CN104120088B - Method for producing HPV58 L1 protein using Hansenula expression system - Google Patents

Abstract

The invention relates to a method for producing HPV58L1 protein by using a hansenula polymorpha expression system. Specifically, the invention discloses a method for generating recombinant hansenula polymorpha cells expressing HPV58L1 protein and the recombinant hansenula polymorpha cells generated by the method. The invention also discloses a method for producing the HPV58L1 protein by using the recombinant hansenula polymorpha cell and application of the produced HPV58L1 protein in preparation of a prophylactic vaccine.

Description

Method for producing HPV58 L1 protein using Hansenula expression system

field of invention

The invention belongs to the technical field of medical bioengineering, and relates to a method for producing HPV58L1 protein, in particular to a method for producing HPV58L1 protein with a Hansenula expression system.

Background technique

Human papillomavirus (human papillomavirus, HPV) is a non-enveloped closed-circle double-stranded DNA virus, belonging to the Papovaviridae polyomavirus subfamily, which mainly invades human epithelial mucosal tissues, and then induces various benign and malignant diseases. Proliferative lesions.

At present, more than 200 different subtypes of HPV have been identified. HPV infection has obvious tissue specificity. Different types of HPV have different tropisms for skin and mucous membranes, and can induce different papillary lesions. There are about 30 types. HPV types are associated with reproductive tract infections, and more than 20 of them are associated with tumors. According to the difference between benign and malignant HPV-induced lesions, HPV can be roughly divided into two categories: 1) High-risk types (such as HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, etc.) : High-risk HPV is closely related to various human tissue malignancies, mainly causing severe dysplasia and invasive cancer. In the world, the HPV types with the top two detection rates are HPV16 and HPV18; in mainland China The latest epidemiological statistics show that the infection rate of HPV52 and HPV58 has exceeded that of HPV18; 2) Low-risk types (such as HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV72, HPV81, etc.): low Dangerous HPV can cause benign proliferative venereal diseases of epidermal cells, such as condyloma acuminatum and flat condyloma plana, among which genital warts induced by HPV6 and HPV11 account for more than 90%.

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

The coat protein of HPV virus is capable of self-assembly, and the L1 protein expressed alone or co-expressed with the L1 protein and L2 protein in various expression systems can self-assemble into virus-like particles (virus-like particle, VLP). The VLP produced by yeast system, baculovirus insect expression system and mammalian cell expression system is closer to the structure of natural virus. The VLP produced by the exogenous expression system can induce the production of neutralizing antibodies in vivo after immunization, and obtain a good immune protection effect.

Hansenula Polymorpha, also known as Pichia augusta, is currently recognized as one of the most ideal exogenous gene expression systems. As a single-cell eukaryotic microorganism, Hansenula polymorpha not only has the characteristics of fast growth and easy genetic manipulation of prokaryotes, but also has the functions of post-translational processing and modification of eukaryotic cells. In addition, Hansenula has the advantages of good safety, easy cultivation, low cost, high expression level and genetic stability, and can overcome the instability of Saccharomyces cerevisiae strain, low yield and long glycosylation side chain And the low copy number of exogenous gene integration in Pichia Pastoris. At present, drugs (such as insulin, trade name Wosulin) and HBV vaccine (trade name Hepavax-Gene) produced using the Hansenula expression system have been marketed.

Chinese invention patent publications CN102586287A and CN102719453A disclose methods for expressing HPV16 and HPV18 type L1 VLPs using the Hansenula expression system. However, there is no public report on whether the L1 protein of HPV58 can be highly expressed in the Hansenula system and assembled into VLP particles. In the above two published patent applications, the result of the integrated copy number of the exogenous gene of HPV16 and HPV18L1 and the method for increasing the copy number of the exogenous gene were not given. In terms of the induced expression of foreign proteins, the induction time of HPV16 and HPV18L1 in the fermenter needs to exceed 20 hours, and no specific information on the protein expression level is provided. In addition, as an important indicator of protein purification, there is no disclosure about the purification process and the purity of the exogenous proteins HPV16 and HPV18L1 when the purification is completed.

