CN110305807B - Method for producing HPV33L1 protein by Hansenula polymorpha expression system - Google Patents

Abstract

The present invention relates to a method for producing HPV33L1 protein using Hansenula polymorpha expression system. Specifically, the present invention discloses a method for producing recombinant hansenula polymorpha cells expressing HPV33L1 protein and the recombinant hansenula polymorpha cells produced by the method. The invention also discloses a method for producing HPV33L1 protein by utilizing the recombinant hansenula polymorpha cells and application of the produced HPV33L1 protein in preparation of prophylactic vaccines.

Description

Method for producing HPV33L1 protein by Hansenula polymorpha expression system

The present application is a divisional application of the invention patent application of which the application date is 2013, 5, 17, application number is 201310183593.5 and the invention name is "method for producing HPV33L1 protein by Hansenula polymorpha expression system".

Technical Field

The invention belongs to the technical field of medical bioengineering, and relates to a method for producing HPV33L1 protein, in particular to a method for producing HPV33L1 protein by using a Hansenula polymorpha expression system.

Background

Human papillomavirus (human papillomavirus, HPV) is a non-enveloped, closed-loop, double-stranded DNA virus belonging to the subfamily polyomaviridae of papovaviridae, which mainly invades the epithelial mucosal tissue of the human body, thereby inducing various benign and malignant proliferative lesions.

Over 200 types of HPV have been identified at present, HPV infection has obvious tissue specificity, different types of HPV have different tropisms to skin and mucous membrane, different papillary lesions can be induced, about 30 types of HPV are associated with genital tract infection, and about 20 types of HPV are associated with tumors. HPV can be broadly divided into two categories, depending on the benign or malignant nature of the HPV-induced lesions: 1) High risk types (e.g., HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, etc.): high-risk HPV is closely related to human multiple tissue malignancies, mainly causing severe atypical hyperplasia and invasive carcinoma; 2) Low risk types (e.g., HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV72, HPV 81, etc.): low-risk HPV can cause benign proliferative diseases of epidermal cells, such as condyloma acuminatum and condyloma plana.

HPV is mainly composed of viral envelope and genomic DNA. The genome is about 7900bp long, and 8 viral protein coding genes exist. Among them, 6 proteins encoded by ORFs are expressed in the early stages of viral replication, called early proteins; the 2 ORF-encoded proteins are expressed in the late stages of viral replication, termed late stage proteins. Late proteins include major coat protein L1 and minor coat protein L2, and are involved in the formation of viral coat.

HPV viral coat proteins are capable of self-assembly, either as L1 proteins expressed alone in a variety of expression systems or when L1 proteins are co-expressed with L2 proteins, into virus-like particles (VLPs) which are more closely related to the structure of the native virus in the context of yeast systems, baculovirus insect expression systems, mammalian cell expression systems, and the like. The VLP produced by the exogenous expression system can induce and produce neutralizing antibodies in vivo after immunization, and a good immunoprotection effect is obtained.

Hansenula polymorpha (Hansenula Polymorpha), also known as Pichia augusta, is currently one of the most desirable exogenous gene expression systems accepted. Hansenula polymorpha is used as a single-cell eukaryotic microorganism, has the characteristics of rapid growth of prokaryotes, easy genetic operation and the like, and has the functions of post-translational processing, modification and the like of eukaryotic cells. In addition, hansenula polymorpha has the advantages of good safety, easy culture, low cost, high expression level, stable inheritance and the like, and can overcome the problems of unstable strain, low yield, overlong glycosylated side chains and low integrated copy number of exogenous genes of Pichia Pastoris (Pichia Pastoris) such as Saccharomyces cerevisiae (Saccharomyces cerevisiae). At present, drugs (such as insulin, trade name Wosulin) and HBV vaccine (trade name Hepavax-Gene) produced by using Hansenula polymorpha expression system are commercially available.

A method for expressing HPV16 and HPV18 type L1 VLPs using the Hansenula polymorpha expression system is disclosed in Chinese patent publication No. CN102586287A and No. CN 102719453A. However, there is no disclosure of whether the L1 protein of HPV type 33 can be expressed efficiently in Hansenula polymorpha system and assembled into VLP particles. In the above 2 published patent applications, the result of integrating copy numbers of exogenous genes of HPV16 and HPV 18L 1 and a method for increasing copy numbers of exogenous genes are not presented. In terms of the induced expression of the foreign proteins, the induction time of HPV16 and HPV 18L 1 in the fermentor is more than 20 hours, and no specific information on the protein expression level is provided. In addition, as an important protein purification index, purity of the foreign proteins HPV16 and HPV 18L 1 at the time of purification process and purification completion is not disclosed.

In order to achieve efficient expression of HPV33L1 protein in hansenula polymorpha and to enable purification of HPV33 VLP proteins of high purity, in the present invention a dual selection strategy of Zeocin resistance selection combined with G418 resistance selection is applied, in particular recombinant hansenula polymorpha expression strains passaged and stabilized using G418 resistance plates up to a concentration of 16mg/ml. The copy number of HPV33 high expression strain obtained by screening can exceed 60 copies through detection. In the fermentation induction process, the expression level of HPV33L1 protein can be remarkably improved by increasing the induction expression temperature to 35 ℃. In addition, aiming at the characteristic that the common chromatographic medium POROS50HS in the VLP purification process is not easy to elute (50% of target protein is still hung when the target protein is eluted by high-concentration NaCl), the invention uses the POROS XS chromatographic medium, so that the target HPV33L1 protein can be eluted by more than 80% of the protein when the target HPV33L1 protein is eluted by high-concentration NaCl, thereby greatly improving the yield of HPV33L1 protein.

