CN108624614A

CN108624614A - The method for generating HPV11 L1 albumen with expressed by Hansenula yeast system

Info

Publication number: CN108624614A
Application number: CN201810637916.6A
Authority: CN
Inventors: 班靖洋; 刘娟; 王贻杰; 程海; 霍烛; 陈丹; 李鼎锋; 刘勇
Original assignee: BEIJING ABZYMO BIOSCIENCES Co Ltd
Current assignee: BEIJING ABZYMO BIOSCIENCES Co Ltd
Priority date: 2012-03-28
Filing date: 2012-03-28
Publication date: 2018-10-09
Also published as: CN103361280A; CN103361280B

Abstract

The present invention relates to the methods for generating HPV11L1 albumen with expressed by Hansenula yeast system.Specifically, the invention discloses the method for the recombination Hansenula yeast cell for generating expression HPV11L1 albumen and the recombination Hansenula yeast cells generated by the method.The invention also discloses generate the purposes of the method and generated HPV11L1 albumen of HPV11L1 albumen in preparing preventative vaccine using the recombination Hansenula yeast cell.

Description

The method for generating HPV11 L1 albumen with expressed by Hansenula yeast system

Invention field

The invention belongs to medical bioengineering technical field, the method for being related to generating HPV11 L1 albumen, more particularly to use The method that expressed by Hansenula yeast system generates HPV11 L1 albumen.

Background technology

Human papilloma virus (human papillomavirus, HPV) is a kind of nonencapsulated closed loop double-stranded DNA virus, Belong to papovaviridae polyomavirus subfamily, the main epithelium mucous membrane tissue for invading human body, and then induces various benign and pernicious Preneoplastic lesions.

It is more than 200 kinds to have identified the different subtype HPV come at present, and HPV infection has apparent tissue specificity, different The HPV of type is different for the thermophilic tropism of skin with mucous membrane, can induce different papillary lesions, kind of HPV types about more than 30 It is not related with reproductive tract infection, wherein have more than 20 kinds it is related to tumour.The good pernicious difference of lesion is induced according to HPV, HPV can be big Cause is divided into two classes：1) high-risk-type (such as HPV16, HPV18, HPV45, HPV3l, HPV33, HPV52, HPV58, HPV35, HPV59, HPV56, HPV39, HPV51 etc.)：It is closely related with mankind's Various Tissues malignant tumour, mainly severe is caused not to be true to type Hyperplasia and infiltrating carcinoma, especially the closest with the generation of cervical carcinoma with the infection of HPV16 and HPV18 types, the infection of the two types accounts for 70% or more of cervical carcinoma infection, wherein HPV16 accounts for 50% or more；2) low risk (such as HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV72, HPV 81 etc.)：It can cause epidermal cell benign proliferative venereal disease, such as condyloma acuminatum and flat Flat condyloma etc., wherein the condyloma acuminatum induced by HPV6 and HPV11 accounts for 90% or more.

HPV is mainly made of virus coat and genomic DNA.Genome is about 7900bp, there is 8 encoding hiv proteases Gene.The albumen of wherein 6 ORF codings is in the early expression of virus replication, referred to as early protein；The albumen of 2 ORF codings exists The late period of virus replication expresses, referred to as late protein.Late protein includes major cat protein L1 and secondary coat protein L2, and Participate in the formation of virus coat.

HPV viruse coat protein can carry out self assembly, the L1 albumen of single expression or by L1 in a variety of expression systems Albumen and L2 albumen can be self-assembled into virus-like particle (virus-like particle, VLP) when co-expressing, wherein with The VLP of the generations such as Yeast system, Baculovirus insect expression system and mammalian cell expression system is closer to natural viral Structure.Generation neutralizing antibody can be induced in vivo after being immunized using the VLP of heterogenous expression system production, obtained good Immune protective effect.

Multiple-shaped nuohan inferior yeast (Hansenula Polymorpha), also referred to as Pichia augusta are currently generally acknowledged One of ideal heterologous gene expression system.Multiple-shaped nuohan inferior yeast both has protokaryon life as single celled eukaryotic microorganism Object fast growing is easy to the features such as genetic manipulation, and has the functions such as eukaryocyte post translational processing and modification.In addition, the inferior ferment of the Chinese Mother is also equipped with that safety is good, be easy to culture, of low cost, expression quantity is high and the advantages such as inheritance stability, and can overcome such as Saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain is unstable, low output and glycosylation side chain are long and complete red The relatively low problem of yeast (Pichia Pastoris) exogenous origin gene integrator copy number.Currently, being given birth to using expressed by Hansenula yeast system Drug (such as insulin, trade name Wosulin) and HBV vaccines (trade name Hepavax-Gene) the list marketing of production.

Summary of the invention

In the first aspect, the present invention provides a kind of recombination Hansenula yeast cells generating expression HPV11 L1 albumen Method, it includes following steps：

A) by being built including the exogenous polynucleotide insertion carrier for the nucleotide sequence for encoding HPV11 L1 albumen Expression construct；

B) expression construct obtained in step a) is used to convert Hansenula yeast cell；With

C) the Hansenula yeast cell obtained in step b) is screened, obtains the recombination containing the exogenous polynucleotide Hansenula yeast cell.

In the second aspect, the present invention also provides the recombination Hansenula yeast cells generated according to the above method.

In terms of third, the present invention also provides a kind of methods generating HPV11 L1 albumen, include the following steps：

I) the recombination Hansenula yeast cell of the present invention is cultivated under conditions of suitable for HPV11 L1 protein expressions；With

Ii it) is recycled from culture and purifies HPV11 L1 albumen.

The method can also include the steps that carrying out depolymerization to purified HPV11 L1 albumen and meeting again.

In terms of the last one, the present invention also provides the HPV11 L1 albumen generated according to the method for the present invention to make The purposes being ready for use in the vaccine for preventing HPV11 infection.

Description of the drawings

Fig. 1 is using MOX promoter fragments as the Southern trace testing results of probe.With ATCC26012 genomic DNAs For control.1：(applied sample amount is for control：1000ng)；2：(applied sample amount is for control：500ng)；3：(applied sample amount is for control： 250ng)；4：(applied sample amount is for control： 125ng)；5：(applied sample amount is for control：62.5ng)；6：HP/pRMHP2.1-11wt genes (applied sample amount is group DNA：15.625ng, brightness is between 500ng and 1000ng controls)；7： HP/pRMHP2.1-11sc (applied sample amount is genomic DNA：15.625ng, brightness is between 500ng and 1000ng controls)；8：HP/pRMHP2.1- (applied sample amount is 11hp genomic DNAs：15.625ng, brightness is between 500ng and 1000ng controls).

The SDS-PAGE results of Fig. 2A HPV11 L1 albumen induced expression in Escherichia coli.1：The control sample not induced Product；2,3,4：IPTG induced expression samples；5：Protein marker.

The purification result of the HPV11 L1 albumen of Fig. 2 B prokaryotic expressions.1：Protein marker；2：Solubilization of inclusion bodies liquid；3：Stream Wear liquid；4：Eluent.

Fig. 2 C rabbit polyclonal antibody purification results.1：Antiserum；2：Flow through liquid；3：Antibody elution 4：Protein marker.

The western blot detection of recombination Hansenula yeast cellular expression levels different Fig. 3.1：Pre-dyed protein marker； 2：Standard items (30 μ g/L)；3：HP/pRMHP2.1-11sc；4,5,6： HP/pRMHP2.1-11hp；7：HP/pRMHP2.1- 11wt。

The detection of expression of HPV11 L1 albumen in Fig. 4 fermentation process.1：Induction 0 hour；2：Standard items (30 μ g/L)；3：In advance Contaminate protein marker；4：Induction 2 hours；5：Induction 4 hours；6：Induction 6 hours；7：Induction 8 hours；8：Induction 10 hours.

The POROS 50HS of Fig. 5 A HPV11 L1 albumen purify electrophoretogram.1：Upper prop sample；2：Flow through liquid；3~9：It is different NaCl concentration elutes.

The CHT of Fig. 5 B HPV11 L1 albumen purifies electrophoretogram.1：Upper prop sample；2：Flow through liquid； 3-5：Different phosphate Concentration elutes.

The transmission electron microscope observing result of the HPV11 L1 albumen of Fig. 6 purifying.

Detailed description of the invention

The present inventor has been successfully set up the method for generating HPV11 L1 albumen using Hansenula yeast, generated HPV11 L1 albumen can be self-assembled into virus-like particle, can be used for preparing the vaccine for preventing HPV infection.

Present invention firstly provides a kind of method for the recombination Hansenula yeast cell generating expression HPV11 L1 albumen, packets Containing following steps：

The invention also includes the recombination Hansenula yeast cells generated according to the method.

Amino acid sequence from the HPV11 L1 albumen of different HPV11 Strain can have differences.The present inventor is logical It crosses and HPV11 L1 protein sequences all in database is compared, chosen on each amino acid position of HPV11 L1 albumen The highest amino acid residue of the frequency of occurrences obtains and is shown in SEQ ID NO:1 amino acid sequence, the sequence are HPV11 L1 The most representative consensus sequence of albumen.Therefore, the HPV11 L1 albumen in the present invention preferably has SEQ ID NO:Shown in 1 Amino acid sequence.