In order to achieve high-efficiency expression of HPV58L1 protein in Hansenula and to purify and obtain high-purity HPV58VLP protein, in the present invention, a double screening strategy of Zeocin resistance screening combined with G418 resistance screening was applied, and a concentration as high as 16mg/ ml of G418 resistance plates to screen passaged and stabilized recombinant Hansenula expression strains. Through detection, the copy number of the highly expressed HPV58 strain obtained through screening can exceed 60 copies. During the fermentation induction process, the expression level of HPV58L1 protein can be significantly increased by increasing the induction expression temperature to 35°C. In addition, in view of the characteristic that POROS50HS, a commonly used chromatography medium in the VLP purification process, is not easy to elute when purifying HPV58 VLP (50% of the target protein still hangs on the column when the target protein is eluted with high concentration NaCl), the present invention uses POROS XS chromatography medium, The target HPV58L1 protein can achieve more than 80% protein elution when eluted with high concentration NaCl, thereby greatly increasing the yield of HPV58L1 protein.

Summary of the invention

In a first aspect, the present invention provides a method for producing recombinant Hansenula cells expressing HPV58L1 protein, comprising the steps of:

a) constructing an expression construct by inserting an exogenous polynucleotide comprising a nucleotide sequence encoding an HPV58L1 protein into a vector;

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

c) Screening the Hansenula cells obtained in step b), to obtain recombinant Hansenula cells containing the exogenous polynucleotide.

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

In a third aspect, the present invention also provides a method for producing HPV58L1 protein, comprising the following steps:

i) culturing the recombinant Hansenula cells of the present invention under conditions suitable for expression of the HPV58L1 protein; and

ii) recovering and purifying HPV58L1 protein from the culture.

In the last aspect, the present invention also provides the use of the HPV58L1 protein produced according to the method of the present invention in the preparation of a vaccine for preventing HPV58 infection.

Description of drawings

Figure 1 SDS-PAGE detection of recombinant Hansenula cell expression level. 1-8: Induced expression of 8 different recombinant Hansenula strains; 9: Standard product (10 μg/mL); 10: Protein molecular weight marker.

Fig. 2 Detection results of Southern blot using MOX promoter fragment as probe. Genomic DNA from ATCC26012 was used as a control. 1: Control (loading volume is 1000ng); 2: Control (loading volume is 500ng); 3: Control (loading volume is 250ng); 4: Control (loading volume is 125ng); 5: Highly expressed recombinant bacteria Genomic DNA of HP-1#/pRMHP2.1-58hp (the loading amount is 7.8ng, and its brightness is between 500ng and 1000ng control); 6: Highly expressed recombinant strain HP-2#/pRMHP2.1-58hp Genomic DNA (7.8ng loaded with brightness between 500ng and 1000ng controls).

Figure 3A: Western Blot detection of HPV58L1 protein expression during fermentation (expression induced at 30°C). 1: Pre-stained protein molecular weight marker; 2: Standard (10 μg/mL); 3: Induction for 1 hour; 4: Induction for 3 hours; 5: Induction for 5 hours; 6: Induction for 7 hours; 7: Induction for 9 hours; 8 : Induction for 10 hours.

B: Western Blot detection of HPV58L1 protein expression during fermentation (expression induced at 35°C). 1: Pre-stained protein molecular weight marker; 2: Standard (10 μg/mL); 3: Induction for 1 hour; 4: Induction for 3 hours; 5: Induction for 5 hours; 6: Induction for 7 hours; 7: Induction for 8 hours; 8 : 9 hours of induction; 9: 10 hours of induction.

Figure 4A: POROS50HS purified electropherogram of HPV58L1 protein. 1: Column sample; 2: Flow-through solution; 3-5: NaCl eluent with different concentrations; 6: Cleaning solution.

B: Electropherogram of POROS XS purification of HPV58L1 protein. 1: Column sample; 2: Flow-through solution; 3-5: NaCl eluent with different concentrations; 6: Cleaning solution; 7: Protein molecular weight marker.

C: Electropherogram of CHT purification of HPV58L1 protein. 1: Column sample; 2: Flow-through solution; 3-6: Phosphate eluent with different concentrations; 7: Cleaning solution; 8: Protein molecular weight marker.

Fig. 5 TEM observation results of purified HPV58L1 protein.

Detailed description of the invention

The present inventors have successfully established a method for producing HPV58L1 protein by using Hansenula yeast, and the produced HPV58L1 protein can self-assemble into virus-like particles, which can be used to prepare vaccines for preventing HPV infection.

The present invention firstly provides a method for producing recombinant Hansenula cells expressing HPV58L1 protein, which comprises the following steps:

a) constructing an expression construct by inserting an exogenous polynucleotide comprising a nucleotide sequence encoding an HPV58L1 protein into a vector;

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

c) Screening the Hansenula cells obtained in step b), to obtain recombinant Hansenula cells containing the exogenous polynucleotide.