Summary of The Invention

In a first aspect, the present invention provides a method of producing a recombinant hansenula polymorpha cell expressing HPV33L1 protein, comprising the steps of:

a) Constructing an expression construct by inserting an exogenous polynucleotide comprising a nucleotide sequence encoding HPV33L1 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 step b) to obtain recombinant hansenula polymorpha cells containing the exogenous polynucleotide.

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

In a third aspect, the invention also provides a method of producing HPV33L1 protein, comprising the steps of:

i) Culturing the recombinant hansenula polymorpha cells of the invention under conditions suitable for HPV33L1 protein expression; and

ii) recovering and purifying HPV33L1 protein from the culture.

In a final aspect, the invention also provides the use of HPV33L1 protein produced according to the method of the invention in the manufacture of a vaccine for preventing HPV33 infection.

Drawings

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

FIG. 2 shows the result of Southern blotting detection using the MOX promoter fragment as a probe. ATCC26012 genomic DNA was used as a control. 1: control (1000 ng loading); 2: control (500 ng loading); 3: control (250 ng loading); 4: control (125 ng loading); 5: genomic DNA of high-expression recombinant bacteria HP-1#/pRMHP2.1-33HP (loading amount is 7.8ng, and brightness is between 500ng and 1000ng control); 6: genomic DNA of recombinant bacteria HP-2#/pRMHP2.1-33HP (loading 7.8ng, brightness between 500ng and 1000ng control).

FIG. 3 Western Blot detection of HPV33L1 protein expression during fermentation (induced expression at 35 ℃). 1: induction for 0 hour; 2: induction for 1 hour; 3: inducing for 3 hours; 4: induction for 5 hours; 5: induction for 7 hours; 6: induction for 8 hours; 7: induction for 9 hours; 8: induction for 10 hours; 9: standard (10. Mu.g/mL); 10: pre-staining protein molecular weight markers.

Fig. 4A: POROS50HS purification electrophoresis pattern of HPV33L1 protein. 1: loading a column sample; 2: flowing through liquid; 3-5: naCl eluents with different concentrations; 6: cleaning liquid; 7: protein molecular weight markers.

B: POROS XS purification of HPV33L1 protein. 1: loading a column sample; 2: flowing through liquid; 3-6: naCl eluents with different concentrations; 7: cleaning liquid; 8: protein molecular weight markers.

C: CHT purification electrophoretogram of HPV33L1 protein. 1: loading a column sample; 2: flowing through liquid; 3,4: phosphate eluents of different concentrations; 5: cleaning liquid; 6: protein molecular weight markers.

FIG. 5 transmission electron microscopy of purified HPV33L1 protein.

Detailed Description

The present inventors have successfully established a method for producing HPV33L1 protein using Hansenula polymorpha, and the produced HPV33L1 protein can self-assemble into virus-like particles, which can be used for preparing vaccines for preventing HPV infection.

The present invention first provides a method for producing a recombinant hansenula polymorpha cell expressing HPV33L1 protein, comprising the steps of:

a) Constructing an expression construct by inserting an exogenous polynucleotide comprising a nucleotide sequence encoding HPV33L1 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 step b) to obtain recombinant hansenula polymorpha cells containing the exogenous polynucleotide.

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

The amino acid sequences of HPV33L1 proteins from different HPV33 strains may differ. The present inventors have obtained the amino acid residues with highest occurrence frequency at each amino acid position of HPV33L1 protein by aligning all HPV33L1 protein sequences in the database, as shown in SEQ ID NO:1, which is the most representative consensus sequence for HPV33L1 protein. Thus, HPV33L1 protein of the present invention preferably has the amino acid sequence of SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.

In order to efficiently express HPV33L1 protein using hansenula polymorpha, the inventors have identified the following sequences according to SEQ ID NO:1, and codon optimization of the nucleotide sequence against Hansenula polymorpha. The optimization principle comprises the following steps: a) The frequency table (http: /(www.kazusa.or.jp/codon/cgi-bin/showcode. Cgispies=4905) the highest or higher frequency of use codons; b) Negative regulatory elements such as PolyAT region, polyGC region, terminator region, and internal splice site, which avoid potential influence on gene transcription or protein translation; c) Comprehensively analyzing mRNA secondary structures comprising a 5 '-end UTR, an HPV33L1 coding region and a 3' -end UTR, avoiding the formation of complex RNA secondary structures and reducing the free energy of the mRNA secondary structures; d) A 5' UTR region which is identical as far as possible to the natural sequence downstream of the Hansenula promoter is employed upstream of the coding region; e) The usual restriction enzyme recognition sites are eliminated. The optimized nucleotide sequence is shown in SEQ ID NO:2. the nucleotide sequence encoding HPV33L1 protein used in the present invention is preferably SEQ ID NO:2, and a sequence shown in seq id no.

The Hansenula polymorpha expression vector which can be used in the invention is a Hansenula polymorpha expression vector pRMHP2.1 (comprising the sequence shown in SEQ ID NO: 9) described in the Chinese patent application with the application number 201210021524. X. The expression construct of the present invention can be obtained by cloning an exogenous polynucleotide comprising a nucleotide sequence encoding HPV33L1 protein into the prmhp2.1 vector. It will be appreciated by those skilled in the art that the expression constructs of the present invention may also be constructed with other vectors, such as those described in the issued chinese patent CN 100400665C.