In order to efficiently express HPV11 L1 albumen using Hansenula yeast, inventor is according to SEQ ID NO:Ammonia shown in 1 Base acid sequence carries out the codon optimization of nucleotide sequence for Hansenula yeast.Optimization principles include：A) it is lost according to Hansenula yeast Pass password frequency of use table (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgispecies =4905) frequency of use highest or higher codon are selected；B) avoid having potential impact to genetic transcription or protein translation Negative regulatory element, such as the areas PolyAT, the areas PolyGC, the silencer area (Sliencer) and internal splice site；C) to including 5 ' It holds the mRNA secondary structures including the code area UTR, HPV11L1 and 3 ' end UTR to carry out comprehensive analysis, avoids complicated RNA two levels knot The formation of structure makes the free energy of mRNA secondary structures reduce；D) it is used and Hansenula yeast promoter as far as possible in upstream of coding region 5 ' the completely the same areas UTR of downstream native sequences；E) common restriction enzyme enzyme recognition site is eliminated.By the nucleosides of optimization Acid sequence is shown in SEQ ID NO:4.The nucleotide sequence of coding HPV11 L1 albumen used in the present invention is preferably SEQ ID NO:Sequence shown in 4.

The adaptable polymorpha expression vector of the present invention is that application No. is the Chinese patent Shens of 201210021524.X Please described in polymorpha expression vector pRMHP2.1 (comprising being shown in SEQ ID NO:15 sequence).By that will include to compile The exogenous polynucleotide of the nucleotide sequence of code HPV11 L1 albumen clones the table that the present invention can be obtained into pRMHP2.1 carriers Expression constructs.It will be understood by those skilled in the art that the expression construct of the present invention can also use other vector constructions, such as Carrier described in the Chinese patent CN100400665C of mandate.

In order to be expressed in Hansenula yeast, exogenous polynucleotide in the expression construct operably with promoter It is connected with terminator.

As used herein, the function connects for referring at least two polynucleotides " are operably connected ".For example, operably connecting It connects including the connection between promoter and another polynucleotides, wherein the promoter sequence originates and mediates this another is more The transcription of nucleotide.Operably connection includes the connection between terminator and another polynucleotides, wherein the terminator Terminate the transcription of another polynucleotides.

It includes but not limited to MOX, FMD, AOX1 and DHAS promoter to be suitable for the invention promoter.In some implementations In scheme, the promoter used in the present invention is the MOX promoters from Hansenula yeast.It is suitable for the invention termination attached bag It includes but is not limited to the MOX terminators from Hansenula yeast.

Expression construct is converted to Hansenula yeast cell and can be carried out with a variety of methods known in the art, including but not It is limited to the conversion that electroporation and PEG are mediated.

In addition, developed in this field it is a variety of for expressing foreign protein Hansenula yeast bacterial strain, including but not limited to CGMCC2.2498 Hansenula yeasts, ATCC34438 Hansenula yeasts and ATCC26012 Hansenula yeast cells.These Hansenula yeast bacterium Strain can also be applied to the present invention.In some embodiments, the Hansenula yeast of the expression construct for converting the present invention is thin Born of the same parents are ATCC26012 Hansenula yeast cells.

In post-conversion, recombination Hansenula yeast cell can be selected according to the resistant gene carried on carrier.It is suitable anti- Property gene includes but not limited to Kan resistant genes and Zeocin resistant genes.According to used carrier, it is also possible to use nutrition Defect culture medium recombinates Hansenula yeast cell to screen.

Expression of the exogenous polynucleotide in Hansenula yeast and its copy number positive correlation in Hansenula yeast.Cause This, further screening can also be carried out by the methods of Southern traces or quantitative PCR and contains multicopy external source multinuclear glycosides The recombination Hansenula yeast cell of acid.It is preferred that exogenous polynucleotide copy number is more than 5 recombination Hansenula yeast cell, more preferable external source Polynucleotide copies number is more than 30 recombination Hansenula yeast cell.

On the other hand, the present invention also provides a kind of method generating HPV11 L1 albumen, include the following steps：

Ii it) is recycled from culture and purifies HPV11 L1 albumen.

The various culture mediums known in the art that can be used for cultivating Hansenula yeast and basic condition of culture, those skilled in the art It can be selected or be changed as needed.The culture of the recombination expressed by Hansenula yeast bacterial strain of the present invention can according to required protein content To carry out in flask or be carried out in the bioreactor (fermentation tank of such as 30L) of different scales.According to selected promoter, Suitable inducer can be added in culture to induce the expression of the HPV11 L1 albumen.Using MOX or FMD promoters In the case of, methanol is added as inducer.

The purifying of generated HPV11 L1 albumen can use various protein purification modes known in the art, such as salt The combination of analysis, ultrafiltration, precipitation, chromatography etc. or these modes.In one embodiment, chromatography media POROS 50HS are used first (Applied Biosystems) carries out preliminary purification, followed by chromatography media Macro-Prep ceramic hydroxyapatites (Type II, 40 μm) is further purified.

The HPV11 L1 albumen of the purifying prepared using the method for the present invention can be self-assembled into virus-like particle (embodiment 9, Fig. 6) good immunogenicity (embodiment 10), and in mouse is shown, therefore present invention provides the HPV11 Purposes of the L1 albumen in preparing the vaccine for preventing HPV11 infection.

Embodiment

It will be further illustrated the present invention, but therefore do not limited the present invention to described by way of embodiment below Scope of embodiments in.

Embodiment 1：The analysis of HPV11 L1 consensus amino acid sequences

The HPV11 L1 albumen of overall length is made of 501 amino acid, is retrieved by GenBank, obtains contain 501 ammonia altogether The overall length HPV11 L1 protein sequences 87 of base acid.Amino acid sequence ratio is carried out using Vector NTI software AlignX functions To analysis, obtain most representative HPV11 L1 consensus amino acid sequences (consensus amino acid sequence, The sequence of the highest amino acid residue of the frequency of occurrences is all made of in each amino acid positions of HPV11 L1), sequence such as SEQ ID NO:Shown in 1.

Embodiment 2：HPV11 L1 encoding genes it is artificial synthesized

The present invention has synthesized 3 kinds of different HPV11 L1 nucleotide sequences altogether, is referred to as 11wt, 11sc and 11hp：

1) 11wt-nucleotide identical with natural HPV11 L1 gene orders shown in GenBank accession number M14119.1 Sequence is shown in SEQ ID NO:2；

2) 11sc-the present inventor is according to SEQ ID NO:Amino acid sequence shown in 1, for saccharomyces cerevisiae genetic code The nucleotide sequence of preferences brand-new design, sequence are shown in SEQ ID NO:3；

3) 11hp-the present inventor is according to SEQ ID NO:Amino acid sequence shown in 1, for Hansenula yeast genetic code The nucleotide sequence of preferences brand-new design, sequence are shown in SEQ ID NO:4.

According to the above nucleotide sequence, commission Sinogenomax Co., Ltd. carry out respectively 11wt, The complete sequence of 11sc and 11hp is artificial synthesized, is cloned in carrier T and (is respectively designated as T-11wt, T-11sc and T-11hp), and Sequence verification is carried out to it.

Embodiment 3：Generate the expression construct for carrying different HPV11 L1 nucleotide sequences

The polymorpha expression vector that the present invention is applied is that application No. is the Chinese patent applications of 201210021524.X Described in polymorpha expression vector pRMHP2.1 (it includes SEQ ID NO:Sequence shown in 15).

(1) MOX promoters and the PCR amplification of MOX terminators

Using the mixed genomic DNA of Hansenula yeast strains A TCC26012 and ATCC34438 as template, with following primer pair The MOX promoters that size is 1518bp are obtained, while NotI restriction enzyme sites are introduced in upstream；

MOX promoter primers：5’-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3’(SEQ ID NO:16)

MOX promoter primers：5’-TTTGTTTTTGTACTTTAGATTGATGTC-3’(SEQ ID NO:17)

Using the mixed genomic DNA of Hansenula yeast strains A TCC26012 and ATCC34438 as template, with following primer pair The MOX terminators that size is 311bp are obtained, while BglII restriction enzyme sites are introduced in downstream；

MOX terminator primers：5’-GGAGACGTGGAAGGACATACCGC-3’(SEQ ID NO:18)

MOX terminator primers：5’-GAagatctCAATCTCCGGAATGGTGATCTG-3’(SEQ ID NO:19)

(2) expression construct for carrying different HPV11 L1 nucleotide sequences is generated

To carry the recombinant plasmid T-11wt of 11wt as template, it is 1554bp's to obtain size with primer 1 and primer 2 amplification HPV11 L1wt genes, while the overlap at 3 ' end of MOX promoter regions is introduced in upstream, introduce MOX terminators 5 ' in downstream The overlay region at end；

Primer 1：5’-CATCAATCTAAAGTACAAAAACAAAATGTGGCGGCCTAGCGACAGCACAG-3’(SEQ ID NO:5) primer 2：5’-GCGGTATGTCCTTCCACGTCTCCTTACTTTTTGGTTTTGGTACGTT-3’(SEQ ID NO:6)

