CN110592133A

CN110592133A - Method for producing HPV31L1 protein by using hansenula polymorpha expression system

Info

Publication number: CN110592133A
Application number: CN201910880015.4A
Authority: CN
Inventors: 霍烛; 于跃; 程海; 刘娟; 王贻杰; 陈丹; 李鼎锋; 刘勇
Original assignee: JIANGSU RUIKE BIOTECHNOLOGY Co Ltd; BEIJING ABZYMO BIOSCIENCES Co Ltd
Current assignee: JIANGSU RUIKE BIOTECHNOLOGY Co Ltd; BEIJING ABZYMO BIOSCIENCES Co Ltd
Priority date: 2013-05-17
Filing date: 2013-05-17
Publication date: 2019-12-20
Also published as: CN104164374B; CN104164374A

Abstract

The invention relates to a method for producing HPV31L1 protein by using a hansenula polymorpha expression system. Specifically, the invention discloses a method for generating recombinant hansenula polymorpha cells expressing HPV31L1 protein and recombinant hansenula polymorpha cells generated by the method. The invention also discloses a method for producing the HPV31L1 protein by using the recombinant hansenula polymorpha cell and application of the produced HPV31L1 protein in preparation of a preventive vaccine.

Description

Method for producing HPV31L1 protein by using hansenula polymorpha expression system

The application is a divisional application of an invention patent application with the application date of 2013, 5 and 17 months and the application number of 201310185027.8 and the name of a method for producing HPV31L1 protein by using a hansenula polymorpha expression system.

Technical Field

The invention belongs to the technical field of medical biological engineering, relates to a method for producing HPV31L1 protein, and particularly relates to a method for producing HPV31L1 protein by using a hansenula polymorpha expression system.

Background

Human Papilloma Virus (HPV) is a non-enveloped closed-loop double-stranded DNA virus, belongs to the family of papovaviridae, the sub-family of polyomaviruses, and mainly invades epithelial mucosal tissues of a human body, thereby inducing various benign and malignant hyperplasia lesions.

Over 200 different subtypes of HPV have been identified, HPV infection is clearly tissue specific, different types of HPV have different tropisms for skin and mucosa and can induce different papillary lesions, and approximately 30 HPV types are associated with genital tract infections, and more than 20 of them are associated with tumors. Depending on the malignancy and the quality of the HPV-induced lesions, HPV can be roughly divided into two categories: 1) high risk types (e.g. HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, etc.): the high-risk HPV is closely related to various human tissue malignant tumors, and mainly causes severe atypical hyperplasia and invasive cancer; 2) low risk types (e.g. HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV72, HPV 81, etc.): the low-risk HPV can cause benign proliferative diseases of epidermal cells, such as condyloma acuminatum and flat condyloma.

HPV is composed primarily of viral coat and genomic DNA. The genome is about 7900bp in length and has 8 virus protein coding genes. Wherein the 6 ORF encoded proteins are expressed early in viral replication, referred to as early proteins; the 2 ORF-encoded proteins are expressed late in viral replication and are referred to as late proteins. The late proteins include major coat protein L1 and minor coat protein L2, and are involved in the formation of the viral coat.

The HPV viral coat protein can self-assemble, and can self-assemble into virus-like particles (VLPs) when the L1 protein alone is expressed in various expression systems or when the L1 protein and the L2 protein are co-expressed, wherein VLPs produced in yeast systems, baculovirus insect expression systems, mammalian cell expression systems, and the like more closely approximate to the structure of natural viruses. After being immunized by the VLP produced by the exogenous expression system, the VLP can induce and generate a neutralizing antibody in vivo, and a good immune protection effect is obtained.

Hansenula Polymorpha (Hansenula Polymorpha), also known as Pichia augusta, is currently recognized as one of the most desirable exogenous gene expression systems. The hansenula polymorpha serving as a unicellular eukaryotic microorganism has the characteristics of rapid growth of prokaryotes, easiness in genetic operation and the like, and also has the functions of post-translational processing and modification of eukaryotic cells and the like. In addition, the hansenula polymorpha has the advantages of good safety, easy culture, low cost, high expression level, stable heredity and the like, and can overcome the problems of unstable strains of Saccharomyces cerevisiae (Saccharomyces cerevisiae), low yield, overlong glycosylated side chain and low integrated copy number of exogenous genes of Pichia Pastoris (Pichia Pastoris). Currently, drugs (e.g., insulin, trade name Wosulin) and HBV vaccines (trade name Hepavax-Gene) produced using the Hansenula polymorpha expression system are commercially available.

Methods for expressing HPV16 and HPV18 type L1 VLPs by using a Hansenula polymorpha expression system are disclosed in Chinese patent publications CN102586287A and CN 102719453A. However, there is no report on whether the L1 protein of HPV31 type can be efficiently expressed in Hansenula polymorpha system and assembled into VLP particles. In the above 2 published patent applications, the results of integrating copy numbers of foreign genes of HPV16 and HPV 18L 1 and methods for increasing copy numbers of foreign genes are not given. In terms of the induced expression of foreign proteins, the induction time of both HPV16 and HPV 18L 1 in the fermenter was more than 20 hours, and no specific information on the protein expression level was provided. In addition, as an important protein purification index, there is no disclosure about the purification process and the purity of the foreign proteins HPV16 and HPV 18L 1 at the completion of the purification.

