttcgagat tgctcactgt tggacaccca tacttcaagg 1680
tccctatgtc tggtggcaga aagcaggaca tccctaaggt gtccgcttac cagtacagag 1740
ttttcagaat ctccctgcct gacccaaaca agttctcgct cccagagtcc accttgtaca 1800
accctgacac gcagagactg gtctgggcct gtgttggtgt ggaaattggc agaggacagc 1860
cacttggtgt tggcctgtct ggtcacccat tgtacaacag attggacgat accgagaact 1920
ctccattctc ctcgaacaag aatcctaagg actccagaga caacgtctct gtggactaca 1980
agcagaccca actgtgcatt atcggctgtg ttccagctat tggagagcac tgggccaagg 2040
gcaagtcgtg caagccttcc aacgtccagc caggtgactg tcctccattg gagttggtga 2100
acacccctat ccaggatggc gatatgatcg acactggata cggtgctatg gacttttcga 2160
cccttcagga gaccaagtcc gaggttcctc tcgacatttg ccagtctgtt tgtaagtacc 2220
ctgattactt gcagatgtcg gcagacgtgt acggagactc catgttcttt tgcctgagaa 2280
gagagcagct cttcgccaga cacttctgga acagaggtgg catggttgga gacactatcc 2340
caaccgagtt gtacattaag ggcactgaca tcagagactc gccttcttcc tatgtctacg 2400
ctccatctcc ttcaggttct atggttagct ccgactcgca gctgttcaac aagccatact 2460
ggcttcacaa ggcccaggga cacaacaatg gcatctgctg gcacaaccag ctcttcctga 2520
cggttgtcga caccactaga tccacaaact tcaccttgtc gactacgacc gagtctgcag 2580
ttccaaacat ctacgaccct aacaagttca aggagtatat tagacacgtt gaggaatatg 2640
acctgcagtt catcttccag ctctgcacca ttacattgtc cactgacgtg atgtcctaca 2700
tccatacgat gaacccagcc attctggatg actggaactt tggtgttgca ccacctccat 2760
ctgcttcctt ggtcgacacc tacagatacc tgcaatcggc cgctatcacg tgtcagaagg 2820
acgccccagc acctaccaag aaggaccctt acgacggatt gaacttctgg aatgtcaacc 2880
tcaaggagaa gttctcgtct gaactggacc agtttccatt gggcagaaag ttcctgttgc 2940
aggccggtgt cagacgcaga cctaccatcg gtccaagaaa gagacctgcc acagcaacta 3000
cggcttctac ctccaagcac aagcgtaaga gagtgtccaa 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 ccttgtggag atcgtctgac aacatggtct 1560
acctgccacc tccatctgtt gctaaggtcg ttaacactga cgattacgtc acgagaaccg 1620
gtatctacta ctatgctgga acctccagac tgttgaccgt cggtcaccct tacttcaagg 1680
tgccaatgtc cggaggtaga aagcaggaca tcccaaaggt ttctgcctac cagtacagag 1740
tcttcagaat ctctttgcca gaccctaaca agttctccct gcctgagtcg accctgtaca 1800
acccagacac tcagagattg gtttgggctt gcgtcggcgt tgagatcggt agaggccaac 1860
ctctgggagt gggtctctcc ggccaccctc tgtacaacag actcgatgac accgagaact 1920
cccctttctc ttccaataag aacccaaagg actccagaga taacgtttcc gtcgactaca 1980
agcagactca gctttgtatc attggttgcg tgcctgccat cggtgaacac tgggcaaagg 2040
gaaagtcctg taagccatcg aacgttcagc caggagactg cccacctctg gagcttgtta 2100
acactccaat ccaagacggt gacatgattg acaccggcta cggagctatg gacttctcta 2160
ccttgcagga gacgaagtct gaggtcccat tggacatctg ccagtccgtg tgcaagtacc 2220
cagactacct tcaaatgtcc gctgacgttt acggcgactc tatgttcttc tgtttgagaa 2280
gagagcagct gttcgctaga cacttttgga acagaggcgg tatggttggt gacaccattc 2340
ctacggaact ctacatcaag ggtacagaca tcagagactc tccatccagc tacgtttatg 2400
ccccttcccc atcgggctca atggtctctt ccgactcgca gttgttcaac aagccttact 2460