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

The amino acid sequences of HPV58L1 proteins from different HPV58 virus strains will be different. The inventors compared all the HPV58L1 protein sequences in the database, selected the amino acid residue with the highest frequency of occurrence at each amino acid position of the HPV58L1 protein, and obtained the amino acid sequence shown in SEQ ID NO: 1, which is HPV58L1 The most representative consensus sequence of the protein. Therefore, the HPV58L1 protein in the present invention preferably has the amino acid sequence shown in SEQ ID NO:1.

In order to efficiently express the HPV58L1 protein using Hansenula, the inventors optimized the codons of the nucleotide sequence for Hansenula according to the amino acid sequence shown in SEQ ID NO:1. The optimization principles include: a) According to the Hansenula genetic code usage frequency table (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4905), select the most frequently used or higher codons; b) Avoid negative regulatory elements that have potential impact on gene transcription or protein translation, such as PolyAT region, PolyGC region, silencer (Sliencer) region and internal splicing sites, etc.; c) Contain 5' end UTR, HPV58L1 Comprehensive analysis of the mRNA secondary structure including the coding region and the 3' UTR to avoid the formation of complex RNA secondary structures and reduce the free energy of the mRNA secondary structure; The 5'UTR region of the natural sequence downstream of the promoter is completely consistent; e) Eliminate commonly used restriction endonuclease recognition sites. The optimized nucleotide sequence is shown in SEQ ID NO:2. The nucleotide sequence encoding HPV58L1 protein used in the present invention is preferably the sequence shown in SEQ ID NO:2.

The Hansenula expression vector applicable to the present invention is the Hansenula expression vector pRMHP2.1 (comprising the sequence shown in SEQ ID NO: 9) described in the Chinese patent application with application number 201210021524.X. The expression construct of the present invention can be obtained by cloning the exogenous polynucleotide comprising the nucleotide sequence encoding the HPV58L1 protein into the pRMHP2.1 vector. Those skilled in the art should understand that the expression construct of the present invention can also be constructed with other vectors, such as the vectors described in the authorized Chinese patent CN100400665C.

To enable expression 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 link between a promoter and another polynucleotide where the promoter sequence initiates and mediates transcription of the other polynucleotide. Operably linked includes 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, AOX1 and DHAS promoters. In some embodiments, the promoter used in the 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 expression constructs into Hansenula cells can be performed by 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 for expressing foreign proteins have been developed in the art, 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 cells used to transform the expression constructs of the invention are ATCC26012 Hansenula cells.

After transformation, recombinant Hansenula cells can be selected based on 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 auxotrophic media for 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 screening concentration of Zeocin may be 0.25, 0.5, 0.75, 1.0 or 1.5 mg/ml, preferably 0.5 mg/ml. The screening concentration of G418 used may be 2, 4, 8, 10, 12, 14, 16, 18 or 20 mg/ml, preferably 16 mg/ml.

The expression level of exogenous polynucleotide in Hansenula is positively correlated with its copy number in Hansenula. Therefore, recombinant Hansenula cells containing multiple copies of exogenous polynucleotides can also be further screened by methods such as Southern blotting or quantitative PCR. The recombinant Hansenula cells with exogenous polynucleotide copy number greater than 15 are preferred, and the recombinant Hansenula cells with exogenous polynucleotide copy number greater than 60 are more preferred.

The present inventors have surprisingly found that the combination of Zeocin and G418 to select recombinant Hansenula cells, especially the use of G418 up to 16 mg/ml for selection can obtain exogenous polynucleotide copy numbers greater than 15, especially greater than 60 Stable recombinant Hansenula cells.

In another aspect, the present invention also provides a method for producing HPV58L1 protein, comprising the following steps:

i) culturing the recombinant Hansenula cells of the present invention under conditions suitable for expression of the HPV58L1 protein; and

ii) recovering and purifying HPV58L1 protein from the culture.

Known in the art can be used for cultivating various media and basic culture conditions of Hansenula, the cultivation of the recombinant Hansenula expression strain of the present invention can be carried out in flasks or in bioreactors of different scales (such as 30L fermenter). According to the selected promoter, an appropriate inducer can be added in the culture to induce the expression of the HPV58L1 protein. In the case of using the MOX or FMD promoter, methanol can be added as an inducer. Yeast fermentation is usually carried out at 30°C, but the inventors surprisingly found that inducing protein expression in recombinant Hansenula cells of the present invention at 35°C can significantly increase the expression of foreign proteins.

The resulting HPV58L1 protein can be purified using various protein purification methods known in the art, such as salting out, ultrafiltration, precipitation, chromatography, etc. or a combination of these methods. In an optimized experimental scheme, the chromatographic medium POROSXS was used for preliminary purification, and then the chromatographic medium Macro-Prep ceramic hydroxyapatite (Type II, 40 μm) was used for further purification.