In order to realize the high-efficiency expression of the HPV31L1 protein in Hansenula polymorpha and purify and obtain high-purity HPV31 VLP protein, the invention applies a dual-screening strategy of Zeocin resistance screening and G418 resistance screening, and particularly applies a G418 resistance plate with the concentration as high as 16mg/ml to screen a recombinant Hansenula polymorpha expression strain after passage and stabilization. Through detection, the copy number of the HPV31 high-expression strain obtained by screening can exceed 60 copies. During fermentation induction, the expression level of HPV31L1 protein can be obviously improved by increasing the temperature for inducing expression to 35 ℃. In addition, aiming at the characteristic that a common chromatography medium POROS 50HS is not easy to elute when purifying HPV31 VLP (50% of target protein hanging columns are still existed when target protein is eluted by high-concentration NaCl) in the VLP purification process, the invention uses the POROS XS chromatography medium, so that the target HPV31L1 protein can be eluted by more than 80% of the protein when the target HPV31L1 protein is eluted by high-concentration NaCl, and the yield of the HPV31L1 protein is greatly improved.

Summary of The Invention

In a first aspect, the present invention provides a method of producing a recombinant hansenula polymorpha cell expressing HPV31L1 protein, comprising the steps of:

a) constructing an expression construct by inserting an exogenous polynucleotide comprising a nucleotide sequence encoding HPV31L1 protein into a vector;

b) transforming Hansenula polymorpha cells with the expression construct obtained in step a); and

c) screening the hansenula polymorpha cells obtained in the step b) to obtain recombinant hansenula polymorpha cells containing the exogenous polynucleotide.

In a second aspect, the present invention also provides recombinant hansenula polymorpha cells produced according to the above method.

In a third aspect, the present invention also provides a method for producing HPV31L1 protein, comprising the steps of:

i) culturing the recombinant hansenula polymorpha cells of the present invention under conditions suitable for expression of HPV31L1 protein; and

ii) recovering and purifying HPV31L1 protein from the culture.

In a final aspect, the invention also provides the use of HPV31L1 protein produced according to the method of the invention in the manufacture of a vaccine for the prevention of HPV31 infection.

Drawings

FIG. 1 SDS-PAGE detection of the expression levels of recombinant Hansenula cells. 1-8: induced expression of 8 different recombinant hansenula polymorpha strains; 9: standard (10. mu.g/mL); 10: a protein molecular weight marker.

FIG. 2 shows the result of Southern blot detection using the MOX promoter fragment as a probe. ATCC26012 genomic DNA was used as a control. 1: control (loading 1000 ng); 2: control (loading 500 ng); 3: control (loading 250 ng); 4: control (loading 125 ng); 5: genomic DNA of high-expression recombinant bacteria HP-1#/pRMHP2.1-31HP (the loading amount is 7.8ng, and the brightness of the loading amount is between 500ng and 1000ng of a control); 6: and (3) highly expressing the genomic DNA of the recombinant bacterium HP-2#/pRMHP2.1-31HP (the loading amount is 7.8ng, and the brightness of the loading amount is between 500ng and 1000ng of a control).

FIG. 3 Western Blot detection of HPV31L1 protein expression during fermentation (induced expression at 35 ℃). 1: inducing for 0 hour; 2: inducing for 1 hour; 3: inducing for 3 hours; 4: inducing for 5 hours; 5: inducing for 7 hours; 6: inducing for 9 hours; 7: inducing for 10 hours; 8: standard (10. mu.g/mL); 9: prestained protein molecular weight markers.

FIG. 4A: POROS 50HS purification electrophoretogram of HPV31L1 protein. 1: loading the column sample; 2: flowing through the liquid; 3-5: NaCl eluent with different concentrations; 6: cleaning fluid; 7: a protein molecular weight marker.

B: POROS XS purification electropherogram of HPV31L1 protein. 1: loading the column sample; 2: flowing through the liquid; 3-5: NaCl eluent with different concentrations; 6: cleaning fluid; 7: a protein molecular weight marker.

C: CHT purification electrophoretogram of HPV31L1 protein. 1: loading the column sample; 2: flowing through the liquid; 3,4: phosphate eluents of different concentrations; 6: cleaning fluid; 7: a protein molecular weight marker.

FIG. 5 Transmission electron microscopy of purified HPV31L1 protein.

Detailed Description

The inventor successfully establishes a method for producing the HPV31L1 protein by using Hansenula polymorpha, and the produced HPV31L1 protein can be self-assembled into virus-like particles and can be used for preparing vaccines for preventing HPV infection.

The invention firstly provides a method for generating recombinant hansenula polymorpha cells expressing HPV31L1 protein, which comprises the following steps:

The present invention also includes recombinant hansenula polymorpha cells produced according to the method.

The amino acid sequences of the HPV31L1 proteins derived from different strains of HPV31 may differ. The present inventors obtained the amino acid residue with the highest frequency of occurrence at each amino acid position of the HPV31L1 protein by aligning all HPV31L1 protein sequences in the database, and obtained the amino acid sequence shown in SEQ ID NO: 1, which is a consensus sequence most representative of HPV31L1 protein. Therefore, the HPV31L1 protein of the present invention preferably has the amino acid sequence of SEQ ID NO: 1.

In order to efficiently express HPV31L1 protein using Hansenula polymorpha, the inventors performed codon optimization of the nucleotide sequence according to the amino acid sequence shown in SEQ ID NO: 1. the optimization principle includes a) selecting codons with the highest or higher frequency of use according to the Hansenula polymorpha genetic code usage table (http:// www.kazusa.or.jp/codon/cgi-bin/showcocodon. cgi? species ═ 4905), b) avoiding negative regulatory elements potentially affecting gene transcription or protein translation, such as PolyAT region, polyGC region, silencer (Slifier) region and internal splice site, etc., c) performing comprehensive analysis of mRNA secondary structures including 5 ' UTR, common UTR 31L1 coding region and 3 ' end, avoiding the formation of complex RNA secondary structures, reducing the free energy of the mRNA secondary structures, d) optimizing the coding sequence of the protein sequence of SEQ ID 462. the coding sequence of the HPV protein sequence of the present invention preferably has been eliminated by restriction enzyme recognition sequence of 5's ID NO: 462.