To carry the recombinant plasmid T-11sc of 11sc as template, it is 1554bp's to obtain size with primer 3 and the amplification of primer 4 HPV11 L1sc genes, while the overlap at 3 ' end of MOX promoter regions is introduced in upstream, introduce MOX terminators 5 ' in downstream The overlay region at end；

Primer 3：5’-CATCAATCTAAAGTACAAAAACAAAATGTGGAGACCAAGCGACAGCACAG-3’(SEQ ID NO:7) primer 4：5’-GCGGTATGTCCTTCCACGTCTCCTTACTTTTTAGTTTTCGTTCTCTTAC-3’(SEQ ID NO:8)

To carry the recombinant plasmid T-11hp of 11hp as template, it is 1554bp's to obtain size with primer 5 and the amplification of primer 6 HPV11 L1hp genes, while the overlap at 3 ' end of MOX promoter regions is introduced in upstream, introduce MOX terminators 5 ' in downstream The overlay region at end；

Primer 5：5’-CATCAATCTAAAGTACAAAAACAAAATGTGGAGACCATCGGACTCGACAG-3’(SEQ ID NO:9) primer 6：5’-GCGGTATGTCCTTCCACGTCTCCTTACTTCTTGGTCTTCGTTCTCTTC-3’(SEQ ID NO: 10)

Respectively using three MOX promoters, 11wt genes/11sc genes/11hp genes, MOX terminators segments as template, with Primer 7 obtains 11wt expression cassettes/11sc expression cassettes/11hp expression cassettes that size is 3.4Kb with the amplification of primer 8, while in upstream NotI restriction enzyme sites are carried, BglII restriction enzyme sites are carried in downstream.

Primer 7：5’-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3’(SEQ ID NO:11) Primer 8：5’-GAagatctCAATCTCCGGAATGGTGATCTG-3’(SEQ ID NO:12)

11wt expression cassettes, 11sc expression cassettes and 11hp expression cassettes are cloned into respectively by NotI+BglII double digestions In pRMHP2.1 carriers, expression construct pRMHP2.1-11wt, pRMHP2.1-11sc and pRMHP2.1-11hp are obtained.

Embodiment 4：Recombinate the generation of Hansenula yeast cell

(1) extraction and digestion of recombinant expression construct constitution grain

Picking is transformed into the E. coli clones of the recombinant expression construct constitution grain obtained in embodiment 3, after expanding culture Plasmid is extracted using E.Z.N.A Plasmid Mini Kit kits (Omega Bio-Tek companies), Bgl II is used in combination to carry out Single endonuclease digestion is recycled using E.Z.N.A Gel Extraction Kit kits (Omega Bio-Tek companies), with 70 μ L It is preheated to 55 DEG C of sterile water to be eluted, by measuring OD₂₆₀It is quantitative to carry out DNA, and the segment of linearisation is diluted to 100ng/ μ l, are stored in -20 DEG C of refrigerators, spare.

(2) processing of Hansenula yeast cell

Picking Hansenula yeast strains A TCC26012 single bacterium colonies access in the small test tube of the YPD fluid nutrient mediums containing 5ml, 37 DEG C culture 12 hours；Bacterium solution 5ml is taken to be forwarded in 200ml YPD culture mediums, 37 DEG C are cultivated 4-6 hours, until OD_600nmAbout 1.0-1.5 centrifuging 10min in 5000rpm；With 200ml 0.1mol/L phosphate buffers (DTT containing 25mmol/L, pH7.5) Thalline is resuspended, mixes well, 30min is incubated in 37 DEG C, 5000rpm centrifuges 10min, abandons supernatant, stay thalline.With the STM of precooling Solution 200ml washes thalline, and thalline pressure-vaccum is uniform, centrifuges 3min in 4 DEG C of 5000rpm, abandons supernatant, stay precipitation.It is molten with ice-cold STM Thalline is resuspended in liquid 100ml, centrifuges 3min in 4 DEG C of 5000rpm, abandons supernatant, stay precipitation.It is ice-cold with 50-200 μ l according to biomass Thalline is resuspended in STM solution, and bacterium solution is transferred in the centrifuge tube after high pressure, and ice bath prepares conversion.

(3) electrotransformation of Hansenula yeast cell

By plasmid:Thalline=1:2 amount is added recombination expressed by Hansenula yeast plasmid 15 μ l, 30 μ l of bacterium solution, and abundant pressure-vaccum is equal It is even, it is placed in be transformed in ice bath；It will be impregnated in advance with alcohol, and after ultraviolet irradiation, take out, add in the electric revolving cup of -20 DEG C of refrigeration Enter plasmid thalline mixed liquor；It shocks by electricity by the condition of voltage 2500V, 150 Ω of resistance, 50 μ F of capacitance；It is rapidly added after electric shock 1ml has been balanced to the YPD solution of room temperature, is gently transferred to after mixing in EP pipes；Thalline after electricity is turned is put in 37 DEG C of water-baths 1h is set, is gently overturned 3 times at interval of 15min；It will be incubated the bacterium solution of 2h, has centrifuged 10min in 5000rpm, abandon supernatant；With 200 μ Thalline is resuspended in l YPD solution, is coated in the YPD tablets of the Zeocin containing 0.25mg/ml with 100 μ l/ plates, and training is inverted in 37 DEG C It supports 3-7 days.

(4) passing on, stablizing for expressed by Hansenula yeast bacterial strain is recombinated

The recombinant bacterial strain single bacterium colony grown in picking Zeocin resistant panels, is inoculated in 5ml Zeocin containing 0.25mg/ml YPD fluid nutrient mediums in, in 37 DEG C, 200rpm shaking table cultures 24-48 hours, until OD values are up to after 50, with 1:1000 ratio It transfers in the YPD fluid nutrient mediums of 5ml Zeocin containing 0.25mg/ml, culture to OD values is up to after 50, then with 1:1000 ratio Example is transferred in the YPD fluid nutrient mediums of 5ml Zeocin containing 0.25mg/ml, and so on, it is continuous to pass 10 times and carry out strain Preservation.Preservation system is bacterium solution:60% glycerine=1:1, it measures preserve strain as needed, usually+500 μ l of 500 μ l bacterium solutions 60% glycerine；

The recombination expressed by Hansenula yeast bacterial strain access 5ml for reaching 10 times is free of to the YPD fluid nutrient mediums of Zeocin resistances In, in 37 DEG C, 200rpm shaking table cultures to OD values up to after 50, with 1:1000 ratio is transferred in 5ml YPD fluid nutrient mediums, And so on, it is continuously passed in the YPD fluid nutrient mediums without Zeocin resistances 5 times.

Bacterium solution after stabilization is coated with to the YPD tablets of the G418 containing 10mg/ml, culture 2-3 days is inverted in 32 DEG C, is put down from each The eugonic single bacterium colony of picking carries out copy number detection in plate.

Embodiment 5：Recombinate the exogenous polynucleotide copy number detection of Hansenula yeast cell

(1) extraction of pastoris genomic dna and quantitative

The eugonic yeast strain single bacterium that inoculation embodiment 4 obtains, which is fallen in the YPD fluid nutrient mediums of 5ml, cultivates Base, 37 DEG C of cultures 16~for 24 hours；2ml Yeast Cultivation liquid, room temperature 4500g centrifugations 3min is taken to collect thalline；Using 500 μ l SCED Solution (1mol/L sorbierites, 10mmol/L sodium citrates, 10mmol/L EDTA, 10mmol/L DTT dithiothreitol (DTT)s) is resuspended Thalline, and 50mg bead fully shaking 5min are added, 50 μ l 10mg/ml lywallzymes, 37 DEG C of warm bath 1h are added；60 μ are added The 10%SDS of l, the Proteinase K of 30 μ l, the RNaseA enzymes of 10 μ l are cultivated after being placed at room temperature for 10 minutes in 55 DEG C of water-bath 2h；350 μ l saturated phenols and 350 μ l chloroforms are added, in 13000rpm after being sufficiently mixed, centrifuge 10 minutes, collect upper after layering Layer liquid；Isometric (about 700 μ l) chloroform is added, in 13000rpm, centrifuges 10 minutes, collects the upper liquid after layering；Toward upper layer The 3mol/L sodium acetate solutions of 140 μ l are added in liquid, 700 μ l isopropanols are gently added after mixing, are placed at room temperature for after mixing 5min is centrifuged 10 minutes in 13000rpm.Supernatant is abandoned, 70% ethyl alcohol of 1ml is added and is cleaned, in 13000rpm, centrifugation 10 Minute, remove supernatant, DNA precipitations are placed in be placed at room temperature for 30min after, 100 μ l TE dissolvings are added.By measuring OD_260nmCarry out base 100ng/ μ l are diluted to because of quantifying for group DNA, and by the segment of linearisation, are stored in -20 DEG C of refrigerators, it is spare.