The purified HPV58L1 protein prepared by the method of the present invention can self-assemble into virus-like particles (Example 9, Figure 5), and shows good immunogenicity in mice (Example 10), so the present invention also provides The application of the HPV58L1 protein in preparing a vaccine for preventing HPV58 infection is disclosed.

Example

The present invention will be further illustrated by means of examples below, but the present invention is not limited to the scope of the described examples.

Example 1: Analysis of HPV58L1 Consensus Amino Acid Sequence

The full-length HPV58L1 protein consists of 498 amino acids. After searching GenBank, use the AlignX function of the Vector NTI software for amino acid sequence alignment analysis to obtain the most representative HPV58L1 consensus amino acid sequence (consensus amino acid sequence, that is, each HPV58L1 Amino acid positions all adopt the sequence of amino acid residues with the highest frequency of occurrence), the sequence of which is shown in SEQ ID NO:1.

Example 2: Optimal Design and Artificial Synthesis of HPV58L1 Encoding Gene

In order to efficiently express the HPV58L1 protein using Hansenula, the inventors optimized the codons of the nucleotide sequence for Hansenula according to the amino acid sequence shown in SEQ ID NO:1. The optimization principles include: a) select codons with the highest or higher frequency of use according to the Hansenula genetic code usage frequency table; b) avoid negative regulatory elements that have potential effects on gene transcription or protein translation, such as PolyAT region, PolyGC region, Silencer region and internal splicing sites, etc.; c) Comprehensive analysis of mRNA secondary structure including 5' end UTR, HPV58L1 coding region and 3' end UTR, to avoid the formation of complex RNA secondary structure, Reduce the free energy of the secondary structure of mRNA; d) Use the 5'UTR region that is completely consistent with the natural sequence downstream of the Hansenula promoter as far as possible upstream of the coding region; e) Eliminate commonly used restriction endonuclease recognition sites. The optimized nucleotide sequence is shown in SEQ ID NO:2.

According to the above nucleotide sequence, Beijing Nuosai Genome Research Center Co., Ltd. was commissioned to carry out the artificial synthesis of the full sequence, cloned it into a T vector (named T-58hp), and verified it by sequencing.

Example 3: Generation of expression constructs carrying the HPV58L1 nucleotide sequence

The Hansenula expression vector used in the present invention is the Hansenula expression vector pRMHP2.1 (SEQ ID NO: 9) described in the Chinese patent application with application number 201210021524.X.

(1) PCR amplification of MOX promoter and MOX terminator

Using the mixed genomic DNA of Hansenula strains ATCC26012 and ATCC34438 as a template, the following primers were used to amplify the MOX promoter with a size of 1518bp, and a NotI restriction site was introduced upstream;

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

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

Using the mixed genomic DNA of Hansenula strains ATCC26012 and ATCC34438 as a template, the following primers were used to amplify the MOX terminator with a size of 311bp, and at the same time, a BglII restriction site was introduced downstream;

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

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

(2) Generate an expression construct carrying the HPV58L1 nucleotide sequence

Using the recombinant plasmid T-58hp carrying 58hp as a template, use the following primers to amplify the HPV58L1hp gene with a size of 1545bp, and at the same time introduce the overlapping sequence at the 3' end of the MOX promoter region upstream, and introduce the overlapping sequence at the 5' end of the MOX terminator downstream Area;

58 upstream primer: 5'-CATCAATCTAAAGTACAAAAACAAAATGTCGGTGTGGAGACCATCTGAAG-3' (SEQ ID NO: 7)

58 downstream primers: 5'-GCGGTATGTCCTTCCACGTCTCCTTATTTCTTGACTTTCTTCCTCTTAGTG-3' (SEQ ID NO: 8)

Using the three fragments of MOX promoter, 58hp gene, and MOX terminator as a mixed template, use the following primers to amplify to obtain a 58hp expression cassette with a size of 3.4Kb. The amplified product carries a NotI restriction site in the upstream and a BglII in the downstream Restriction sites.

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

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

The 58hp expression cassette was cloned into the pRMHP2.1 vector by NotI+BglII double enzyme digestion to obtain the expression construct pRMHP2.1-58hp.