The hansenula polymorpha expression vector which can be applied in the invention is the Hansenula polymorpha expression vector pRMHP2.1 (comprising a sequence shown in SEQ ID NO: 9) described in Chinese patent application with the application number of 201210021524. X. The expression constructs of the invention may be obtained by cloning an exogenous polynucleotide comprising a nucleotide sequence encoding HPV31L1 protein into a prmhp2.1 vector. It will be appreciated by those skilled in the art that the expression constructs of the invention may also be constructed using other vectors, for example as described in the granted chinese patent CN 100400665C.

The exogenous polynucleotide in the expression construct is operably linked to a promoter and a terminator for expression in Hansenula.

As used herein, "operably linked" refers to a functional linkage of at least two polynucleotides. For example, an operable linkage includes a linkage between a promoter and another polynucleotide, wherein the promoter sequence initiates and mediates transcription of the other polynucleotide. The operable linkage comprises a linkage between a terminator and the other polynucleotide, wherein the terminator terminates transcription of the other polynucleotide.

Promoters suitable for use in the present invention include, but are not limited to, the MOX, FMD, AOX1, and DHAS promoters. In some embodiments, the promoter used in the present invention is the MOX promoter from hansenula. Suitable terminators for use in the present invention include, but are not limited to, the MOX terminator from Hansenula.

Transformation of the expression constructs into hansenula polymorpha cells can be performed using a variety of methods known in the art, including but not limited to electroporation and PEG-mediated transformation.

In addition, a variety of strains of Hansenula polymorpha for expression of foreign proteins have been developed in the art, including but not limited to, CGMCC2.2498 Hansenula polymorpha, ATCC34438 Hansenula polymorpha, and ATCC26012 Hansenula polymorpha cells. These Hansenula strains can also be used in the present invention. In some embodiments, the hansenula cells used to transform the expression constructs of the invention are ATCC26012 hansenula cells.

After transformation, recombinant Hansenula polymorpha cells can be selected depending on the resistance gene carried on the vector. Suitable resistance genes include, but are not limited to, Zeocin resistance gene and G418 resistance gene. Depending on the vector used, it is also possible to use nutrient-deficient media for the selection of recombinant Hansenula polymorpha cells. In one embodiment, Zeocin is used to select recombinant hansenula polymorpha cells. In another embodiment, G418 is used to select recombinant Hansenula polymorpha cells. In a preferred embodiment, Zeocin and G418 are used in combination to select recombinant hansenula polymorpha cells. The screening concentration of Zeocin may be 0.25, 0.5, 0.75, 1.0 or 1.5mg/ml, preferably 0.5 mg/ml. The selection concentration of G418 used may be 2, 4, 8, 10, 12, 14, 16, 18 or 20mg/ml, preferably 16 mg/ml.

The level of expression of the exogenous polynucleotide in Hansenula polymorpha was positively correlated with its copy number in Hansenula polymorpha. Therefore, recombinant Hansenula polymorpha cells containing multiple copies of the exogenous polynucleotide can be further screened by Southern blotting, quantitative PCR, or the like. Recombinant hansenula polymorpha cells with a copy number of the exogenous polynucleotide of more than 15 are preferred, and recombinant hansenula polymorpha cells with a copy number of the exogenous polynucleotide of more than 60 are more preferred.

The present inventors have surprisingly found that the combined use of Zeocin and G418 for selection of recombinant hansenula polymorpha cells, in particular using up to 16mg/ml of G418, enables stable recombinant hansenula polymorpha cells with copy numbers of the exogenous polynucleotide of more than 15, in particular more than 60, to be obtained.

In another aspect, the present invention also provides a method for producing HPV31L1 protein, comprising the steps of:

ii) recovering and purifying HPV31L1 protein from the culture.

Various media and basic culture conditions are known in the art for the culture of Hansenula polymorpha, and those skilled in the art can select or modify them as necessary. The culture of the recombinant Hansenula polymorpha expressing strain of the present invention can be performed in flasks or in bioreactors of various sizes (e.g., 30L fermentors) depending on the amount of protein desired. Depending on the promoter selected, an appropriate inducer may be added to the culture to induce expression of the HPV31L1 protein. In case of using MOX or FMD promoter, methanol may be added as inducer. Yeast fermentation culture is conventionally performed at 30 ℃, but the inventors surprisingly found that induction of protein expression by the recombinant Hansenula polymorpha cells of the present invention at 35 ℃ can significantly increase the expression level of foreign proteins.

Purification of the HPV31L1 protein produced may use various protein purification means known in the art, such as salting out, ultrafiltration, precipitation, chromatography, and the like, or a combination of these means. In an optimized protocol, a preliminary purification was first carried out with the chromatography medium POROS XS, followed by a further purification with the chromatography medium Macro-Prep ceramic hydroxyapatite (Type II, 40 μm).

The purified HPV31L1 protein prepared by the method of the present invention can self-assemble into virus-like particles (example 9, fig. 5) and shows good immunogenicity in mice (example 10), and thus the present invention also provides the use of the HPV31L1 protein in the preparation of vaccines for preventing HPV31 infection.

Examples

The invention will now be further illustrated by way of the following examples, without thereby limiting the invention to the scope of the examples described.

Example 1: analysis of HPV31L1 consensus amino acid sequence

The full-length HPV31L1 protein consists of 504 amino acids, and after GenBank retrieval, the alignment analysis of amino acid sequences is carried out by using Vector NTI software align X function to obtain the most representative HPV31L1 consensus amino acid sequence (i.e. the sequence with the most frequent amino acid residues at each amino acid position of HPV31L 1), wherein the sequence is shown as SEQ ID NO: 1 is shown.