To be able to express in Hansenula, the exogenous polynucleotide in the expression construct is operably linked to a promoter and a terminator.

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

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

Transformation of the expression construct into hansenula 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 hansenula strains have been developed in the art for expressing the foreign proteins including, but not limited to, CGMCC2.2498 Hansenula, ATCC34438 Hansenula, and ATCC26012 Hansenula cells. These hansenula strains can also be used in the present invention. In some embodiments, the hansenula cell used to transform the expression construct of the invention is an ATCC26012 hansenula cell.

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

The expression level of the exogenous polynucleotide in Hansenula is positively correlated with its copy number in Hansenula. Thus, further selection of recombinant Hansenula cells containing multiple copies of the exogenous polynucleotide can also be performed by Southern blotting or quantitative PCR. Preferably, the exogenous polynucleotide has a copy number greater than 15, more preferably, the exogenous polynucleotide has a copy number greater than 60.

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

In another aspect, the present invention also provides a method of producing HPV33L1 protein, comprising the steps of:

i) Culturing the recombinant hansenula polymorpha cells of the invention under conditions suitable for HPV33L1 protein expression; and

ii) recovering and purifying HPV33L1 protein from the culture.

Various media and minimal culture conditions useful for culturing Hansenula are known in the art, and can be selected or modified as desired by those skilled in the art. The cultivation of the recombinant Hansenula polymorpha expressing strain of the invention can be carried out in flasks or in bioreactors of different sizes (e.g.30L fermenters) depending on the amount of protein desired. Depending on the promoter selected, a suitable inducer may be added to the culture to induce expression of the HPV33L1 protein. In case of using MOX or FMD promoter, methanol may be added as inducer. Yeast fermentation culture is conventionally carried out at 30 ℃, and the inventors have surprisingly found that the induction of protein expression by the recombinant Hansenula polymorpha cells of the invention at 35 ℃ can significantly increase the expression level of exogenous proteins.

Purification of the HPV33L1 protein produced may use various protein purification means known in the art, such as salting out, ultrafiltration, precipitation, chromatography, etc., or a combination of these means. In an optimized protocol, a preliminary purification is first performed with the chromatographic medium POROS XS, followed by a further purification using the chromatographic medium Macro-Prep ceramic hydroxyapatite (Type II,40 μm).

The purified HPV33L1 protein prepared by the method of the present invention can self-assemble into virus-like particles (example 9, fig. 5) and show good immunogenicity in mice (example 10), and thus the present invention also provides the use of the HPV33L1 protein in the preparation of a vaccine for preventing HPV33 infection.

Examples

The invention will be further illustrated by means of examples which are not intended to limit the invention to the embodiments described.

Example 1: analysis of HPV33L1 consensus amino acid sequence

The full length HPV33L1 protein consists of 499 amino acids, after GenBank retrieval, the amino acid sequence comparison analysis is carried out by using Vector NTI software alignX function, and the most representative HPV33L1 consensus amino acid sequence (consensus amino acid sequence, namely the sequence of the amino acid residue with the highest occurrence frequency is adopted at each amino acid position of HPV33L 1) is obtained, and the sequence is shown as SEQ ID NO: 1.

Example 2: optimal design and artificial synthesis of HPV33L1 coding gene

In order to efficiently express HPV33L1 protein using hansenula polymorpha, the inventors have identified the following sequences according to SEQ ID NO:1, and codon optimization of the nucleotide sequence against Hansenula polymorpha. The optimization principle comprises the following steps: a) Selecting the codons with highest or higher use frequency according to the Hansenula genetic code use frequency table; b) Negative regulatory elements such as PolyAT region, polyGC region, terminator region, and internal splice site, which avoid potential influence on gene transcription or protein translation; c) Comprehensively analyzing mRNA secondary structures comprising a 5 '-end UTR, an HPV33L1 coding region and a 3' -end UTR, avoiding the formation of complex RNA secondary structures and reducing the free energy of the mRNA secondary structures; d) A 5' UTR region which is identical as far as possible to the natural sequence downstream of the Hansenula promoter is employed upstream of the coding region; e) The usual restriction enzyme recognition sites are eliminated. The optimized nucleotide sequence is shown in SEQ ID NO:2.

based on the above nucleotide sequences, the complete sequence synthesis by Beijing nuoxel genome research center Co., ltd was commissioned, cloned into a T vector (designated as T-33 hp), and sequencing verified.

Example 3: production of expression constructs carrying HPV33L1 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

Taking mixed genome DNA of Hansenula polymorpha strains ATCC26012 and ATCC34438 as templates, amplifying by using the following primer pair to obtain a MOX promoter with the size of 1518bp, and introducing a NotI enzyme cutting site at the upstream;

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

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

Taking mixed genome DNA of Hansenula polymorpha strains ATCC26012 and ATCC34438 as templates, 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) produces an expression construct carrying the HPV33L1 nucleotide sequence

Amplifying by using a recombinant plasmid T-33hp carrying 33hp as a template and using the following primer pair to obtain HPV33L1 hp gene with the size of 1548bp, introducing an overlapping sequence at the 3 'end of a MOX promoter region at the upstream side, and introducing an overlapping region at the 5' end of a MOX terminator at the downstream side;

33 upstream primer:

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

33 downstream primer: 5'-GCGGTATGTCCTTCCACGTCTCCTTATTTCTTGACTTTCTTTCTCTTGGC-3' (SEQ ID NO: 8)

The three fragments of MOX promoter, 33hp gene and MOX terminator are used as mixed templates, and the following primer pair is used for amplification to obtain a 33hp expression cassette with the size of 3.4Kb, wherein the amplified product carries a NotI enzyme cutting site at the upstream and a BglII enzyme cutting site at the downstream.