ggctgcacaa ggctcaaggc cacaataacg gtatttgttg gcacaaccag ctgttcctca 2520
ccgtggttga cacaaccaga tccaccaact ttacgctttc caccactacc gaatctgccg 2580
tccctaacat ctacgaccca aacaagttca aggaatacat cagacacgtg gaagagtacg 2640
acctccaatt catcttccag ttgtgcacta tcaccctgtc taccgacgtt atgtcttaca 2700
ttcacacgat gaaccctgca atcttggacg attggaactt cggcgtcgcc cctccacctt 2760
ccgcatctct tgttgacact tacagatact tgcagtccgc agccatcact tgccaaaagg 2820
acgctcctgc cccaactaag aaggacccat acgacggcct gaacttctgg aacgttaact 2880
tgaaggagaa gttctcctcg gaactcgacc agttccctct gggtagaaag ttcctcctgc 2940
aggcaggagt gagacgcaga ccaacgatcg gcccaagaaa gagaccagct accgccacca 3000
ctgcatccac gtctaagcac 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 ctctgtggag atcctctgac aacatggttt 1560
acttgcctcc accttccgtc gccaaggttg tcaacaccga cgactacgtg accagaacgg 1620
gcatctacta ttacgccggt acttcgagat tgctcactgt tggacaccca tacttcaagg 1680
tccctatgtc tggtggcaga aagcaggaca tccctaaggt gtccgcttac cagtacagag 1740
ttttcagaat ctccctgcct gacccaaaca agttctcgct cccagagtcc accttgtaca 1800
accctgacac gcagagactg gtctgggcct gtgttggtgt ggaaattggc agaggacagc 1860
cacttggtgt tggcctgtct ggtcacccat tgtacaacag attggacgat accgagaact 1920
ctccattctc ctcgaacaag aatcctaagg actccagaga caacgtctct gtggactaca 1980
agcagaccca actgtgcatt atcggctgtg ttccagctat tggagagcac tgggccaagg 2040
gcaagtcgtg caagccttcc aacgtccagc caggtgactg tcctccattg gagttggtga 2100
acacccctat ccaggatggc gatatgatcg acactggata cggtgctatg gacttttcga 2160
cccttcagga gaccaagtcc gaggttcctc tcgacatttg ccagtctgtt tgtaagtacc 2220
ctgattactt gcagatgtcg gcagacgtgt acggagactc catgttcttt tgcctgagaa 2280
gagagcagct cttcgccaga cacttctgga acagaggtgg catggttgga gacactatcc 2340
caaccgagtt gtacattaag ggcactgaca tcagagactc gccttcttcc tatgtctacg 2400
ctccatctcc ttcaggttct atggttagct ccgactcgca gctgttcaac aagccatact 2460
ggcttcacaa ggcccaggga cacaacaatg gcatctgctg gcacaaccag ctcttcctga 2520
cggttgtcga caccactaga tccacaaact tcaccttgtc gactacgacc gagtctgcag 2580
ttccaaacat ctacgaccct aacaagttca aggagtatat tagacacgtt gaggaatatg 2640
acctgcagtt catcttccag ctctgcacca ttacattgtc cactgacgtg atgtcctaca 2700
tccatacgat gaacccagcc attctggatg actggaactt tggtgttgca ccacctccat 2760
ctgcttcctt ggtcgacacc tacagatacc tgcaatcggc cgctatcacg tgtcagaagg 2820
acgccccagc acctaccaag aaggaccctt acgacggatt gaacttctgg aatgtcaacc 2880
tcaaggagaa gttctcgtct gaactggacc agtttccatt gggcagaaag ttcctgttgc 2940
aggccggtgt cagacgcaga cctaccatcg gtccaagaaa gagacctgcc acagcaacta 3000
cggcttctac ctccaagcac aagcgtaaga gagtgtccaa 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 ccttgtggag atcgtctgac aacatggtct 1560
acctgccacc tccatctgtt gctaaggtcg ttaacactga cgattacgtc acgagaaccg 1620
gtatctacta ctatgctgga acctccagac tgttgaccgt cggtcaccct tacttcaagg 1680