Example 2: optimized design and artificial synthesis of HPV31L1 encoding gene

In order to efficiently express HPV31L1 protein using hansenula polymorpha, the inventors have expressed HPV31L1 protein according to SEQ ID NO: 1, and carrying out codon optimization of the nucleotide sequence aiming at the Hansenula polymorpha. The optimization principle comprises the following steps: a) selecting codons with highest or higher use frequency according to a hansenula polymorpha genetic code use frequency table; b) negative regulatory elements such as PolyAT regions, PolyGC regions, silencer (Sliener) regions, internal splice sites, etc., which have potential effects on gene transcription or protein translation, are avoided; c) comprehensively analyzing the secondary structure of mRNA containing 5 'end UTR, HPV31L1 coding region and 3' end UTR, avoiding the formation of complex RNA secondary structure and reducing the free energy of the secondary structure of mRNA; d) the 5' UTR region which is completely consistent with the natural sequence at the downstream of the Hansenula polymorpha promoter is adopted at the upstream of the coding region as far as possible; e) the commonly used restriction enzyme recognition sites were eliminated. The optimized nucleotide sequence is shown in SEQ ID NO: 2.

based on the above nucleotide sequences, the entire sequence was artificially synthesized by Beijing Nonsula genome research center, Inc., and cloned into a T vector (named T-31hp) for sequence verification.

Example 3: generation of expression constructs carrying HPV31L1 nucleotide sequence

The hansenula polymorpha expression vector applied by the invention is a hansenula polymorpha expression vector pRMHP2.1(SEQ ID NO: 9) described in Chinese patent application with the application number of 201210021524. X.

(1) PCR amplification of MOX promoter and MOX terminator

Using mixed genome DNA of hansenula polymorpha strains ATCC26012 and ATCC34438 as a template, amplifying by using a following primer pair to obtain a 1518bp MOX promoter, and introducing an NotI enzyme cutting site at the upstream;

MOX promoter upstream primer: 5'-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3' (SEQ ID NO: 3)

MOX promoter downstream primer: 5'-TTTGTTTTTGTACTTTAGATTGATGTC-3' (SEQ ID NO: 4)

Using mixed genome DNA of hansenula polymorpha strains ATCC26012 and ATCC34438 as a template, amplifying by using the following primer pair to obtain a MOX terminator with the size of 311bp, and introducing BglII enzyme cutting sites at the downstream;

MOX terminator upstream primer: 5'-GGAGACGTGGAAGGACATACCGC-3' (SEQ ID NO: 5)

MOX terminator downstream primer: 5'-GAAGATCTCAATCTCCGGAATGGTGATCTG-3' (SEQ ID NO: 6)

(2) Generation of expression constructs carrying HPV31L1 nucleotide sequence

Using a recombinant plasmid T-31hp carrying 31hp as a template, amplifying by using a following primer pair to obtain an HPV31L 1hp gene with the size of 1563bp, introducing an overlapping sequence at the 3 'end of an MOX promoter region at the upstream, and introducing an overlapping region at the 5' end of an MOX terminator at the downstream;

31, an upstream primer:

5’-CATCAATCTAAAGTACAAAAACAAAATGTCGCTGTGGAGACCATCTGAAG-3’(SEQ ID NO：7)

31, a downstream primer: 5'-GCGGTATGTCCTTCCACGTCTCCTTATTTCTTGGTCTTCTTGCGCTTCGC-3' (SEQ ID NO: 8)

Three fragments of MOX promoter, 31hp gene and MOX terminator are used as a mixed template, a 31hp expression cassette with the size of 3.4Kb is obtained by amplification of the following primer pairs, and an amplification product carries NotI enzyme digestion site at the upstream and BglII enzyme digestion site at the downstream.

The 31hp expression cassette was cloned into pRMHP2.1 vector by double digestion with NotI + BglII to obtain the expression construct pRMHP2.1-31 hp.

Example 4: production of recombinant Hansenula cells

(1) Extraction and enzyme digestion of recombinant expression construct plasmid

Coli colonies transformed into the recombinant expression construct Plasmid obtained in example 3 were picked, after extensive culture, the Plasmid was extracted using E.Z.N.A Plasmid Mini Kit (Omega Bio-Tek Co., Ltd.), and single-digested with BglII, recovered using E.Z.N.A Gel Extraction Kit (Omega Bio-Tek Co., Ltd.), eluted with 50. mu.L of sterile water preheated to 55 ℃ by measuring OD₂₆₀DNA quantification was performed and the linearized fragments were diluted to 100 ng/. mu.l and stored in a-20 ℃ freezer for use.

(2) Treatment of Hansenula polymorpha cells

Selecting a single colony of Hansenula polymorpha strain ATCC26012, inoculating the single colony into a small test tube containing 5ml of YPD liquid culture medium, and culturing at 30 ℃ for 12 hours; transferring 5ml of the bacterial solution into 200ml of YPD medium, and culturing at 30 deg.C for 4-6 hr to OD_600nmAbout 1.0 to about 1.5, and centrifuging at 5000rpm for 10 min; resuspending the cells in 200ml of 0.1mol/L phosphate buffer (containing 25mmol/L DTT, pH7.5), mixing well, incubating at 30 deg.C for 30min, centrifuging at 5000rpm for 10min, discarding the supernatant, and retaining the cells. 200ml of precooled STM solution is used for washing thalli, the thalli are evenly blown and sucked, the thalli are centrifuged for 3min at the temperature of 4 ℃ and the rpm of 5000, supernatant is discarded, and sediment is remained. The cells were resuspended in 100ml of ice-cold STM solution, centrifuged at 5000rpm at 4 ℃ for 3min, the supernatant was discarded and the pellet was retained. Resuspension with 50-200. mu.l ice-cold STM solution according to the bacterial loadAnd transferring the bacterial liquid into a centrifugal tube with high pressure, and carrying out ice bath to prepare for transformation.