(2) Southern immunoblot methods carry out quantifying for exogenous polynucleotide copy number

a：It is prepared by probe

MOX described in Chinese patent application of the MOX probes that the present invention is applied application No. is 201210021524.X Probe, using DIG DNA Labeling and Detection kit kits (the Cat No of Roche companies： 11093657910) probe preparation is carried out.The specific steps are：The PCR product of 10 μ l MOX promoter regions is added into EP tubules (200ng/ μ l), is used in combination sealed membrane to seal up, and boiling water boils 10 minutes.It is immediately placed in the absolute ethyl alcohol capsule of precooling (- 20 DEG C) (cooling down suddenly).Pipe outer wall ethyl alcohol is dried, centrifuges (about 10s), sequentially adds 5 μ l tiny electrolytic cells water, 2 μ l 10x Hexanucleotide Mix、2μl 10x dTP Labeling Mixture、1μl 7Klenow Enzyme (labeling Grade), mixing, sealed membrane sealing.37 DEG C of water-baths are stayed overnight, -20 DEG C of preservations.

b：Southern traces

Using digoxin hybridization check kit I (the Cat No of Beijing Mei Laibo medical science and technologies Co., Ltd： DIGD- And the efficient hybridization solutions of HyB (Cat No 110)：Hyb-500 Southern trace detections) are carried out according to product description.

(3) copy number of different recombination Hansenula yeast bacterial strains compares

Southern traces testing result (see Fig. 1) is shown：Screen the HP/pRMHP2.1-11wt bacterial strains obtained, HP/ The copy number of pRMHP2.1-11sc bacterial strains and HP/pRMHP2.1-11hp bacterial strains is close, and copy number is all higher than 31 but is less than 63.

Embodiment 6：The prokaryotic expression of HPV11 L1 albumen and preparation (1) HPV11 L1 genes of rabbit polyclonal antibody PCR amplification and expression plasmid clone's structure

Using pRMHP2.1-11hp as template, size is obtained as the HPV11 of 1522bp with primer 9 and the amplification of primer 10 L1hp genetic fragments (without terminator codon), while NdeI restriction enzyme sites are introduced in upstream, introduce SalI digestions position in downstream Point；

Primer 9：5’-ggaattccatATGTGGAGACCATCGGACTCGACAG-3’(SEQ ID NO:13)

Primer 10：5’-CCGGTCGACCTTCTTGGTCTTCGTTCTCTTCCTC-3’(SEQ ID NO:14)

11hp gene fragment clones are entered to the pET43.1a carriers of NdeI+XhoI double digestions by NdeI+SalI double digestions In (Novagen companies), the correct recombinant plasmid of sequence verification is named as pET43.1a-11hp.

(2) induced expression of the HPV11 L1 albumen in Escherichia coli

Recombinant plasmid pET43.1a-11hp converts E. coli expression strains BL21-CodonPlus (DE3)-RIPL, obtains Engineering bacteria BL21-CodonPlus (DE3)-RIPL/pET43.1a-11hp must be expressed.Picking single bacterium colony accesses the liquid of LB containing 5ml In the test tube of body culture medium (the 50 μ g/ml containing Amp), 37 DEG C of cultures to OD₆₀₀For IPTG is added when 0.6-0.8 to final concentration 0.5mmol/L, induction time 6h, while the control tube that setting does not induce.Bacterium solution is collected, SDS-PAGE detects recombinant protein Expression (Fig. 2A).

(3) purifying of HPV11 L1 albumen

Preparation of samples：Picking expresses engineering bacteria BL21-CodonPlus (DE3)-RIPL/pET43.1a-11hp single bacterium colonies It accesses in the test tube of LB liquid medium containing 5ml (the 50 μ g/ml containing Amp), 37 DEG C of overnight incubations, next day presses 1% inoculum concentration It accesses in the 1L triangular flasks of LB liquid medium containing 400ml (the 50 μ g/ml containing Amp), until OD₆₀₀When about 0.8, IPTG is added To final concentration 0.5mmol/L, collects bacterium solution after inducing 6h and centrifuge, bacterial sediment is pressed 1:The ratio of 10 (g/ml) is resuspended in PBS solution, it is heavy that carrying out ultrasonic bacteria breaking (5s, 5s, 400W, the 60cycles) under ice bath, 10000r/min centrifugation 10min are collected by centrifugation It forms sediment.

Purification process (Fig. 2 B)：Ultrasound precipitation is pressed 1:The ratio of 10 (g/ml) be added solution (100mM phosphate, 500mM NaCl, 20mM imidazoles, 8M ureas, pH8.0) in be sufficiently stirred dissolving, 10000r/min centrifuges 10min, collects supernatant 0.45 μm of membrane filtration.Chelating Sepharose FF chromatography medias are splined on, with solution (100mM phosphate, 150mM NaCl, 500mM imidazoles, 8M ureas, pH8.0) elution, collect destination protein eluting peak.Dialysis removal imidazoles.

(4) prepared by rabbit polyclonal antibody

Rabbit is immunized and serum collection：With 2 new zealand rabbits of HPV11 L1 protein immunizations of the prokaryotic expression of purifying, exempt from It is the 0th, 3,6,8 week each immune primary that epidemic disease method, which uses back multi-point injection method, immune programme, and immunizing dose is 250 μ g/ Secondary, antigen need to again be injected after Freund's complete adjuvant (FCA) or incomplete Freund's adjuvant (FIA) are fully emulsified, exempt from for the first time Epidemic disease application FCA, follow-up immunization application FIA were taken a blood sample by arteria carotis in the 9th week and detach serum, and every rabbit can detach serum 40-60ml。

Antibody purification (Fig. 2 C)：Antiserum is after 0.45 μm of filtering, and upper prop is in chromatography media rProtein A Sepharose FF carry out affinitive layer purification.Under pH neutrallty conditions, antibody is incorporated on chromatography media, with citric acid-lemon Lemon acid sodium buffer solution (pH4.0) elutes, and collects antibody elution peak, is neutralized to pH neutrality with 1M Tris.Cl, pH9.0.

Embodiment 7：The expression study of difference recombination Hansenula yeast bacterial strain

(1) induced expression of Hansenula yeast bacterial strain is recombinated

Picking recombinates Hansenula yeast single bacterium colony from tablet, accesses in the small test tube of the YPG fluid nutrient mediums containing 5ml, 37 DEG C culture 24 hours；By bacterium solution switching in the 100ml triangular flasks of the inducing cultures of YPM containing 30ml, initial density OD₆₀₀ =1, it is induced 72 hours in 37 DEG C of shaking tables, final concentration of 0.5% methanol solution was added in bacterium solution every 12 hours (i.e. 0.15ml/ bottles).

Bacterium solution after taking 25ml to induce is transferred in the centrifuge tube of 50ml, is centrifuged 10min in 10000rpm, is abandoned supernatant；With Bacterial sediment is resuspended in the cell cracking of 50ml, after mixing well, is pressed in Ultrasound Instrument and " power 60%, time 20min, opens 5s, closes The ultrasonication program of 5s " carries out bacterial cell disruption, needs to keep ice bath in shattering process；By the bacterium solution of ultrasonication, in 10000rpm centrifuges 10min, and collection supernatant freezes spare in -20 DEG C.

(2) detection of expression of Hansenula yeast bacterial strain HPV11 L1 albumen is recombinated

The detection of HPV11 L1 protein expression levels is carried out using western blot semi-quantitative method

Glue and electrophoresis：12% polyacrylamide gel is prepared, measuring samples and standard items are added, upper layer glue is with 8V/ The voltage of cm, separation gel carry out electrophoresis with the voltage of 15V/cm, until when bromophenol blue reaches separation gel bottom, stop electrophoresis, cut Separation gel；

Semidry method transferring film：Pvdf membrane first uses methyl alcohol process 15 seconds, and positive liquid is soaked into again extremely after being rinsed 3 times with deionized water Few 5min；Running gel is soaked in negative electrode solution, installs electrotransfer device in order, and the electric current of 150mA carries out electricity and turns 35- 45min；After the completion of transferring film, check whether pre-dyed protein marker band is completely transferred.

Closing：Film addition is had in the valve bag of 5% milk of 20ml with tweezers, bubble is squeezed out, uses plastic film sealing Machine seals.Shaking table shakes closing 1 hour or (should be put into refrigerator cold-storage preservation overnight) overnight slowly.

Primary antibody is incubated：The rabbit-anti HPV11 L1 albumen prepared mostly anti-be added with 1/200 ratio is filled into 5% milk Valve bag in, film is put into after mixing, squeeze out bubble, sealed with plastic film sealing machine.Shaking table shakes incubation 1.5 hours slowly.

Wash film：It takes the film out and is washed 5 times with TBST, about 100ml, time are 5 minutes every time.Last time is 10 minutes.

Secondary antibody is incubated：The goat-anti rabbit secondary antibody of HRP labels is added to the valve bag for filling 5% milk with 1/1000 ratio In, film is put into after mixing, bubble is squeezed out, is sealed with plastic film sealing machine.Shaking table shakes incubation 2 hours slowly.

Colour developing：Film is put into developing solution, shaking table, which is shaken slowly to band, to be occurred, and is rinsed color development stopping with flowing water, is dried laggard Row is taken pictures.