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

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

The expression construct pRMHP2.1-33hp was obtained by cloning the 33hp expression cassette into pRMHP2.1 vector by NotI+BglII double cleavage.

Example 4: production of recombinant Hansenula polymorpha cells

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

Coli colonies transformed into the recombinant expression construct Plasmid obtained in example 3 were picked up, the Plasmid was extracted using E.Z.N.A Plasmid Mini Kit (Omega Bio-Tek Co.) after the amplification culture, and single cleavage was performed using BglII, recovery was performed using E.Z.N.A Gel Extraction Kit Kit (Omega Bio-Tek Co.), elution was performed with 50. Mu.L of sterile water preheated to 55℃and OD was measured ₂₆₀ DNA quantification was performed and linearized fragments were diluted to 100 ng/. Mu.l and stored in a-20℃refrigerator for further use.

(2) Treatment of Hansenula polymorpha cells

Selecting Hansenula polymorpha strain ATCC26012 single colony, inoculating into a small test tube containing 5ml of YPD liquid culture medium, and culturing at 30 ℃ for 12 hours; transferring 5ml of the bacterial liquid into 200ml YPD culture medium, culturing at 30deg.C for 4-6 hr to OD _600nm About 1.0 to about 1.5, and centrifuging at 5000rpm for 10min; the cells were resuspended in 200ml of 0.1mol/L phosphate buffer (containing 25mmol/L DTT, pH 7.5) and thoroughly mixedEven, incubate at 30℃for 30min, centrifuge at 5000rpm for 10min, discard supernatant, leave the cells. The thalli are washed with 200ml of precooled STM solution, the thalli are blown and sucked uniformly, the thalli are centrifuged for 3min at 5000rpm at 4 ℃, and the supernatant is discarded, and the sediment is left. The cells were resuspended in 100ml ice-cold STM solution and centrifuged at 5000rpm for 3min at 4℃and the supernatant was discarded leaving a pellet. The cells were resuspended in 50-200. Mu.l of ice-cold STM solution according to the cell amount, and the cells were transferred to a centrifuge tube after high pressure, and ice-bath was performed to prepare transformation.

(3) Electrotransformation of Hansenula polymorpha cells

15 μl of recombinant Hansenula polymorpha expression plasmid and 30 μl of bacterial liquid are added according to the mass material and the thallus=1:2, and the mixture is fully and uniformly blown and sucked and placed in an ice bath for transformation; soaking in alcohol in advance, irradiating with ultraviolet, taking out in an electric rotating cup refrigerated at-20deg.C, and adding plasmid thallus mixed solution; electric shock is carried out according to the conditions of 2500V, 150 omega resistance and 50 mu F capacitance; after electric shock, 1ml of YPD solution which is balanced to room temperature is quickly added, and the mixture is gently mixed and then transferred into an EP tube; placing the thallus after electric transformation in a water bath at 30 ℃ for 1h, and gently reversing for 3 times every 15 min; centrifuging the bacterial liquid incubated for 2 hours at 5000rpm for 10min, and discarding the supernatant; the cells were resuspended in 200. Mu.l of YPD solution and plated at 100. Mu.l/plate on YPD plates containing 0.5mg/ml Zeocin and incubated for 3-7 days at 30℃with inversion.

(4) Passage and stabilization of recombinant hansenula polymorpha expression strains

Single colonies of the recombinant strain growing on the Zeocin resistant plate are selected, inoculated into 5ml of YPD liquid medium containing 0.5mg/ml Zeocin, shake-cultured at 30 ℃ for 24-48 hours at 200rpm, transferred into 5ml of YPD liquid medium containing 0.5mg/ml Zeocin in a ratio of 1:1000 until the OD value reaches 50, transferred into 5ml of YPD liquid medium containing 0.5mg/ml Zeocin in a ratio of 1:1000 after the OD value reaches 50, and continuously transferred 10 times and preserved by analogy. The preservation system is bacterial liquid and 60% glycerol=1:1, and the bacterial strain is preserved according to the required amount, and is usually 500 mu l bacterial liquid and 500 mu l 60% glycerol;

the recombinant Hansenula polymorpha expression strain transferred to 10 times was inoculated into 5ml of a Zeocin-resistant YPD liquid medium, shake-cultured at 30℃at 200rpm until the OD was 50, transferred into 5ml of a YPD liquid medium at a ratio of 1:1000, and similarly, transferred into a Zeocin-resistant YPD liquid medium for 5 times successively.

The stabilized bacterial liquid is coated on YPD plates containing 16mg/ml G418 and is cultured upside down at 30 ℃ for 2-3 days.