Example 4: Production of Recombinant Hansenula Cells

(1) Extraction and digestion of recombinant expression construct plasmid

Pick the Escherichia coli colony transformed into the recombinant expression construct plasmid obtained in Example 3, use the EZNA Plasmid Mini Kit kit (Omega Bio-Tek Company) to extract the plasmid after expansion and culture, and perform single enzyme digestion with BglII, apply EZNA Gel Extraction Kit kit (Omega Bio-Tek Company) was used for recovery, and 50 μL of sterile water preheated to 55°C was used for elution, DNA quantification was carried out by measuring OD ₂₆₀ , and the linearized fragment was diluted to 100 ng/μl, and stored Store in -20°C refrigerator for later use.

(2) Treatment of Hansenula cells

Pick a single colony of Hansenula strain ATCC26012, insert it into a small test tube containing 5ml of YPD liquid medium, and incubate at 30°C for 12 hours; take 5ml of the bacterial liquid and transfer it to 200ml of YPD medium, and incubate at 30°C for 4-6 hours , until the OD _600nm is about 1.0-1.5, centrifuge at 5000rpm for 10min; resuspend the bacteria in 200ml of 0.1mol/L phosphate buffer (containing 25mmol/L DTT, pH7.5), mix well, and incubate at 30°C for 30min , centrifuge at 5000rpm for 10min, discard the supernatant, and keep the bacteria. Wash the bacteria with 200ml of pre-cooled STM solution, blow and aspirate the bacteria evenly, centrifuge at 5000rpm at 4°C for 3min, discard the supernatant, and keep the precipitate. Resuspend the bacteria in 100ml of ice-cold STM solution, centrifuge at 5000rpm at 4°C for 3min, discard the supernatant and keep the precipitate. Resuspend the cells with 50-200 μl of ice-cold STM solution according to the amount of cells, and transfer the cell solution to a high-pressure centrifuge tube, put it in an ice bath, and prepare for transformation.

(3) Electrotransformation of Hansenula cells

According to the amount of plasmid: cell = 1:2, add 15 μl of recombinant Hansenula expression plasmid and 30 μl of bacterial solution, blow and aspirate fully, and place in an ice bath to be transformed; soak in alcohol in advance, and after ultraviolet irradiation, in - Take out the electric cup refrigerated at 20°C, and add the mixture of plasmid cells; conduct electric shocks under the conditions of voltage 2500V, resistance 150Ω, and capacitance 50μF; after electric shock, quickly add 1ml of YPD solution that has been equilibrated to room temperature, mix gently and transfer to In EP tube; place the electroporated bacteria in a water bath at 30°C for 1 hour, and gently invert 3 times at intervals of 15 minutes; centrifuge the bacteria solution that has been incubated for 2 hours at 5000 rpm for 10 minutes, discard the supernatant; resuspend in 200 μl YPD solution Bacteria were spread on YPD plates containing 0.5 mg/ml Zeocin at 100 μl/plate, and cultured upside down at 30°C for 3-7 days.

(4) Passage and stability of recombinant Hansenula expression strain

Pick a single colony of the recombinant strain grown on the Zeocin-resistant plate, inoculate it in 5ml of YPD liquid medium containing 0.5mg/ml Zeocin, and culture it on a shaker at 30°C and 200rpm for 24-48 hours until the OD value reaches 50 , transferred to 5ml of YPD liquid medium containing 0.5mg/ml Zeocin at a ratio of 1:1000, cultured until the OD value reached 50, and then transferred to 5ml of YPD liquid medium containing 0.5mg/ml Zeocin at a ratio of 1:1000 In the YPD liquid culture medium, by analogy, pass 10 times continuously and carry out the preservation of strain. The preservation system is bacterial liquid: 60% glycerol = 1:1, and the bacterial strain is preserved according to the required amount, usually 500 μl bacterial liquid + 500 μl 60% glycerol;

Inoculate the recombinant Hansenula expression strain that has been passed 10 times into 5ml of YPD liquid medium without Zeocin resistance, culture it on a shaker at 30°C and 200rpm until the OD value reaches 50, and transfer it at a ratio of 1:1000 In 5ml YPD liquid medium, and so on, in the YPD liquid medium without Zeocin resistance, continuously pass 5 times.

Spread the stabilized bacterial solution on a YPD plate containing 16 mg/ml G418, and culture it upside down at 30°C for 2-3 days.

Example 5: Expression studies of recombinant Hansenula strains

Pick multiple recombinant Hansenula single colonies from the YPD plate containing 16mg/ml G418, insert them into a small test tube containing 5ml of YPG liquid medium, and culture at 30°C for 24 hours; transfer the bacterial solution to a medium containing 30ml of YPM Put the induction medium in a 100ml Erlenmeyer flask with an initial density of OD ₆₀₀ =1, and induce it on a shaker at 30°C for 72 hours, and add methanol solution with a final concentration of 0.5% to the bacterial solution every 12 hours.