(3) Electrotransformation of Hansenula polymorpha cells

Adding 15 mul of recombinant hansenula polymorpha expression plasmid and 30 mul of bacterial liquid according to the ratio of the plasmid to the thallus of 1: 2, fully and uniformly blowing and sucking, and placing in an ice bath for conversion; soaking in ethanol, irradiating with ultraviolet, refrigerating at-20 deg.C, taking out, and adding plasmid thallus mixed solution; carrying out electric shock according to the conditions of 2500V voltage, 150 omega resistance and 50 muF capacitance; after electric shock, 1ml of YPD solution which is balanced to room temperature is quickly added, and the YPD solution is gently mixed evenly and then transferred into an EP tube; placing the electrically transformed thallus in a water bath at 30 ℃ for 1h, and slightly reversing 3 times at intervals of 15 min; centrifuging the bacteria liquid incubated for 2h at 5000rpm for 10min, and discarding the supernatant; the cells were resuspended in 200. mu.l of YPD solution, plated on YPD plates containing 0.5mg/ml Zeocin at 100. mu.l/plate, and cultured by inversion at 30 ℃ for 3 to 7 days.

(4) Passage and stability of recombinant hansenula polymorpha expression strain

And (3) selecting a single colony of the recombinant strain growing on the Zeocin resistance plate, inoculating the single colony in 5ml of YPD liquid culture medium containing 0.5mg/ml Zeocin, carrying out shake culture at 30 ℃ and 200rpm for 24-48 hours until the OD value reaches 50, transferring the single colony into 5ml of YPD liquid culture medium containing 0.5mg/ml Zeocin according to the proportion of 1: 1000, culturing until the OD value reaches 50, transferring into 5ml of YPD liquid culture medium containing 0.5mg/ml Zeocin according to the proportion of 1: 1000, and repeating the steps for 10 times and preserving strains. The preservation system is bacterial liquid and 60% glycerol which are 1: 1, and the strains are preserved according to the required quantity, and the preservation system is usually 500 mul of bacterial liquid and 500 mul of 60% glycerol;

the recombinant Hansenula polymorpha expression strain which is passed 10 times is inoculated into 5ml YPD liquid culture medium without Zeocin resistance, after shaking culture is carried out at 30 ℃ and 200rpm until the OD value reaches 50, the strain is transferred into 5ml YPD liquid culture medium in the proportion of 1: 1000, and the like, and 5 times of continuous transfer is carried out in the YPD liquid culture medium without Zeocin resistance.

The stabilized bacterial solution was spread on YPD plates containing 16mg/ml G418, and cultured in an inverted state at 30 ℃ for 2 to 3 days.

Example 5: expression study of recombinant Hansenula polymorpha strains

Picking a plurality of recombinant hansenula polymorpha single colonies from YPD plates containing 16mg/ml G418, inoculating the single colonies into small test tubes containing 5ml YPG liquid culture medium, and culturing at 30 ℃ for 24 hours; transferring the bacterial liquid into a 100ml triangular flask containing 30ml YPM induction medium with initial density of OD₆₀₀After shaking at 30 ℃ for 72 hours, a methanol solution with a final concentration of 0.5% was added to the resulting suspension every 12 hours.

Transferring 10ml of induced bacterial liquid into a 50ml centrifuge tube, centrifuging at 10000rpm for 10min, and removing supernatant; 50ml of cell lysis is used for resuspending thalli precipitation, after the thalli precipitation is fully and uniformly mixed, the thalli is crushed in an ultrasonic instrument according to an ultrasonic crushing program of 60% of power, 20min of time, 5s of opening and 5s of closing, and ice bath is kept in the crushing process; the ultrasonicated bacterial liquid was centrifuged at 10000rpm for 10min, and after collecting the supernatant, SDS-PAGE was performed to detect the induction results (shown in FIG. 1) shown: the expression level of the high-expression recombinant hansenula polymorpha strain can reach more than 20 mug/ml.

Example 6: detection of copy number of exogenous polynucleotide in recombinant Hansenula polymorpha cells

(1) Extraction and quantification of Yeast genomic DNA

Inoculating 2 high-expression yeast strains obtained in the example 5 into a 5ml YPD liquid culture medium, and culturing at 30 ℃ for 16-24 h; taking 2ml of yeast culture solution, centrifuging at room temperature of 4500g for 3min, and collecting thalli; resuspending thallus with 500 μ L SCED solution (1mol/L sorbitol, 10mmol/L sodium citrate, 10mmol/L EDTA, 10mmol/L DTT dithiothreitol), adding 50mg glass beads, shaking for 5min, adding 50 μ L10 mg/ml lywallzyme, and warm-bathing at 37 deg.C for 1 h; adding 60 μ l of 10% SDS, 30 μ l of proteinase K, and 10 μ l of RNaseA enzyme, standing at room temperature for 10 minutes, and culturing in a water bath at 55 deg.C for 2 h; adding 350 μ l of saturated phenol and 350 μ l of chloroform, mixing thoroughly, centrifuging at 13000rpm for 10min, and collecting the supernatant; adding equal volume (about 700 μ l) of chloroform, centrifuging at 13000rpm for 10min, and collecting the supernatant after layering; add 140. mu.l of 3mol/L sodium acetate solution into the supernatant, mix gently, add 700. mu.l isopropanol, mix well, stand at room temperature for 5min, centrifuge at 13000rpm for 10 min. Discarding the supernatant, adding 1ml 70% ethanol for cleaningWashing, centrifuging at 13000rpm for 10 minutes, pouring off the supernatant, standing the DNA precipitate at room temperature for 30min, and adding 100. mu.l TE to dissolve. By measuring OD_260nmQuantification of genomic DNA was performed and the linearized fragments were diluted to 100 ng/. mu.l and stored in a-20 ℃ freezer for use.