Example 5: expression study of recombinant hansenula polymorpha Strain

Multiple single recombinant Hansenula polymorpha colonies were picked from YPD plates containing 16mg/ml G418 and inoculated into small tubes containing 5ml YPG liquid medium for cultivation at 30℃for 24 hours; the bacterial solution was transferred to a 100ml Erlenmeyer flask containing 30ml YPM induction medium, and the initial density was OD ₆₀₀ =1, shaking table induction was performed at 30 ℃ for 72 hours, and methanol solution with a final concentration of 0.5% was added to the bacterial solution every 12 hours.

Transferring 10ml of the induced bacterial liquid into a 50ml centrifuge tube, centrifuging at 10000rpm for 10min, and discarding the supernatant; re-suspending the bacterial precipitate by 50ml cell lysis, fully mixing, and crushing the bacterial precipitate in an ultrasonic instrument according to an ultrasonic crushing program of 'power 60%, time 20min, 5s on and 5s off', wherein an ice bath is required to be maintained in the crushing process; the sonicated bacterial solution was centrifuged at 10000rpm for 10min, and the supernatant was collected and subjected to SDS-PAGE detection, and the induction results (shown in FIG. 1) showed that: the expression level of the recombinant hansenula polymorpha strain with high expression can reach more than 50 mug/ml.

Example 6: exogenous polynucleotide copy number detection of recombinant hansenula cells

(1) Extraction and quantification of yeast genomic DNA

Inoculating 2 high-expression yeast strains obtained in the example 5 to a 5ml YPD liquid culture medium, and culturing at 30 ℃ for 16-24 h; collecting 2ml yeast culture solution, centrifuging 4500g at room temperature for 3min, and collecting thallus; the cells were resuspended using 500. Mu.l of SCED solution (1 mol/L sorbitol, 10mmol/L sodium citrate, 10mmol/L EDTA, 10mmol/L DTT dithiothreitol), and 50mg of glass beads were added and thoroughly shaken for 5min, 50. Mu.l of 10mg/ml muramidase were added, and the incubation was performed at 37℃for 1h; mu.l of 10% SDS, 30. Mu.l of proteinase K, 10. Mu.l of RNaseA enzyme were added, and the mixture was left at room temperature for 10 minutes and then incubated in a water bath at 55℃for 2 hours; adding 350 μl of saturated phenol and 350 μl of chloroform, thoroughly mixing, centrifuging at 13000rpm for 10minCollecting the layered supernatant; adding equal volume (about 700 μl) of chloroform, centrifuging at 13000rpm for 10min, and collecting the supernatant after delamination; to the supernatant, 140. Mu.l of 3mol/L sodium acetate solution was added, followed by gentle mixing, 700. Mu.l of isopropyl alcohol was added, and after mixing, the mixture was left at room temperature for 5 minutes and centrifuged at 13000rpm for 10 minutes. The supernatant was discarded, 1ml of 70% ethanol was added for washing, the mixture was centrifuged at 13000rpm for 10 minutes, the supernatant was removed, and after the DNA precipitate was left at room temperature for 30 minutes, 100. Mu.l of TE was added for dissolution. By measuring OD _260nm Quantification of genomic DNA was performed and linearized fragments were diluted to 100 ng/. Mu.l and stored in a-20℃refrigerator for further use.

(2) Southern blotting method for quantification of copy number of exogenous polynucleotide

a: probe preparation

The MOX probe described in the Chinese patent application No. 201210021524.X of the MOX probe applied by the invention is prepared by using a DIG DNA Labeling and Detection kit kit (Cat No: 11093657910) of Roche company. The method comprises the following specific steps: to the EP vial, 10. Mu.l of PCR product (200 ng/. Mu.l) of the MOX promoter region was added, and the mixture was sealed with a sealing film and boiled in boiling water for 10 minutes. Immediately put into a pre-chilled (-20 ℃) anhydrous ethanol capsule (quench cooling). The outer wall of the tube was wiped dry with ethanol, centrifuged (about 10 s), and 5. Mu.l of endotoxin test water, 2. Mu.l of 10x Hexanucleotide Mix, 2. Mu.l of 10x dTP Labeling Mixture, 1. Mu.l of 7KlenoW Enzyme (1 absing grade), mixed well, and sealed with a sealing film. Water bath at 37 deg.c overnight and preservation at-20 deg.c.

b: southern blotting

Southern blot detection was performed using digoxin hybridization detection kit I (Cat No: DIGD-110) from Beijing Laibo medical science and technology Co., ltd.) and HyB high-efficiency hybridization solution (Cat No: hyb-500) according to the product specification.

Southern blot detection results (shown in FIG. 2) showed that: the high-expression recombinant Hansenula polymorpha HP-1#/pRMHP2.1-33HP strain obtained by screening on a g418 resistant plate at 16mg/ml had more than 60 exogenous polynucleotide copies of the HP-2#/pRMHP2.1-33HP strain. The use of zeocin antibodies to screen for dual selection strategies combined with g418 resistance screening, in particular g418 resistance plates at concentrations up to 16mg/ml, has been shown to help obtain recombinant strains with high copy integration and high expression of HPV33L1 protein.

Example 7: fermentation process of HPV33L1 recombinant Hansenula polymorpha expression strain

Fermenting seed liquid: taking 1 frozen glycerol strain (HP-1 #/pRMHP2.1-33 HP), thawing, sucking 50 μl, inoculating into 5ml YPD culture medium, shake culturing at 30deg.C and 200rpm for 20-24hr, A _600nm About 2-5, sucking 1ml of each of the qualified products into 2 bottles of 500ml YPD medium, shaking at 30deg.C and 200rpm for 20-24hr to A _600nm About 15 to 20, and the qualified fermentation seed liquid is used for standby.