(3) result

By comparing influence of the different HPV11 L1 nucleotide sequences for protein expression, as a result (Fig. 3) is shown：HPV days The 11sc that right gene 11 wt was substantially not detectable albumen expression (the 7th duct), is optimized according to saccharomyces cerevisiae codon-bias Gene has the expression (the 3rd duct) of certain level in Hansenula yeast, but its expression is far below according to Hansenula yeast password Therefore the gene 11 hp (ducts 4-6) of sub- preferences optimization design is integrated for Hansenula yeast host genetic codon bias Optimization realizes that high level expression is vital for HPV11 L1 albumen in Hansenula yeast bacterial strain.

Embodiment 8：The zymotechnique of HPV11 L1 recombination expressed by Hansenula yeast bacterial strains and purifying process research

Fermentation seed liquid：1 is taken to freeze glycerol stock (HP/pRMHP2.1-11hp).50 μ l accesses 5ml are drawn after thawing In YPD culture mediums, in 37 DEG C, 200rpm shaking table cultures 20-24hr, A_600nmAbout 2-5 respectively draws 1ml accesses 2 after assay approval In bottle 500ml YPD culture mediums, in 37 DEG C, 200rpm shaking table cultures 20-24hr to A_600nmAbout 15-20, conduct after assay approval Fermentation seed liquid is for use.

Ferment control：Fermentation initial culture medium contains yeast powder 300g, peptone 150g, glycerine 100g, basic salt (K₂SO₄ 273g, MgSO₄100g, 85%H₃PO₄400ml, KOH 62g), 10L purified waters fully dissolve, and are added in 30L fermentation tanks, pure Change water and be settled to 14L, 121 DEG C, 60ml PTM1 liquid microelements (CuSO is added in 30min sterilizings after being cooled to 30 DEG C₄·5H₂O 6.0g, KI 0.088g, MnSO₄·H₂O 3.0g, Na₂MoO₄·2H₂O 0.2g, H₃BO₃0.02g, CoCl₂·6H₂O 0.5g, ZnCl₂20.0g FeSO₄·7H₂O 65.0g, Biotin 0.2g, dense H₂SO₄5.0ml, purified water are settled to 1L, 0.22 μm of filter Membrane filtration degerming), ammonium hydroxide adjusts pH5.6, is inoculated with 1 bottle of 500ml fermentation seed liquid, and fermentation volume is 15L at this time.Starting stirring Rotating speed is 200rpm, air mass flow 0.5Nm3/hr, tank press 0.5bar, and it is 20-80% that oxygen dissolving value is controlled in fermentation.Initial growth After phase maintains about 25hr, bacterium solution A_600nmReach 20 or so, dissolved oxygen starts rapid increase, starts to add benefit with 100ml/hr flow velocity streams Expect culture medium (50% glycerine (W/V), 12ml PTM1), adds growth period into becoming a mandarin at this time.After cultivating about 6-8hr, bacterium solution A_600nmIt reaches To 90 or so, stop stream and add, ammonium hydroxide adjusts pH value to 6.0.Start stream after dissolved oxygen bottom out plus methanol (contains 12ml/L PTM1) enter the induced expression phase, methanol initial flow rate of acceleration is 50ml/hr, is sampled per hour, methanol determination of electrode methanol is dense Degree by adjusting methanol feeding rate control methanol concentration ＜ 5g/L, and is used for Western after collecting wet thallus ultrasonication Trace detection (5 times of dilutions of sample), western blot testing result is as shown in Figure 4.

Lower tank centrifugation：Tank is played after inducing 10hr, centrifuging 30min under the conditions of 4 DEG C with 5000rpm collects wet thallus.Harvest - 20 DEG C of wet thallus freezes.

Bacterial cell disruption：The HPV11 expression wet thallus for taking -20 DEG C of preservations, 0.9% is added according to the ratio of 10ml/g wet thallus Physiological saline cleans.After thalline washing, by 20ml buffer solutions/g wet thallus addition disruption buffer (NaCl containing 0.5mol/L, 0.02%Tween-80,0.05mol/L MOPS) fully dissolving, cracking pressure 1500bar broken using high-pressure homogenization, cycle It is 5-8 times broken, microscopy percentage of damage ＞ 90%.Bacterial cell disruption liquid centrifuges 30min under the conditions of 4 DEG C with 10000rpm, collects supernatant Liquid adds 50% ammonium sulfate, and after precipitating 30min, 10000rpm centrifuges 30min under the conditions of 4 DEG C, collects supernatant.

Chromatographic purifying：The bacterial cell disruption supernatant filtered through 1 μm, is splined on chromatography media POROS 50HS (Applied Biosystems), destination protein is adsorbed onto on chromatography media, with 0.5M~1.5M NaCl gradient elutions, destination protein and impurity Initial gross separation is obtained, the HPV11 L1 albumen (Fig. 5 A) of elution is collected.Just pure HPV11 L1 albumen is splined on chromatography media Macro-Prep ceramic hydroxyapatites (Type II, 40 μm), destination protein is incorporated on chromatography media, with 20~200mM phosphorus Hydrochlorate concentration gradient elutes, and destination protein is detached with impurity, collects the HPV11 L1 albumen (Fig. 5 B) of elution.

The depolymerization and reunion of HPV11 L1 VLP：HPV11 L1 albumen after purification is added 25mM DTT, 0.05% Polysorbate80, under the conditions of pH8.2, in room temperature depolymerization 2 hours.Dialysis removal DTT, system of dialysing：1.0M NaCl, 20mM phosphate, 0.05%Polysorbate80, pH 7.2.By 1:100 ratios are dialysed, and are changed 3 times, each 30min.It dialyses again Into reunion buffer solution, system of dialysing：1M NaCl,5mM Ca²⁺, 60mM sodium citrates, pH6.2,0.05% Polysorbate80.In 4 DEG C, dialysis obtains the HPV11 L1 VLP of reunion after 20-24 hours.

The accelerated stability of HPV11 L1 albumen compares before and after meeting again：Light dissipates in solution caused by aggregation meeting due to albumen It penetrates and changes, therefore the detection of albumen aggregation can be carried out by the absorbance value at 350nm.Under the conditions of 37 DEG C, compare Place after a certain period of time, the aggregation extent of the HPV11 L1 albumen for the HPV11 L1 albumen and reunion that do not meet again, by table 1 as it can be seen that The accelerated stability of HPV11 L1 albumen is significantly better than the HPV11 L1 albumen that do not meet again after reunion.

Table 1 meet again front and back HPV11 L1 albumen accelerated stability analysis

Embodiment 9：The HPV11 L1 recombinant proteins of transmission electron microscope observing purifying

With the HPV11 L1 protein samples of 3 times of dilutions of sterile water after purification, one droplet of drop is on cured disk.Copper mesh is taken to make have branch The surface for holding film is contacted with sample liquid surface, is stood 1min and is taken out, takes out copper mesh, extra drop is absorbed with filter paper item, is slightly dried in the air It is dry.2% acetic acid uranium solution is taken, one droplet of drop is on cured disk.The copper mesh for being adsorbed with sample is positioned over dye liquor surface (sample and dye liquor Contact), stand 2min.Copper mesh is taken out, extra drop is absorbed with filter paper item, is dried under incandescent lamp.Using JEOL-1400 models Transmission electron microscope observing VLP particle shapes are simultaneously taken pictures (shown in result figure 6).

Embodiment 10：With the immunogenicity research for the HPV11 L1 VLP that Hansenula yeast is prepared by recombinant

Using measurement humoral immunity effect ED₅₀The immunogenicity of the method evaluation HPV11 L1 VLP of (median effective dose)

(1) mouse is immune：70 6 week old Balb/c female mices (are purchased from Chinese Academy of Medical Sciences's Animal Experimental Study Institute), cleaning grade raising.It is divided into 6 groups, including 5 experimental groups and 1 control group, by required immunizing dose by HPV11 L1 Protein sample is diluted (table 2).Immune programme is：0th, 3,6 week each immune primary, and mouse is killed simultaneously within 14 days after last time is immune Detach serum.

The grouping of 2 mouse of table

(2) ELISA method measures the serological conversion rate that HPV11 L1 VLP are immunized after mouse, the specific steps are：It is slow with coating Escherichia coli recombination HPV11 L1 albumen is diluted to 0.5 μ g/ml by fliud flushing, 0.1ml is added per hole, 4 DEG C overnight.Next day washing buffer Liquid washs 3 times, gets rid of most residual liquid.It is closed 30 minutes with antibody diluent, washing buffer is washed 3 times, is detected after drying, Or 4 DEG C of damp proof preservations after drying.With sample diluting liquid by each mice serum sample with 1:10000 are diluted, take 0.1ml in In the above-mentioned reacting hole being coated with, sets 37 DEG C and be incubated 1 hour, wash 5 times.(while doing blank, negative hole control).In reacting hole Middle addition antibody diluent 1:The sheep anti-mouse igg secondary antibody 0.1ml of the HRP labels of 10000 diluted fresh, 37 DEG C are incubated 30 points Clock washs 5 times, last time is washed with distilled water.It is added the tmb substrate solution 0.1ml of Extemporaneous in each reacting hole, 37 DEG C colour developing 10 minutes.50 μ l 2M sulfuric acid 0.05ml are added in each reacting hole to terminate reaction.In microplate reader, in 450nm Locate (630nm is reference wavelength), each hole OD values are surveyed after returning to zero with blank control wells.Cutoff values calculate and positive findings judgement： Cutoff values=negative control value × 2.1；Sample OD values>Cutoff values are then judged to the positive.