tgccaatgtc cggaggtaga aagcaggaca tcccaaaggt ttctgcctac cagtacagag 1740
tcttcagaat ctctttgcca gaccctaaca agttctccct gcctgagtcg accctgtaca 1800
acccagacac tcagagattg gtttgggctt gcgtcggcgt tgagatcggt agaggccaac 1860
ctctgggagt gggtctctcc ggccaccctc tgtacaacag actcgatgac accgagaact 1920
cccctttctc ttccaataag aacccaaagg actccagaga taacgtttcc gtcgactaca 1980
agcagactca gctttgtatc attggttgcg tgcctgccat cggtgaacac tgggcaaagg 2040
gaaagtcctg taagccatcg aacgttcagc caggagactg cccacctctg gagcttgtta 2100
acactccaat ccaagacggt gacatgattg acaccggcta cggagctatg gacttctcta 2160
ccttgcagga gacgaagtct gaggtcccat tggacatctg ccagtccgtg tgcaagtacc 2220
cagactacct tcaaatgtcc gctgacgttt acggcgactc tatgttcttc tgtttgagaa 2280
gagagcagct gttcgctaga cacttttgga acagaggcgg tatggttggt gacaccattc 2340
ctacggaact ctacatcaag ggtacagaca tcagagactc tccatccagc tacgtttatg 2400
ccccttcccc atcgggctca atggtctctt ccgactcgca gttgttcaac aagccttact 2460
ggctgcacaa ggctcaaggc cacaataacg gtatttgttg gcacaaccag ctgttcctca 2520
ccgtggttga cacaaccaga tccaccaact ttacgctttc caccactacc gaatctgccg 2580
tccctaacat ctacgaccca aacaagttca aggaatacat cagacacgtg gaagagtacg 2640
acctccaatt catcttccag ttgtgcacta tcaccctgtc taccgacgtt atgtcttaca 2700
ttcacacgat gaaccctgca atcttggacg attggaactt cggcgtcgcc cctccacctt 2760
ccgcatctct tgttgacact tacagatact tgcagtccgc agccatcact tgccaaaagg 2820
acgctcctgc cccaactaag aaggacccat acgacggcct gaacttctgg aacgttaact 2880
tgaaggagaa gttctcctcg gaactcgacc agttccctct gggtagaaag ttcctcctgc 2940
aggcaggagt gagacgcaga ccaacgatcg gcccaagaaa gagaccagct accgccacca 3000
ctgcatccac gtctaagcac 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

Claims (10)

1. A polynucleotide encoding HPV68L1 protein, wherein the polynucleotide is as set forth in SEQ ID NO: 2, 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 HPV68L1 protein, comprising the steps of: constructing a polypeptide integrated with or containing a nucleotide sequence shown as SEQ ID NO: 2, culturing, collecting thalli, crushing the thalli to obtain lysate, and separating and purifying the lysate to obtain the HPV68L1 protein.
6. The method of producing HPV68L1 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 HPV68L1 protein obtainable by a method of producing an HPV68L1 protein according to any one of claims 5 to 6.
8. Use of the polynucleotide of claim 1 for encoding an HPV68L1 protein, or the recombinant expression vector of claim 2, or the host cell of claim 3, or the HPV68L1 protein of claim 7, in the preparation of an HPV vaccine.
9. A preparation method of an anti-HPV vaccine comprises the following steps: the method for producing HPV68L1 protein according to any one of claims 5-6 is used to prepare HPV68L1 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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