The fermentation process comprises the following steps: the fermentation initiation medium contained 300g of yeast powder, 150g of peptone, 100g of glycerin, and basic salt (K) ₂ SO ₄ 273g，MgSO ₄ 100g，85％H ₃ PO ₄ 400ml, KOH 62 g), 10L of purified water is fully dissolved, the purified water is added into a 30L fermentation tank, the purified water is fixed to 14L, the temperature is 121 ℃ for 30min for sterilization, 60ml of PTM1 trace element liquid (CuSO) is added after cooling to 30 DEG C ₄ ·5H ₂ O 6.0g，KI 0.088g，MnSO ₄ ·H ₂ O 3.0g，Na ₂ MoO ₄ ·2H ₂ O 0.2g，H ₃ BO ₃ 0.02g，CoCl ₂ ·6H ₂ O 0.5g，ZnCl ₂ 20.0g，FeSO ₄ ·7H ₂ O65.0g,Biotin 0.2g, dense H ₂ SO ₄ 5.0ml of purified water to a volume of 1L, filtration sterilization with a 0.22 μm filter membrane), pH adjustment with ammonia water of 5.6, inoculation of 1 bottle of 500ml of fermentation seed liquid, and fermentation volume of 15L. The initial stirring speed is 200rpm, the air flow is 0.5Nm3/hr, the tank pressure is 0.5bar, and the dissolved oxygen value is controlled to be 20-80% in fermentation. After the initial growth period is maintained for about 25hr, bacterial liquid A _600nm When the amount reaches about 20, dissolved oxygen starts to rise rapidly, and the feed medium (50% glycerol (W/V), 12ml PTM1) starts to flow at a flow rate of 100ml/hr, and the feed medium enters the feed growth phase. Culturing for about 6-8hr to obtain bacterial liquid A _600nm Stopping feeding until the pH reaches about 90, and regulating the pH value to 6.0 by ammonia water. After dissolved oxygen starts to rise, methanol (containing 12ml/L PTM 1) starts to flow into the induction expression period, the temperature is adjusted to 35 ℃ in the induction period, the initial flow rate of methanol is 50ml/hr, and the methanol is taken every hourThe methanol concentration was measured by the methanol electrode and was controlled to < 5g/L by adjusting the methanol feed rate.

Centrifuging the lower tank and detecting the expression quantity: after induction for 10hr, the cells were placed in a jar and centrifuged at 5000rpm for 30min at 4℃to collect wet cells, which were stored at-20℃for further use. In addition, 10ml of fermentation broth was transferred to a 50ml centrifuge tube, centrifuged at 10000rpm for 10min, the supernatant was discarded, and the cell pellet was resuspended in 50ml of cell lysate, and after sufficient mixing, subjected to ultrasonication, and the ultrasonicated broth was subjected to Western blotting detection, the Western blotting detection results are shown in FIG. 3: the whole induction period can be ended within 10 hours, and the expression level of the fermentation liquor of HPV33 can reach more than 150 mug/mL.

Example 8: purification process research and optimization of HPV33L1 recombinant protein

And (3) thallus crushing: taking HPV33 expression wet thalli preserved at-20 ℃, adding 0.9% physiological saline into the mixture according to the proportion of 10ml/g wet thalli, and cleaning. After the thalli are washed, crushing buffer solution (containing 0.5mol/L NaCl,0.02 percent Tween-80 and 0.05mol/L MOPS) is added into 20ml buffer solution/g wet thalli for full dissolution, high-pressure homogenization and crushing are adopted, the crushing pressure is 1500bar, the cyclic crushing is carried out for 5-8 times, and the microscopic examination crushing rate is more than 90 percent. The cell disruption solution was centrifuged at 10000rpm for 30min at 4℃to collect the supernatant, and 50% ammonium sulfate was added thereto to precipitate for 30min, followed by centrifugation at 10000rpm for 30min at 4℃to collect the supernatant.

The first step of chromatographic purification: 1) The conventional process comprises the following steps: crushing the supernatant by filtering thalli with 1 μm, loading the supernatant on a chromatography medium POROS50HS, adsorbing target protein on the chromatography medium, and performing gradient elution by 0.5M-1.5M NaCl; 2) The optimization process comprises the following steps: the supernatant was crushed by 1 μm-filtered cells, applied to a chromatography medium POROS XS, and eluted with a 0.5M-1.5M NaCl gradient. HPV33L1 protein was adsorbed using the above 2 different chromatographic media, and the column was washed with 0.5M NaOH after gradient elution with different concentrations of NaCl. The purification of HPV33L1 protein during SDS-PAGE detection chromatography is shown in FIGS. 4A and 4B: 1) After the chromatography medium POROS50HS is used for eluting high-concentration NaCl, more than 50% of HPV33L1 protein still remains without eluting; 2) Using the chromatographic medium POROS XS, only less than 20% of HPV33L1 protein was not eluted after elution with high concentration of NaCl. The application of the chromatography medium POROS XS is favorable for the elution of HPV33L1 protein when the preliminary chromatography purification of HPV33L1 protein is carried out, thereby greatly improving the yield of HPV33L 1.