Take 10ml of the induced bacterial solution and transfer it to a 50ml centrifuge tube, centrifuge at 10,000rpm for 10min, discard the supernatant; use 50ml of cell lysate to resuspend the bacterial pellet, mix well, press "power 60%, Time 20min, on 5s, off 5s" ultrasonic crushing program to crush the bacteria, keep the ice bath during the crushing process; centrifuge the ultrasonic crushed bacterial liquid at 10000rpm for 10min, collect the supernatant and perform SDS-PAGE detection, and induce the results (As shown in Figure 1 ) shows: The expression level of the highly expressed recombinant Hansenula strain can reach above 25 μg/ml.

Example 6: Exogenous polynucleotide copy number detection of recombinant Hansenula cells

(1) Extraction and quantification of yeast genomic DNA

Inoculate 2 high-expression yeast strains obtained in Example 5 into 5ml of YPD liquid medium, culture at 30°C for 16-24h; take 2ml of yeast culture solution, and centrifuge at room temperature for 3min to collect the bacteria; apply 500μl of SCED solution (1mol /L sorbitol, 10mmol/L sodium citrate, 10mmol/L EDTA, 10mmol/L DTT dithiothreitol) to resuspend the bacteria, and add 50mg glass beads to fully shake for 5min, add 50μl 10mg/ml lysozyme, 37 Incubate at ℃ for 1 hour; add 60 μl of 10% SDS, 30 μl of proteinase K, and 10 μl of RNaseA enzyme, place at room temperature for 10 minutes and incubate in a water bath at 55°C for 2 hours; add 350 μl of saturated phenol and 350 μl of chloroform, mix thoroughly, and centrifuge at 13,000 rpm After 10 minutes, collect the stratified supernatant; add an equal volume (about 700 μl) of chloroform, centrifuge at 13,000 rpm for 10 minutes, collect the stratified supernatant; add 140 μl of 3 mol/L sodium acetate solution to the supernatant, gently After mixing gently, 700 μl isopropanol was added, after mixing, it was left at room temperature for 5 minutes, and centrifuged at 13000 rpm for 10 minutes. Discard the supernatant, add 1ml of 70% ethanol to wash, centrifuge at 13000rpm for 10 minutes, pour off the supernatant, leave the DNA pellet at room temperature for 30min, add 100μl TE to dissolve. Genomic DNA was quantified by measuring OD _260nm , and the linearized fragment was diluted to 100 ng/μl, and stored in a -20°C refrigerator for future use.

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

a: Probe preparation

The MOX probe used in the present invention is described in the Chinese patent application No. 201210021524.X, and the DIG DNA Labeling and Detection kit (Cat No: 11093657910) of Roche Company was used for probe preparation. The specific steps are as follows: add 10 μl of the PCR product (200ng/μl) of the MOX promoter region to the EP small tube, seal it with a parafilm, and boil it in boiling water for 10 minutes. Immediately put into a pre-cooled (-20°C) absolute ethanol small box (sudden cooling). Dry the outer wall of the tube with ethanol, centrifuge (about 10s), add 5 μl endotoxin test water, 2 μl 10x Hexanucleotide Mix, 2 μl 10x dTP Labeling Mixture, 1 μl 7Klenow Enzyme (labeling grade), mix well, and seal with parafilm. Store in a water bath at 37°C overnight, and store at -20°C.

b: Southern blot

Digoxigenin hybridization detection kit I (Cat No: DIGD-110) and HyB high-efficiency hybridization solution (Cat No: Hyb-500) from Beijing Milebo Medical Technology Co., Ltd. were used for Southern blot detection according to the product instructions.

The results of Southern blotting (as shown in Figure 2) show that: the highly expressed recombinant Hansenula HP-1#/pRMHP2.1-58hp strain and HP-2#/pRMHP2. 1-58hp strains have more than 60 copies of exogenous polynucleotides. It shows that the dual screening strategy of applying Zeocin antibody screening combined with G418 resistance screening, especially the use of G418 resistance plate screening with a concentration of up to 16 mg/ml helps to obtain recombinant strains with high copy integration and high expression of HPV58L1 protein.

Example 7: Fermentation process and optimization of HPV58L1 recombinant Hansenula expression strain

Fermentation seed liquid: Take 1 tube of frozen glycerol bacteria (HP-1#/pRMHP2.1-58hp), absorb 50μl into 5ml YPD medium after thawing, culture at 30℃, 200rpm shaker for 20-24hr, A _600nm approx. 2-5. After passing the test, take 1ml each and put it into two bottles of 500ml YPD medium, culture it on a shaker at 30°C and 200rpm for 20-24hrs until the A _600nm is about 15-20, and use it as a fermented seed solution after passing the test.