(3) humoral immunity effect ED₅₀Calculating

According to 3 result of calculation of table, the humoral immunity effect ED of HPV11 L1 VLP₅₀Value is 0.568 μ g, it is shown that HPV11 L1 VLP have good immunogenicity.

The humoral immunity effect detection case of 3 mouse of table

Wherein：It is 1 to turn dilution higher than 50% sun, and it is 2 to turn dilution less than 50% sun.It is obtained by calculating：Distance than The logarithm for being 0, ED50 for 0.8160, the log10 dilutions turned higher than 50% sun is 0.2456, and 50% sun turns dilution and is 1.76.So ED50 values are 0.568 μ g.

Sequence table

<110>Beijing Abzymo Biosciences Technology Co., Ltd.

<120>The method for generating HPV11 L1 albumen with expressed by Hansenula yeast system

<130> I2012TC136CB

<160> 19

<170> PatentIn version 3.3

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<223> HPV11 L1 consensus sequence

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Met Trp Arg Pro Ser Asp Ser Thr Val Tyr Val Pro Pro Pro Asn Pro

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Tyr Gln Tyr Arg Val Phe Lys Val Val Leu Pro Asp Pro Asn Lys Phe

65 70 75 80

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

85 90 95

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

100 105 110

Gly Val Ser Gly His Pro Leu Leu Asn Lys Tyr Asp Asp Val Glu Asn

115 120 125

Ser Gly Gly Tyr Gly Gly Asn Pro Gly Gln Asp Asn Arg Val Asn Val

130 135 140

Gly Met Asp Tyr Lys Gln Thr Gln Leu Cys Met Val Gly Cys Ala Pro

145 150 155 160

Pro Leu Gly Glu His Trp Gly Lys Gly Thr Gln Cys Ser Asn Thr Ser

165 170 175

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

180 185 190

Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asn Phe Ala

195 200 205

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

210 215 220

Val Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ala Ala Asp Pro Tyr Gly

225 230 235 240

Asp Arg Leu Phe Phe Tyr Leu Arg Lys Glu Gln Met Phe Ala Arg His

245 250 255

Phe Phe Asn Arg Ala Gly Thr Val Gly Glu Pro Val Pro Asp Asp Leu

260 265 270

Leu Val Lys Gly Gly Asn Asn Arg Ser Ser Val Ala Ser Ser Ile Tyr

275 280 285

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

290 295 300

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

305 310 315 320

Cys Trp Gly Asn His Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser

325 330 335

Thr Asn Met Thr Leu Cys Ala Ser Val Ser Lys Ser Ala Thr Tyr Thr

340 345 350

Asn Ser Asp Tyr Lys Glu Tyr Met Arg His Val Glu Glu Phe Asp Leu

355 360 365

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

370 375 380

Ala Tyr Ile His Thr Met Asn Pro Ser Val Leu Glu Asp Trp Asn Phe

385 390 395 400

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

405 410 415

Val Gln Ser Gln Ala Ile Thr Cys Gln Lys Pro Thr Pro Glu Lys Glu

420 425 430

Lys Gln Asp Pro Tyr Lys Asp Met Ser Phe Trp Glu Val Asn Leu Lys

435 440 445

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

450 455 460

Leu Leu Gln Ser Gly Tyr Arg Gly Arg Thr Ser Ala Arg Thr Gly Ile

465 470 475 480

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

485 490 495

Thr Lys Thr Lys Lys

500

<210> 2

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<212> DNA

<213> Human Papillomavirus

<400> 2

atgtggcggc ctagcgacag cacagtatat gtgcctcctc ccaaccctgt atccaaggtt 60

gttgccacgg atgcgtatgt taaacgcacc aacatatttt atcatgccag cagttctaga 120

ctccttgctg tgggacatcc atattactct atcaaaaaag ttaacaaaac agttgtacca 180

aaggtgtctg gatatcaata tagagtgttt aaggtagtgt tgccagatcc taacaagttt 240

gcattacctg attcatccct gtttgacccc actacacagc gtttagtatg ggcgtgcaca 300

gggttggagg taggcagggg tcaaccttta ggcgttggtg ttagtgggca tccattgcta 360

aacaaatatg atgatgtaga aaatagtggt gggtatggtg gtaatcctgg tcaggataat 420

agggttaatg taggtatgga ttataaacaa acccagctat gtatggtggg ctgtgctcca 480

ccgttaggtg aacattgggg taagggtaca caatgttcaa atacctctgt acaaaatggt 540

gactgccccc cgttggaact tattaccagt gttatacagg atggggacat ggttgataca 600

ggctttggtg ctatgaattt tgcagactta caaaccaata aatcggatgt tccccttgat 660

atttgtggaa ctgtctgcaa atatcctgat tatttgcaaa tggctgcaga cccttatggt 720

gataggttgt ttttttattt gcgaaaggaa caaatgtttg ctagacactt ttttaatagg 780

gccggtactg tgggggaacc tgtgcctgat gacctgttgg taaaaggggg taataacaga 840

tcatctgtag ctagtagtat ttatgtacat acacctagtg gctcattggt gtcttcagag 900

gctcaattat ttaataaacc atattggctt caaaaggctc agggacataa caatggtatt 960

tgctggggaa accacttgtt tgttactgtg gtagatacca cacgcagtac aaatatgaca 1020

ctatgtgcat ctgtgtctaa atctgctaca tacactaatt cagattataa ggaatacatg 1080

cgccatgtgg aggagtttga tttacagttt atttttcaat tgtgtagcat tacattatct 1140

gcagaagtca tggcctatat acacacaatg aatccttctg ttttggagga ctggaacttt 1200

ggtttatcgc ctccaccaaa tggtacactg gaggatactt atagatatgt acagtcacag 1260

gccattacct gtcagaaacc cacacctgaa aaagaaaaac aggatcccta taaggatatg 1320

agtttttggg aggttaactt aaaagaaaag ttttcaagtg aattagatca gtttcccctt 1380

ggacgtaagt ttttattgca aagtggatat cgaggacgga cgtctgctcg tacaggtata 1440

aagcgcccag ctgtgtctaa gccctctaca gcccccaaac gaaaacgtac caaaaccaaa 1500

aagtaa 1506

<210> 3

<211> 1506

<212> DNA

<213> Artificial

<220>

<223> 11sc

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atgtggagac caagcgacag cacagtatat gtgcctccac ctaacccagt atccaaagtt 60