And a second step of chromatographic purification: the initially purified HPV33L1 protein is loaded on a chromatography medium Macro-Prep ceramic hydroxyapatite (Type II,40 μm), the target protein is bound on the chromatography medium, the target protein is eluted with a 20-200 mM phosphate concentration gradient, the target protein is separated from impurities, and the eluted HPV33L1 protein is collected (see FIG. 4C).

Example 9: purified HPV33L1 recombinant protein observed by transmission electron microscope

The purified HPV33L1 protein sample was diluted 3-fold with sterile water and placed drop-by-drop on a wax dish. And (3) taking the copper net, enabling the surface with the support film to be in contact with the surface of the sample liquid, standing for 1min, taking out the copper net, sucking excessive liquid drops by using a filter paper strip, and slightly airing. Taking 2% uranium acetate solution, and dripping one droplet on a wax tray. The copper mesh with the adsorbed sample is placed on the surface of the dye liquor (the sample is contacted with the dye liquor) and is kept stand for 2min. The copper net is taken out, redundant liquid drops are sucked by a filter paper strip, and the copper net is dried under an incandescent lamp. VLP particle morphology was observed and photographed using a JEOL-1400 model transmission electron microscope (results shown in FIG. 5).

Example 10: immunogenicity study of HPV33L 1VLP recombinantly produced with Hansenula polymorpha

Use of measuring humoral immune efficacy ED ₅₀ Method of evaluating immunogenicity of HPV33L1 VLPs (half effective dose)

(1) Immunization of mice: 85 female Balb/c mice (purchased from the institute of laboratory animals, national academy of sciences of medicine) were kept in clean grade. The HPV33L1 protein samples were diluted at the required immunizing dose (table 1) into 6 groups, including 5 experimental groups and 1 control group. The immunization procedure was: immunization was performed once at weeks 0, 3, and 6, mice were killed and serum was isolated 14 days after the last immunization.

TABLE 1 grouping of mice

(2) ELISA method for measuring serum positive transfer rate of HPV33L 1VLP immunized mice comprises the following specific steps: the E.coli recombinant HPV33L1 protein was diluted to 0.5. Mu.g/ml with coating buffer, added 0.1ml per well, overnight at 4 ℃. The next day the wash buffer was washed 3 times, and the residual liquid was thrown away. Blocking for 30min with antibody diluent, washing with washing buffer solution for 3 times, and detecting after spin-drying or preserving at 4deg.C after air-drying. Each mouse serum sample was diluted 1:10000 with the sample dilution, 0.1ml was placed in the coated reaction well, incubated at 37℃for 1 hour, and washed 5 times. (blank, negative Kong Duizhao simultaneously). An HRP-labeled goat anti-mouse IgG secondary antibody (0.1 ml) was added to the reaction wells in a fresh dilution of 1:10000, incubated at 37℃for 30 minutes, washed 5 times, and washed with double distilled water in the last pass. To each reaction well was added 0.1ml of a TMB substrate solution prepared temporarily, and the mixture was developed at 37℃for 10 minutes. To each reaction well was added 50. Mu.l of 2M sulfuric acid (0.05 ml) to terminate the reaction. The OD of each well was measured on a microplate reader at 450nm (630 nm is the reference wavelength) with the blank well zeroed. Calculating a Cutoff value and judging a positive result: cutoff value = negative control value x 2.1; and judging that the OD value of the sample is larger than the Cutoff value as positive.

(3) Humoral immunity efficacy ED ₅₀ Calculation of (2)

Humoral immune efficacy ED of HPV33L1 VLPs was calculated from the mouse positive rate at each of the different dose levels ₅₀ A value of 0.145. Mu.g indicates that HPV33L1 VLPs have good immunogenicity.

Sequence listing

<110> Beijing Anbaisheng biotechnology Co., ltd

JIANGSU RUIKE BIOTECHNOLOGY Co.,Ltd.

<120> method for producing HPV33L1 protein using Hansenula polymorpha expression system

<130> I2019TC3489CB

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 499

<212> PRT

<213> Artificial

<220>

<223> HPV33 L1 consensus sequence

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Phe Ser Ile Lys Asn Pro Asn Asn Ala Lys Lys Leu Leu Val Pro

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

65 70 75 80

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

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

100 105 110

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

115 120 125

Asp Thr Glu Thr Ser Asn Lys Tyr Pro Gly Gln Pro Gly Ala Asp Asn

130 135 140

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

145 150 155 160

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

165 170 175

Thr Asn Ala Ala Pro Ala Asn Asp Cys Pro Pro Leu Glu Leu Ile Asn

180 185 190

Thr Ile Ile Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met

195 200 205

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

210 215 220

Cys Gly Ser Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Thr Ser Glu

225 230 235 240

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

245 250 255

Val Arg His Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro

260 265 270

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

275 280 285

Ser Ala Phe Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Glu Ser

290 295 300

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

305 310 315 320

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

325 330 335

Thr Arg Ser Thr Asn Met Thr Leu Cys Thr Gln Val Thr Ser Asp Ser

340 345 350

Thr Tyr Lys Asn Glu Asn Phe Lys Glu Tyr Ile Arg His Val Glu Glu

355 360 365

Tyr Asp Leu Gln Phe Val Phe Gln Leu Cys Lys Val Thr Leu Thr Ala

370 375 380

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

385 390 395 400

Trp Gln Phe Gly Leu Thr Pro Pro Pro Ser Ala Ser Leu Gln Asp Thr

405 410 415

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

420 425 430

Pro Lys Glu Lys Glu Asp Pro Leu Gly Lys Tyr Thr Phe Trp Glu Val

435 440 445

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

450 455 460

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

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

485 490 495

Val Lys Lys

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<223> 33hp

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atgtcggtgt ggagaccatc tgaagccacc gtctacctgc cacctgttcc agtgtccaaa 60