Fermentation process: 1) Conventional process: The initial fermentation medium contains 300g of yeast powder, 150g of peptone, 100g of glycerin, basic salt (K ₂ SO ₄ 273g, MgSO ₄ 100g, 85%H ₃ PO ₄ 400ml, KOH 62g), 10L purified Fully dissolve the water, add it to a 30L fermenter, set the volume to 14L with purified water, sterilize at 121°C for 30 minutes, cool to 30°C and add 60ml of PTM1 trace element solution (CuSO ₄ 5H ₂ O6.0g, KI0.088g, 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 O 65.0g, Biotin0 .2g, concentrated H ₂ SO ₄ 5.0ml, purified water to 1L, 0.22μm membrane filter to sterilize), ammonia water to adjust the pH to 5.6, inoculate a bottle of 500ml fermentation seed liquid, and the fermentation volume at this time is 15L. The initial stirring speed is 200rpm, the air flow rate is 0.5Nm3/hr, the tank pressure is 0.5bar, and the dissolved oxygen value is controlled at 20-80% during fermentation. After the initial growth period was maintained for about 25 hours, the A _600nm of the bacterial solution reached about 20, the dissolved oxygen began to rise rapidly, and the feed medium (50% glycerol (W/V), 12ml PTM1) was added at a flow rate of 100ml/hr. enters the fed-batch growth period. After cultivating for about 6-8 hours, the A _600nm of the bacterial solution reaches about 90, stop feeding, and adjust the pH value to 6.0 with ammonia water. After the dissolved oxygen starts to rise, start to add methanol (containing 12ml/LPTM1) to enter the induction expression period. The induction temperature is set to 30°C, the initial flow acceleration rate of methanol is 50ml/hr, and samples are taken every hour. The methanol electrode is used to measure the concentration of methanol. Methanol flow acceleration rate to control methanol concentration at <5g/L. 2) Optimizing process: The parameters of the fermentation process are basically the same as those described in the "conventional process", the difference is that the induction temperature is raised to 35°C under the induction condition.

Centrifugation in tanks and detection of expression levels: After 10 hours of induction, they were placed in tanks, centrifuged at 5000 rpm for 30 min at 4°C to collect wet cells, and stored at -20°C for later use. In addition, take 10ml of fermentation broth and transfer it to a 50ml centrifuge tube, centrifuge at 10000rpm for 10min, discard the supernatant, resuspend the bacterial pellet with 50ml of cell lysate, mix well and perform ultrasonication, and the ultrasonication solution is used for Western blot detection The results of Western blot detection are shown in Figures 3A and 3B: 1) Under normal process conditions, that is, induction at 30°C, the expression level of HPV58L1 protein was only about half of that of the control sample (10 μg/mL). times, so the expression level of HPV58L1 in the fermentation broth was 25 μg/mL. 2) Under the optimized process conditions, that is, induction at 35°C, the expression level of HPV58L1 protein was more than 2 times that of the control sample (10 μg/mL). Since the sample was diluted 5 times, the expression level of HPV58L1 in the fermentation broth was 100 μg/mL above. Therefore, during the fermentation induction process, the yield of HPV58L1 protein can be significantly increased by increasing the induction expression temperature to 35°C.

Example 8: Purification process research and optimization of HPV58L1 recombinant protein

Cell crushing: take HPV58 expressed wet cells stored at -20°C, and add 0.9% saline to wash at a ratio of 10ml/g wet cells. After the cells are washed, add 20ml of buffer solution/g of wet cells to the crushing buffer (containing 0.5mol/L NaCl, 0.02% Tween-80, 0.05mol/L MOPS) to fully dissolve, and use high-pressure homogenate to crush with a crushing pressure of 1500bar , cyclic crushing 5-8 times, and the crushing rate of microscopic inspection is >90%. The broken cell solution was centrifuged at 10,000 rpm for 30 min at 4°C, the supernatant was collected, and then 50% ammonium sulfate was added to precipitate for 30 min, then centrifuged at 10,000 rpm for 30 min at 4°C, and the supernatant was collected.