(2) Quantification of copy number of exogenous polynucleotide by Southern blotting method

a: probe preparation

The MOX probe described in the Chinese patent application with the MOX probe application number of 201210021524.X applied in the invention is prepared by applying DIG DNA Labeling and Detection kit (Cat No: 11093657910) of Roche company. The method comprises the following specific steps: to an EP vial, 10. mu.l of PCR product (200 ng/. mu.l) of the MOX promoter region was added, sealed with a sealing film, and boiled in boiling water for 10 minutes. Immediately put into a small box of anhydrous ethanol (cooled suddenly) which was precooled (-20 ℃). Ethanol on the outer wall of the tube was wiped off, centrifuged (about 10 seconds), and 5. mu.l of water for endotoxin detection, 2. mu.l of 10XHexanucleotide Mix, 2. mu.l of 10 XdTP Labeling Mix, and 1. mu.l of 7Klenow Enzyme (labelinggrade) were sequentially added thereto, mixed well, and sealed with a sealing film. Water bath at 37 ℃ overnight, and storage at-20 ℃.

b: southern blotting

Southern blot detection was carried out using digoxin hybridization detection kit I (Cat No: DIGD-110) and HyB high-performance hybridization solution (Cat No: Hyb-500) from Beijing Meilebo medical science and technology, Inc., according to the product instructions.

The Southern blot assay results (as shown in FIG. 2) show that: the copy number of exogenous polynucleotides of high-expression recombinant hansenula polymorpha HP-1#/pRMHP2.1-31HP strains and HP-2#/pRMHP2.1-31HP strains obtained by screening a g418 resistant plate with the concentration of 16mg/ml exceeds 60. The dual screening strategy using zeocin antibody screening in combination with g418 resistance screening, particularly using g418 resistance plates at concentrations up to 16mg/ml, was shown to help obtain recombinant strains with high copy integration and high expression of HPV31L1 protein.

Example 7: fermentation process of HPV31L1 recombinant hansenula polymorpha expression strain

Fermenting the seed liquid: thawing 1 frozen glycerol strain (HP-1#/pRMHP2.1-31HP)Then, 50. mu.l of the solution is taken out and inoculated into 5ml of YPD medium, and shake cultivation is carried out at 30 ℃ and 200rpm for 20-24hr, A_600nmAbout 2-5, sucking 1ml of qualified YPD medium, inoculating into 2 bottles of 500ml YPD medium, shake culturing at 30 deg.C and 200rpm for 20-24hr to A_600nmAbout 15-20, and using the qualified product as fermentation seed liquid for later use.

The fermentation process comprises the following steps: the fermentation initiation medium contains 300g of yeast powder, 150g of peptone, 100g of glycerol and basic salt (K)₂SO₄273g，MgSO₄ 100g，85％H₃PO₄400ml of KOH 62g), 10L of purified water is fully dissolved, the solution is added into a 30L fermentation tank, the volume of the purified water is constant to 14L, the solution is sterilized at 121 ℃ for 30min, 60ml of PTM1 microelement liquid (CuSO) is added after the solution is cooled to 30 DEG C₄·5H₂O 6.0g，KI 0.088g，MnSO₄·H₂O 3.0g，Na₂MoO₄·2H₂O 0.2g，H₃BO₃ 0.02g，CoCl₂·6H₂O 0.5g，ZnCl₂ 20.0g，FeSO₄·7H₂O65.0 g, Biotin 0.2g, concentrated H₂SO₄5.0ml, purified water to 1L, 0.22 μm filter membrane filtration sterilization), adjusting pH to 5.6 with ammonia water, inoculating 1 bottle of 500ml fermented seed liquid, wherein the fermentation volume is 15L. The initial stirring speed is 200rpm, air flow rate is 0.5Nm3/hr, tank pressure is 0.5bar, and dissolved oxygen value is controlled to be 20-80% during fermentation. After the initial growth period is maintained for about 25hr, bacterial liquid A_600nmWhen the concentration reached about 20, the dissolved oxygen began to rise rapidly, and feeding of the feed medium (50% glycerol (W/V), 12ml PTM1) was started at a flow rate of 100ml/hr, at which time the fed-batch phase was started. Culturing for about 6-8hr to obtain bacterial liquid A_600nmWhen the pH value reaches about 90, the feeding is stopped, and the pH value is adjusted to 6.0 by ammonia water. After the dissolved oxygen begins to rise again, beginning to add methanol (containing 12ml/L PTM1) to enter an induction expression period, adjusting the temperature to 35 ℃ in the induction period, adjusting the initial methanol adding rate to be 50ml/hr, sampling every hour, measuring the methanol concentration by a methanol electrode, and controlling the methanol concentration to be less than 5g/L by adjusting the methanol adding rate.

Centrifuging in a lower tank and detecting expression quantity: inducing for 10hr, placing into tank, centrifuging at 4 deg.C and 5000rpm for 30min, and freezing at-20 deg.C. In addition, 10ml of fermentation liquid is taken and transferred into a 50ml centrifuge tube, centrifugation is carried out for 10min at 10000rpm, the supernatant is discarded, 50ml of cell lysate is used for resuspending thalli precipitation, ultrasonic disruption is carried out after full mixing, the ultrasonic disruption solution is used for carrying out Western blot detection, and the detection result of the Western blot is shown in figure 3: the whole induction period can be finished within 10 hours, and the expression quantity of the fermentation liquor of HPV31 can approach 100 mu g/mL.

Example 8: research and optimization of purification process of HPV31L1 recombinant protein

And (3) crushing thalli: taking HPV31 expression wet thallus stored at-20 ℃, and adding 0.9% physiological saline according to the proportion of 10ml/g wet thallus for cleaning. After the thalli is washed, 20ml of buffer solution/g of wet thalli is added into a crushing buffer solution (containing 0.5mol/L NaCl, 0.02% Tween-80 and 0.05mol/L MOPS) to be fully dissolved, high-pressure homogenate is used for crushing, the crushing pressure is 1500bar, the circular crushing is carried out for 5-8 times, and the microscopic examination crushing rate is more than 90%. Centrifuging the thallus crushed solution at 4 deg.C at 10000rpm for 30min, collecting supernatant, adding 50% ammonium sulfate, precipitating for 30min, centrifuging at 4 deg.C at 10000rpm for 30min, and collecting supernatant.