gttgccacgg atgcttatgt taaaagaacc aacatattct atcatgccag cagttctagg 120

cttcttgcag tgggtcatcc ttactattcc atcaaaaagg ttaacaaaac tgttgtgcca 180

aaggtgtcag gatatcaata cagagtattt aaggtggtgt taccagatcc taacaaattt 240

gcattgccag actcgtctct tttcgatccc acaactcaac gtttggtatg ggcatgcaca 300

ggcctagagg tgggcagagg acagccatta ggtgtgggtg taagtggaca tcctttacta 360

aataaatatg atgatgttga aaattcaggg ggttacggtg gtaacccagg acaggacaac 420

agggttaatg taggtatgga ttacaaacaa acacaattat gcatggttgg atgtgccccc 480

ccattgggcg agcactgggg taaaggtaca cagtgtagta atacatctgt acagaatggt 540

gactgcccgc ccttagaact tattacgagt gttatacagg atggcgatat ggttgacaca 600

ggctttggtg ctatgaattt tgctgatttg cagaccaata aatcagatgt tcctcttgac 660

atctgtggca ctgtatgcaa atatccagat tacttacaaa tggctgcaga cccatatggt 720

gatagattat tcttttatct aagaaaggaa caaatgtttg ccagacattt ctttaacagg 780

gctggtactg tgggggaacc tgtgcctgat gatcttttag ttaagggtgg taacaataga 840

tcgtctgtag cgagtagtat ctatgttcac actccaagcg gctctttggt gtcctctgag 900

gcacaattgt tcaataagcc atactggcta caaaaagccc agggacacaa caatggtatt 960

tgttggggta atcatctgtt tgttactgtg gtagatacca caagaagtac caacatgaca 1020

ttatgtgcat ccgtatctaa atctgccaca tacacgaatt ctgactataa agagtacatg 1080

cgtcatgtgg aagagtttga tttacaattc atttttcaat tatgtagcat tacattgtct 1140

gctgaagtaa tggcctatat tcacacaatg aatccctctg ttctcgaaga ctggaacttt 1200

gggttatcgc ctcccccaaa tggtacactc gaggatacct acaggtatgt gcagtcacag 1260

gccattacct gtcaaaagcc cactccagaa aaggaaaagc aagatcccta taaggacatg 1320

agtttctggg aggttaattt aaaagaaaag ttttctagtg aattggatca gtttcctttg 1380

ggaagaaagt tcttgttaca aagtggatac aggggaagaa cctctgctcg tactggtatt 1440

aagagaccag ctgtttccaa accctctact gcccctaaac gtaagagaac gaaaactaaa 1500

aagtaa 1506

<210> 4

<211> 1506

<212> DNA

<213> Artificial

<220>

<223> 11hp

<400> 4

atgtggagac catcggactc gacagtgtac gttccacctc cgaatccagt cagcaaagtt 60

gtggccactg atgcgtatgt caagagaacc aacatctttt accatgcctc gtcttccaga 120

ctcttggctg ttggtcaccc ttactacagc attaagaaag tcaataagac tgtggtgccc 180

aaggtgtctg gataccaata tcgcgtcttc aaagtggttc tgccggaccc taacaagttc 240

gcattgccag actcctcact gtttgatccg acgactcaga gacttgtctg ggcctgcaca 300

ggtctggaag tgggacgtgg ccagcctctg ggtgttggcg tgtctggcca tccactcttg 360

aacaagtatg atgacgtgga gaacagtggt ggctacggag gcaatccagg gcaagacaac 420

agagtcaatg ttggaatgga ttacaaacag actcagctgt gcatggttgg ctgtgctcct 480

ccacttggtg aacactgggg caaaggcacc caatgctcca acacgtcggt ccagaatggt 540

gactgtccac cgttggagct cataacgtct gtgattcaag atggagacat ggttgacaca 600

ggatttggtg cgatgaactt cgctgatctt cagacgaaca agtcagacgt tcctctggac 660

atctgcggga ctgtctgtaa atatccggac tacctccaga tggcagctga tccctatgga 720

gaccggttgt tcttctacct gcgcaaggaa cagatgtttg caagacactt cttcaacaga 780

gctggcactg ttggagagcc tgtgccagat gacctgttgg tcaaaggtgg caacaatcga 840

agctctgtgg cctcctcgat ttacgttcac actccgtctg gcagcctcgt gtcctctgaa 900

gcacaactgt tcaacaaacc ttattggctt cagaaagctc agggacataa caatgggatt 960

tgctggggta atcacctgtt tgtcaccgtt gtggacacga ccaggagcac caacatgact 1020

ctgtgtgcga gtgtgtcgaa gtctgccacc tacacgaact cagactacaa agagtacatg 1080

agacatgttg aagagttcga cttgcagttt atcttccaac tgtgctccat caccctgtcg 1140

gctgaggtca tggcctacat tcacaccatg aacccgtcag ttcttgagga ctggaacttt 1200

ggtctctctc cacctcccaa tggaactctg gaggacacct atcgctacgt tcagtcacaa 1260

gccatcacct gtcagaagcc gactccagag aaagagaagc aggatcccta caaagacatg 1320

agcttctggg aggtcaacct gaaagagaag ttctccagtg aactggatca gtttccactt 1380

ggtcggaagt ttctcttgca atccggctat cgtggcagaa cctcggccag gacaggaatc 1440

aagagaccag cagtgagcaa accttcgaca gcgccgaaga ggaagagaac gaagaccaag 1500

aagtaa 1506

<210> 5

<211> 50

<212> DNA

<213> Artificial

<220>

<223> primer1

<400> 5

catcaatcta aagtacaaaa acaaaatgtg gcggcctagc gacagcacag 50

<210> 6

<211> 46

<212> DNA

<213> Artificial

<220>

<223> primer2

<400> 6

gcggtatgtc cttccacgtc tccttacttt ttggttttgg tacgtt 46

<210> 7

<211> 50

<212> DNA

<213> Artificial

<220>

<223> primer3

<400> 7

catcaatcta aagtacaaaa acaaaatgtg gagaccaagc gacagcacag 50

<210> 8

<211> 49

<212> DNA

<213> Artificial

<220>

<223> primer4

<400> 8

gcggtatgtc cttccacgtc tccttacttt ttagttttcg ttctcttac 49

<210> 9

<211> 50

<212> DNA

<213> Artificial

<220>

<223> primer5

<400> 9

catcaatcta aagtacaaaa acaaaatgtg gagaccatcg gactcgacag 50

<210> 10

<211> 48

<212> DNA

<213> Artificial

<220>

<223> primer6

<400> 10

gcggtatgtc cttccacgtc tccttacttc ttggtcttcg ttctcttc 48

<210> 11

<211> 41

<212> DNA

<213> Artificial

<220>

<223> primer7

<400> 11

aaggaaaaaa gcggccgcaa cgatctcctc gagctgctcg c 41

<210> 12

<211> 30

<212> DNA

<213> Artificial

<220>

<223> primer8

<400> 12

gaagatctca atctccggaa tggtgatctg 30

<210> 13

<211> 35

<212> DNA

<213> Artificial

<220>

<223> primer9

<400> 13

ggaattccat atgtggagac catcggactc gacag 35

<210> 14

<211> 34

<212> DNA

<213> Artificial

<220>

<223> primer10

<400> 14

ccggtcgacc ttcttggtct tcgttctctt cctc 34

<210> 15

<211> 6210

<212> DNA

<213> Artificial

<220>

<223> pRMHP2.1

<400> 15

cgcggccgct tttttcctta gatcttacgg ttatccacag aatcagggga taacgcagga 60

aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 120

gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 180

aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 240

gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 300

ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 360

cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 420

ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 480

actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 540

tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 600

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 660

ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 720

cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 780

ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 840

tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaggt 900

acccctcgtg aagaaggtgt tgctgactca taccaggcct gaatcgcccc atcatccagc 960

cagaaagtga gggagccacg gttgatgaga gctttgttgt aggtggacca gttggtgatt 1020

ttgaactttt gctttgccac ggaacggtct gcgttgtcgg gaagatgcgt gatctgatcc 1080

ttcaactcag caaaagttcg atttattcaa caaagccgcc gtcccgtcaa gtcagcgtaa 1140

tgctctgcca gtgttacaac caattaacca attctgatta gaaaaactca tcgagcatca 1200

aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt 1260

tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc 1320

ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa 1380

taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa 1440

gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat 1500

cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc 1560

gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg 1620

ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg 1680

ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct 1740

tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa 1800

catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc 1860

catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc 1920

catataaatc agcatccatg ttggaattta atcgcggcct cgagcaagac gtttcccgtt 1980

gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc 2040

atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac acaacgtggc 2100

tttcagatcc gatatcctat cctgcaggtc gactcccgcg actcggcgtt cactttcgag 2160

ctattatcaa cgccggaata cgtcagaaac agccgtgccc cagggaccag aaagcctact 2220

ggtgagtatg ttctttcgtg tgatttttcc gaggatgaga acgacgataa cgagcacaac 2280

tcggagtcgg aggacacgct tattgcgttg aacgcagcca catcagcagg ctgtcaagac 2340

tgagtatggc cacagagctg gattctcggc ctcatactca agacgttagt aaactccgtc 2400

tgccagaaat tgctgacgag gatgtataat aatagatgaa ttacgaacaa ttgtagttca 2460

aaaaaattta gtaacaatat tgtctagatg acagatgtgc tgaaaccagt gaactccaat 2520

aaaccactca ccgctaccca agagaaacag atcagagtgc tagggccttg tttcagagta 2580

ctacaacgtt taccagaagc ttgagcaagt tctcaaacgc gggtttgtcg acatatgcac 2640

ccactaatag ggaacgtgag ctgggtttag accgtcgtga gacaggttag ttttacccta 2700

ctgatgaatg ttatcgcaat agtaattgaa cttagtacga gaggaaccgt tcattcagat 2760

aattggtttt tgcggctgtc tgatcaggca ttgccgcgaa gctaccatct gctggataat 2820

ggctgaacgc ctctaagtca gaatccatgc tagaaagcga tgatttcttg ccctcgcaca 2880

ttttagttgg atacgaataa ggcactctgt gtcgctgaac catagcaggc tggcaatggt 2940

gcttttaacg gaaaggtttt aagtgcttgc cggtggatag caatgtcatt atgcgcgagg 3000