gtggtctcga ccgatgagta tgtgagcagg acgtcgatct actactatgc tggctcctcg 120

agactcttgg cagttggtca tccgtacttc agcatcaaga accccaacaa tgccaagaaa 180

ctgcttgtgc caaaggtttc tggcttgcag tacagagtct tcagagtgag actgcctgat 240

ccaaacaagt ttggctttcc ggacacatcg ttctacaatc ctgacactca gagactggtc 300

tgggcctgtg ttggcttgga gattggcaga ggacaaccac tgggtgtggg catctctgga 360

catccactgc tcaacaagtt tgatgacaca gagacgtcca acaagtatcc tggtcagcca 420

ggtgctgaca acagagagtg tctgtcgatg gactacaagc agacacaact gtgcttgctc 480

ggctgcaaac cacctactgg agaacactgg ggcaaaggag ttgcctgcac caatgctgca 540

cctgcgaatg actgtccacc gcttgagctg atcaacacga tcattgagga tggtgacatg 600

gtggacactg gctttggatg catggacttc aagaccttgc aagccaacaa gtcggatgtt 660

ccgatcgaca tttgtggtag cacctgcaag tatcctgatt acctgaagat gacgtctgag 720

ccatatggag actccctgtt cttctttctc cgcagagaac agatgtttgt cagacacttc 780

ttcaacagag ctggcaaact tggtgaggca gttcctgacg atctgtacat caaaggctct 840

ggaaccactg cgtcgattca gtcctcggcc ttctttccga ccccaagcgg ctcgatggtg 900

acgtcggagt cccagttgtt caacaagccc tactggcttc agagagctca aggtcacaac 960

aatggcatct gttggggaaa ccaggtcttt gtgaccgttg tggacactac cagaagcacg 1020

aacatgacac tgtgcactca agtcacctcg gactccacgt acaagaatga gaacttcaaa 1080

gagtacattc gccacgttga agagtatgat ctccagttcg tgtttcagtt gtgcaaagtg 1140

accctgactg cagaagtgat gacctacatc catgccatga atccagacat tctggaagac 1200

tggcagttcg gtctgacacc tccaccgtct gcctcgttgc aggacacgta caggtttgtc 1260

acctcgcaag caatcacctg tcagaagaca gttcctccaa aggagaaaga agatccactt 1320

ggcaagtaca ccttctggga ggtggacctg aaagagaagt tctctgccga cctggatcag 1380

tttccgcttg gaaggaagtt tctgctccaa gctggcttga aagcgaagcc aaagctgaag 1440

agagctgcac caacttccac acgcacctcg tctgccaaga gaaagaaagt caagaaa 1497

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<212> DNA

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<223> MOX promoter forward primer

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aaggaaaaaa gcggccgcaa cgatctcctc gagctgctcg c 41

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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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<223> HPV33 forward primer

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catcaatcta aagtacaaaa acaaaatgtc ggtgtggaga ccatctgaag 50

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gcggtatgtc cttccacgtc tccttatttc ttgactttct ttctcttggc 50

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<223> pRMHP2.1

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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 (7)

1. A method of producing HPV33L1 protein, comprising the steps of:

i) Producing recombinant hansenula polymorpha cells expressing human papillomavirus type 33L1 (HPV 33L 1) protein;

ii) culturing said recombinant hansenula polymorpha cells under conditions suitable for expression of said HPV33L1 protein; and

iii) Recovering and purifying the HPV33L1 protein from the culture;

wherein step i) comprises the steps of:

a) Constructing an expression construct by inserting an exogenous polynucleotide comprising a nucleotide sequence encoding HPV33L1 protein into a vector having the sequence set forth in SEQ ID No. 9;

b) Transforming hansenula polymorpha cells with the expression construct obtained in step a); and

c) Screening the hansenula polymorpha cells obtained in step b) with a Zeocin resistance screen followed by a G418 resistance screen to obtain recombinant hansenula polymorpha cells containing the exogenous polynucleotide with a copy number greater than 60;

wherein the Hansenula polymorpha cells in step b) are ATCC26012 Hansenula polymorpha cells wherein the concentration of Zeocin is 0.5mg/ml and the concentration of G418 is 16mg/ml;

wherein in step iii) a preliminary purification is firstly carried out with a chromatography medium POROSXS, followed by a further purification with a chromatography medium Macro-Prep ceramic hydroxyapatite,

wherein the nucleotide sequence encoding HPV33L1 protein is shown in SEQ ID NO. 2.

2. The method of claim 1, wherein step i) comprises inducing expression of said HPV33L1 protein at 35 ℃.

3. The method of claim 1, wherein the amino acid sequence of HPV33L1 protein is set forth in SEQ ID No. 1.

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 claim 4, wherein the promoter is a MOX promoter.

6. The method of claim 4, wherein the terminator is a MOX terminator.

7. The method of any one of claims 1-3, 5 and 6, wherein the hansenula polymorpha cells are transformed by electroporation in step b).