A first step of chromatographic purification: 1) the conventional process comprises the following steps: the supernatant fluid of the crushed thallus filtered by 1 mu M is loaded on a chromatographic medium POROS 50HS, and the target protein is adsorbed on the chromatographic medium and is eluted by 0.5-1.5M NaCl in a gradient way; 2) the optimization process comprises the following steps: the supernatant of the cell disruption filtered at 1 μ M was applied to a chromatographic medium POROS XS and eluted with a gradient of 0.5M to 1.5M NaCl. The 2 different chromatographic media were used to adsorb HPV31L1 protein, and the column was washed with 0.5M NaOH after gradient elution with NaCl at different concentrations. SDS-PAGE detects the purification of HPV31L1 protein during chromatography, and the results are shown in FIG. 4A and FIG. 4B: 1) by applying a chromatography medium POROS 50HS, more than 50% of HPV31L1 protein is still remained and is not eluted after high-concentration NaCl is eluted; 2) using the chromatography medium POROS XS, only less than 20% of the HPV31L1 protein was not eluted after elution with high NaCl concentrations. The primary chromatography purification of the HPV31L1 protein is shown, and the chromatography medium POROS XS is favorable for the elution of the HPV31L1 protein, so that the yield of the HPV31L1 is greatly improved.

A second step of chromatographic purification: and loading the HPV31L1 protein after primary purification on a chromatography medium Macro-Prep ceramic hydroxyapatite (Type II, 40 mu m), binding the target protein on the chromatography medium, eluting by using a phosphate concentration gradient of 20-200 mM, separating the target protein from impurities, and collecting the eluted HPV31L1 protein (see figure 4C).

Example 9: purified HPV31L1 recombinant protein observed by transmission electron microscope

The purified HPV31L1 protein sample was diluted 3-fold with sterile water and dropped in a small drop onto a wax dish. And (3) taking the copper net to enable the surface with the supporting film to be in contact with the surface of the sample liquid, standing for 1min, taking out the copper net, absorbing the redundant liquid drops by using a filter paper strip, and slightly drying. A2% uranium acetate solution was dropped on a wax dish. The copper mesh with the adsorbed sample is placed on the surface of the dye solution (the sample is contacted with the dye solution), and is kept stand for 2 min. The copper mesh was removed, excess drops were removed with a filter paper strip, and dried under an incandescent lamp. The VLP particle morphology was observed using a JEOL-1400 model transmission electron microscope and photographed (results are shown in FIG. 5).

Example 10: immunogenicity study of HPV31L1 VLPs recombinantly prepared with Hansenula polymorpha

Use of the determination of the humoral immune potency ED₅₀(half effective dose) method of assessing immunogenicity of HPV31L1 VLPs

(1) Immunization of mice; 85 Balb/c female mice (purchased from institute of laboratory animals, national academy of medical sciences) 6 weeks old were kept at a clean level. The HPV31L1 protein samples were diluted at the required immunization doses into 6 groups, including 5 experimental groups and 1 control group (table 1). The immunization program is as follows: mice were sacrificed once each at weeks 0, 3, and 6, and 14 days after the last immunization and sera were isolated.

TABLE 1 grouping of mice

(2) The ELISA method for determining the serum positive conversion rate of HPV31L1 VLP immunized mice comprises the following specific steps: the E.coli recombinant HPV31L1 protein was diluted to 0.5. mu.g/ml with coating buffer, 0.1ml per well and overnight at 4 ℃. The next day wash buffer was washed 3 times and the residual liquid was drained off. Blocking with antibody diluent for 30min, washing with washing buffer solution for 3 times, drying, and detecting, or air drying and storing at 4 deg.C in a damp-proof manner. Diluting each mouse serum sample by 1: 10000 with sample diluent, taking 0.1ml to the coated reaction well, incubating for 1 hour at 37 ℃, and washing for 5 times. (blank, negative well control at the same time). 0.1ml of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1: 10000 in fresh is added to the reaction well, incubated at 37 ℃ for 30 minutes, washed 5 times, and washed with double distilled water for the last time. 0.1ml of the temporarily prepared TMB substrate solution was added to each reaction well, and color development was carried out at 37 ℃ for 10 minutes. The reaction was terminated by adding 50. mu.l of 0.05ml of 2M sulfuric acid to each reaction well. The OD of each well was measured on a microplate reader at 450nm (630nm as the reference wavelength) after zeroing with a blank control well. Cutoff value calculation and positive result judgment: cutoff value ═ negative control value × 2.1; the sample OD value > Cutoff value was judged to be positive.

(3) Humoral immune efficacy ED₅₀Is calculated by

Calculation of the humoral immune potency, ED, of HPV31L1 VLP based on the mouse positivity at various dose levels₅₀The value was 0.093. mu.g, showing that HPV31L1 VLP has good immunogenicity.