ataaatcctt tgcatacgac ttaaatgtac aacggggtat tgtaagcagt agagtagcct 3060

tgttgttacg atctgctgag attaagcctc agttgtccga tttgtttgtg tttacacaac 3120

acaatctccc ctaagagata ttacttggtg gtagccgggt gtcttttaga ggagattttt 3180

tatatttttt tgagtagagg tcgtgcgtgg aacaattttg gatggttgtg tataaaattt 3240

tgaatcgaag agaccaatat tattttttaa ttacttatta ttttgtgttt tttttgtttt 3300

ttgatttaag ttatttaatt cagggataag ttggcttttt aatttttttg tttttatttt 3360

tttggtagca agcctttaat taaattgaat tttcgatgtg tagtggtgta aggatcttta 3420

tccaaaacat ttcggttgat attggccaat agaagtgtgt tgagctctga caaatgacag 3480

atattctgta ccatagtaag attcaaggta aatgagagaa aatgtatggt aattgatggc 3540

tgtcgtatga aaagtgagta cgatacgggc aactaagcta acaagagtag gcagaataga 3600

cagatattct gtgatgcaaa gtgtgtggca cacgtcctgt ttgtcatctg gcgctgagaa 3660

ggaaatttaa gcatagagat ggtggcagag ttgaaagagg tgctacaagg tgcctggaag 3720

agccggcaaa aaaagtggta gtggtaaagg atgaatgaag actttttgcg ccataccact 3780

gttgctgctc ttcctaaagg caggattaca agaagaagtc ggagggatta ggattggcga 3840

ccactcttct ctacgtgctg ggaggaaaaa aaagataggt gtggaagagg tgggcatata 3900

tatatagacg tttggataga caatggattg atgcagaacg cgacctcgag accgcgttgg 3960

actggaggga agggaaaaaa aaataataaa aaaaaaaaag attgcagcac ctgagtttcg 4020

cgtatggtct cccactacac tactcggtca ggctcttagc agcttaacta cagttgatcg 4080

gacgggaaac ggtgctttct gctagatatg gccgcaaccg aaagagtatg gaaacgagtg 4140

ggggatatga attgagaggg gggagaagta ctattcaact gttggttggt ggcagctatt 4200

gtagaacgaa atgcttaagt ataatatttt ttttttctcg gtaggtgaaa ttgtataaga 4260

gagaagacca ctgtagatga aggatgtact gatgtgaaac tgtatgagtg gtttgacgat 4320

tctacgtagg agaattaaaa agttggggaa attggaaatt agaggagaat ctcctgttga 4380

aaaatgaaga tcaagcatga aatgtatgaa tgccaatttg gagatcagca caacgtcatg 4440

ttaaagggga attgactgaa aagcgtagaa agtccatact ccagtgatga acttgtggtt 4500

tttcaagttt ctttaatttt ttcggttgtc gcgtagcagg gtggcacgaa gaaaaaattt 4560

cgtggtacca agaaatatgt cgggggaata atatttggag gcggtaggga attagaatcg 4620

aaatgaaaaa tataaaaagc tggaggaagg tgctaaaaaa aagccaatga ataattatgt 4680

aacttttgga aaaagttata ttagagagga aattttacgt gagagatgcg gatggacttt 4740

catgcgaaag cataaaagaa gatttgaaaa ctgaatgtga agaagcgaaa gcttctgcgc 4800

gtttggggtt cgattttcga aagggagttc tgaaaagagc tcttcaaggc ctcgttgatt 4860

agtggagaga gtaaagttgt tatcgtcaga tacactttct ctcaaggcta attcaacatg 4920

ggtgatcttg ggcggtcaaa aaaaataatt tttgatggtc tgagtagcct gaacagtctc 4980

ataccaaatt tctaattatt tttgtgtgtg agcactgatg gatttagtgg attactagcc 5040

tatggcaaga attcggacca caatccaaat aaagaaagtg catggaagaa gtaatcaaat 5100

aatttttaca tagcaagcaa caatagattt atttatcatg gcagccaagt ttgaaacacg 5160

aaagcgcgta caataataat gacgtatgtt gaactttttt tctccttaac tataaaaatc 5220

agttaagccg taaatatgta gatgaggccg tgctcagtcc tgctcctcgg ccacgaagtg 5280

cacgcagttg ccggccgggt cgcgcagggc gaactcccgc ccccacggct gctcgccgat 5340

ctcggtcatg gccggcccgg aggcgtcccg gaagttcgtg gacacgacct ccgaccactc 5400

ggcgtacagc tcgtccaggc cgcgcaccca cacccaggcc agggtgttgt ccggcaccac 5460

ctggtcctgg accgcgctga tgaacagggt cacgtcgtcc cggaccacac cggcgaagtc 5520

gtcctccacg aagtcccggg agaacccgag ccggtcggtc cagaactcga ccgctccggc 5580

gacgtcgcgc gcggtgagca ccggaacggc actggtcaac ttggccatat tgtttctata 5640

ttatctttgt actaaagagc aattgataat gtgcgagaaa aactggtcct tatatgccgt 5700

ttgcagcact ccctcccgaa ctttacgaaa agtcgtgcgc cacctgattt tcatcacgcc 5760

aaaaacctac acgtatgact actccgggcc agtgttcacc acgagctata tagtgttaat 5820

taattacctt attggttagc tctgcatgta agggtggtgt gagccgggaa ttgggtctac 5880

tctagcgttc agtaaggtga tataaagctc tgtatagcca gaagtggaca tcacccaaca 5940

aggcgtctcg gggacttgcc tgtccgtgca aggttgttcc atggaagctc taccgccgga 6000

gcggcccaaa ggacaataag aagtgctaca ccacctccgc agaggacaca ggcttaaaac 6060

cctctttctc ggtttcggga ccggttcccg gagattgtct ttaccccacg caccgtgctg 6120

gagccatagc agttgttgca actttgcgag ttgtcacctt ttcctccgtg gcccgcctct 6180

tttctggtgc acggatgtag tctagaccaa 6210

<210> 16

<211> 41

<212> DNA

<213> Artificial

<220>

<223> MOX promoter primer

<400> 16

aaggaaaaaa gcggccgcaa cgatctcctc gagctgctcg c 41

<210> 17

<211> 27

<212> DNA

<213> Artificial

<220>

<223> MOX promoter primer

<400> 17

tttgtttttg tactttagat tgatgtc 27

<210> 18

<211> 23

<212> DNA

<213> Artificial

<220>

<223> MOX terminator primer

<400> 18

ggagacgtgg aaggacatac cgc 23

<210> 19

<211> 30

<212> DNA

<213> Artificial

<220>

<223> MOX terminator primer

<400> 19

gaagatctca atctccggaa tggtgatctg 30

Claims

1. a kind of method for the recombination Hansenula yeast cell generating expression Human Papillomavirus Type 11 L1 (HPV11 L1) albumen, It comprises the steps of：

A) by that will include the nucleotide for encoding HPV11 L1 albumen operably being connect with MOX promoters and MOX terminators The exogenous polynucleotide insetion sequence of sequence is shown in SEQ ID NO:15 carrier builds expression construct；

C) the Hansenula yeast cell obtained in step b) is screened with Zeocin and G418, obtains and contains the external source multinuclear The recombination Hansenula yeast cell of thuja acid.

2. the method for claim 1 wherein the amino acid sequences of the HPV11 L1 albumen to be shown in SEQ ID NO:1.

3. the method for claim 2, wherein the nucleotides sequence of the coding HPV11 L1 albumen is shown in SEQ ID NO:4.

4. the method for claim 1 wherein convert Hansenula yeast cell by electroporation in step b).

5. the method for claim 1 wherein the Hansenula yeast cell in step b) is ATCC26012 Hansenula yeast cells.

6. the method for claim 1 wherein the G418 of the Zeocin and 10mg/ml that use 0.25mg/ml in step c) to step b) The Hansenula yeast cell of middle acquisition is screened.

7. the method for claim 1 wherein screenings described in step c) to include

C1) the Hansenula yeast cell obtained in step b) is coated in the YPD tablets of the Zeocin containing 0.25mg/ml, in 37 DEG C It is inverted culture 3-5 days；

C2) picking is inoculated in 5ml and contains 0.25mg/ml in the single bacterium colony grown on the YPD tablets of the Zeocin containing 0.25mg/ml In the YPD fluid nutrient mediums of Zeocin, in 37 DEG C, 200rpm shaking table cultures 24-36 hours, until OD values are up to after 50, with 1:1000 Ratio transfer in the YPD fluid nutrient mediums of 5ml Zeocin containing 0.25mg/ml, culture to OD values is up to after 50, then with 1: 1000 ratio is transferred in the YPD fluid nutrient mediums of 5ml Zeocin containing 0.25mg/ml, continuous to pass 10 times；

C3) the recombination expressed by Hansenula yeast bacterial strain access 5ml for reaching 10 times is free of to the YPD fluid nutrient mediums of Zeocin resistances In, in 37 DEG C, 200rpm shaking table cultures to OD values up to after 50, with 1:1000 ratio is transferred in 5ml YPD fluid nutrient mediums, It is continuously passed in the YPD fluid nutrient mediums without Zeocin resistances 5 times；With

C4) product of acquisition is coated on to the YPD tablets of the G418 containing 10mg/ml, culture 2-3 days, picking growth are inverted in 32 DEG C Vigorous single bacterium colony carries out copy number detection.

8. the method for any one of claim 1-7, wherein the recombination Hansenula yeast cell contains the external source of multicopy Polynucleotides.

9. the method for claim 8, wherein the recombination Hansenula yeast cell contains the external source multinuclear that copy number is more than 30 Thuja acid.

10. a kind of recombination Hansenula yeast cell generated according to the method for any one of claim 1-9.

11. a kind of method generating HPV11 L1 albumen, includes the following steps：

I) the recombination Hansenula yeast cell of claim 10 is cultivated under conditions of suitable for the HPV11 L1 protein expressions；With

Ii it) is recycled from culture and purifies the HPV11 L1 albumen.

12. the method for claim 11 further includes the steps that carrying out depolymerization to purified HPV11 L1 albumen and meeting again.

13. the HPV11 L1 albumen generated according to the method for claim 11 or 12 is preparing the epidemic disease for preventing HPV11 infection Purposes in seedling.

14. encoding the polynucleotides of HPV11 L1 albumen, it includes SEQ ID NO:Nucleotide sequence shown in 4.

15. a kind of expression construct is shown in SEQ by the way that the nucleotide sequence for encoding HPV11 L1 albumen is inserted into sequence ID NO:It is obtained in 15 expression vector pRMHP2.1.