Sequence listing

<110> Beijing Anbaisheng Biotech Co., Ltd

Jiangsu Ruike Biotechnology Ltd

<120> method for producing HPV31L1 protein using hansenula polymorpha expression system

<130> I2019TC3622CB

<160> 9

<170> PatentIn version 3.3

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<211> 504

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<223> HPV31 L1 consensus sequence

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

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Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Thr Arg Thr Asn

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

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

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Lys Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp

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Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Glu Thr

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

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Pro Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Phe Asp

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

130 135 140

Arg Glu Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Leu

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Gly Cys Lys Pro Pro Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys

165 170 175

Ser Asn Asn Ala Ile Thr Pro Gly Asp Cys Pro Pro Leu Glu Leu Lys

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Glu Pro Tyr Gly Asp Thr Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met

245 250 255

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

260 265 270

Pro Thr Asp Leu Tyr Ile Lys Gly Ser Gly Ser Thr Ala Thr Leu Ala

275 280 285

Asn Ser Thr Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp

290 295 300

Ala Gln Ile Phe Asn Lys Pro Tyr Trp Met Gln Arg Ala Gln Gly His

305 310 315 320

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

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Thr Thr Arg Ser Thr Asn Met Ser Val Cys Ala Ala Ile Ala Asn Ser

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Asp Thr Thr Phe Lys Ser Ser Asn Phe Lys Glu Tyr Leu Arg His Gly

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Glu Glu Phe Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu

370 375 380

Ser Ala Asp Ile Met Thr Tyr Ile His Ser Met Asn Pro Ala Ile Leu

385 390 395 400

Glu Asp Trp Asn Phe Gly Leu Thr Thr Pro Pro Ser Gly Ser Leu Glu

405 410 415

Asp Thr Tyr Arg Phe Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Thr

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Ala Pro Gln Lys Pro Lys Glu Asp Pro Phe Lys Asp Tyr Val Phe Trp

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

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

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

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Ala Lys Arg Lys Lys Thr Lys Lys

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atgtcgctgt ggagaccatc tgaagccacc gtgtacctgc cacctgttcc ggtgagcaaa 60

gtcgtgtcca ctgatgagta tgtcacgaga accaacatct actaccacgc tggctcggcc 120

agacttctca cagttggtca tccatattac tcgattccga agtcggacaa tcccaagaag 180

attgtggttc cgaaagtctc tggcttgcag tacagagtgt tcagagtcag actgcctgat 240

ccaaacaagt ttggcttccc agacacctcc ttctacaatc cagagactca gagactggtc 300

tgggcatgtg ttggcttgga agtgggcaga ggtcaacctc ttggagttgg catctctgga 360

catccactcc tgaacaagtt tgatgacacc gagaactcga acaggtatgc tggtggacct 420

ggcaccgaca acagagagtg cattagcatg gactacaagc agacacaact gtgcttgctg 480

ggctgcaaac caccgatcgg tgaacactgg ggcaaaggat ctccatgctc caacaatgcc 540

atcactccag gtgactgtcc acctcttgag ctcaagaact cggtcattca ggatggagac 600

atggtggaca ctggctttgg tgccatggac tttacagcct tgcaagacac gaagagcaat 660

gttccactgg acatctgcaa ctcgatttgc aagtatcctg actacctgaa gatggtggct 720

gagccatatg gtgacacctt gttcttctac ctgagacgcg aacagatgtt tgtcagacac 780

ttcttcaaca ggtctggcac agttggtgag tcggtgccca ccgatctgta catcaaaggc 840

tcgggatcca ctgcaacctt ggcgaactcg acgtactttc cgacaccttc gggcagcatg 900

gttacctccg atgctcaaat cttcaacaaa ccatactgga tgcagagagc acaaggtcac 960

aacaatggca tctgttgggg aaatcagctg tttgtgactg ttgtcgacac cacgagatcg 1020

accaacatga gcgtgtgtgc tgccattgcg aactcggata ctacgttcaa gtcctcgaac 1080

ttcaaagagt atctcagaca tggagaagag tttgatcttc agttcatctt tcaactgtgc 1140

aagatcacat tgtctgcaga catcatgacc tacattcact ccatgaatcc tgccattctg 1200

gaggactgga acttcggtct cacgacccca ccttctggct cgcttgaaga cacataccgc 1260

tttgtgacct cgcaagccat cacgtgtcag aagactgctc cacagaaacc aaaggaagat 1320

ccattcaaag actatgtctt ttgggaggtg aatctgaaag agaagttctc tgcagacttg 1380

gatcagtttc ctcttggaag gaagttcctg ctccaagctg gctacagagc cagaccgaag 1440

ttcaaagcag gcaagagatc ggctccaagc gcctccacca cgacaccagc gaagcgcaag 1500

aagaccaaga aa 1512

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tttgtttttg tactttagat tgatgtc 27

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ggagacgtgg aaggacatac cgc 23

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gaagatctca atctccggaa tggtgatctg 30

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catcaatcta aagtacaaaa acaaaatgtc gctgtggaga ccatctgaag 50

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<223> HPV31 L1 reverse primer

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gcggtatgtc cttccacgtc tccttatttc ttggtcttct tgcgcttcgc 50

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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

Claims

1. A method of producing a recombinant hansenula polymorpha cell expressing human papillomavirus type 31L 1(HPV 31L 1) protein comprising the steps of:

2. The method of claim 1, wherein the amino acid sequence of the HPV31L1 protein is set forth in SEQ ID NO: 1.

3. the method of claim 2, wherein the nucleotide sequence encoding HPV31L1 protein is set forth in SEQ ID NO: 2.

4. the method of any one of claims 1-3, wherein the exogenous polynucleotide is operably linked to a promoter and a terminator in the expression construct.

5. The method of any one of claims 1-4, wherein the recombinant Hansenula polymorpha cells contain multiple copies of the exogenous polynucleotide.

6. A recombinant Hansenula polymorpha cell produced according to the method of any one of claims 1 to 5.

7. A method of producing HPV31L1 protein comprising the steps of:

i) culturing the recombinant hansenula polymorpha cell of claim 6 under conditions suitable for expression of the HPV31L1 protein; and

ii) recovering and purifying the HPV31L1 protein from the culture.

8. The method of claim 7, wherein step i) comprises inducing expression of said HPV31L1 protein at 35 ℃.

9. The method according to claim 7 or 8, wherein said HPV31L1 protein is purified in step ii) using POROS XS chromatography medium.

10. Use of the HPV31L1 protein produced according to the method of any one of claims 7-9 in the manufacture of a vaccine for the prevention of HPV31 infection.