The first step of chromatographic purification: 1) Conventional process: the supernatant of the crushed bacteria filtered by 1 μm is loaded on the chromatographic medium POROS50HS, the target protein is adsorbed on the chromatographic medium, and eluted with a gradient of 0.5M to 1.5M NaCl ; 2) Optimizing process: The supernatant of the crushed bacteria filtered by 1 μm is loaded on the chromatographic medium POROS XS, and eluted with a gradient of 0.5M to 1.5M NaCl. The HPV58L1 protein was adsorbed with the above two different chromatographic media, and the column was washed with 0.5M NaOH after gradient elution with different concentrations of NaCl. SDS-PAGE was used to detect the purification of HPV58L1 protein during the chromatography process, and the results are shown in Figure 4A and Figure 4B: 1) After using the chromatography medium POROS50HS, after elution with high concentration NaCl, more than 50% of the HPV58L1 protein remained. 2) Using the chromatographic medium POROS XS, after elution with high concentration NaCl, less than 20% of the HPV58L1 protein was not eluted. It shows that in the preliminary chromatographic purification of HPV58L1 protein, the application of chromatographic medium POROS XS can facilitate the elution of HPV58L1 protein, thereby greatly increasing the yield of HPV58L1.

The second step of chromatographic purification: the HPV58L1 protein after preliminary purification is loaded on the chromatographic medium Macro-Prep ceramic hydroxyapatite (Type II, 40μm), and the target protein is bound to the chromatographic medium, and the protein is washed with 20-200mM phosphate Concentration gradient elution, the target protein was separated from impurities, and the eluted HPV58L1 protein was collected (see Figure 4C).

Embodiment 9: Observation of purified HPV58L1 recombinant protein by transmission electron microscope

Dilute the purified HPV58L1 protein sample 3 times with sterile water, and drop a small drop on the wax dish. Take the copper grid to make the surface with the supporting film contact with the surface of the sample liquid, let it stand for 1min, take it out, take out the copper grid, absorb excess liquid droplets with a filter paper strip, and let it dry slightly. Take 2% uranyl acetate solution and drop a small drop on the wax dish. The copper grid with the sample adsorbed is placed on the surface of the dye solution (the sample is in contact with the dye solution) and allowed to stand for 2 minutes. Take out the copper mesh, absorb the excess droplet with a filter paper strip, and dry it under an incandescent lamp. The morphology of VLP particles was observed and photographed with a JEOL-1400 transmission electron microscope (results are shown in Figure 5).

Example 10: Study on Immunogenicity of HPV58L1 VLP Prepared Recombinantly with Hansenula

Evaluation of immunogenicity of HPV58L1 VLP by measuring humoral immunity potency ED ₅₀ (half effective dose)

(1) Immunization of mice: 85 6-week-old Balb/c female mice (purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences) were kept in clean grade. They were divided into 6 groups, including 5 experimental groups and 1 control group, and the HPV58L1 protein samples were diluted according to the required immunization dose (Table 1). The immunization procedure was as follows: each immunization was performed once at 0, 3 and 6 weeks, and the rats were killed 14 days after the last immunization and the serum was separated.

Table 1 Grouping of mice

(2) ELISA was used to measure the seroconversion rate of mice immunized with HPV58L1 VLP. The specific steps were: dilute E. coli recombinant HPV58L1 protein to 0.5 μg/ml with coating buffer, add 0.1ml to each well, and leave overnight at 4°C. The next day, wash with washing buffer 3 times, and shake off the residual liquid. Block with antibody diluent for 30 minutes, wash with washing buffer 3 times, spin dry for detection, or dry and store at 4°C against humidity. Dilute each mouse serum sample at 1:10000 with sample diluent, take 0.1ml into the above-mentioned coated reaction well, incubate at 37°C for 1 hour, and wash 5 times. (At the same time, do blank and negative well controls). Add 0.1ml of HRP-labeled goat anti-mouse IgG secondary antibody freshly diluted in antibody diluent 1:10000 to the reaction well, incubate at 37°C for 30 minutes, wash 5 times, and wash with double distilled water for the last time. Add 0.1 ml of temporarily prepared TMB substrate solution to each reaction well, and develop color at 37°C for 10 minutes. 0.05 ml of 50 μl of 2M sulfuric acid was added to each reaction well to terminate the reaction. On the microplate reader, at 450nm (630nm is the reference wavelength), measure the OD value of each well after zeroing the blank control well. Cutoff value calculation and positive result judgment: Cutoff value = negative control value × 2.1; sample OD value > Cutoff value is judged as positive.

(3) Calculation of humoral immunity ED ₅₀

According to the calculation results of the positive rate of mice at different dose levels, the humoral immunity ED ₅₀ value of HPV58L1 VLP was 0.134 μg, showing that HPV58L1 VLP has good immunogenicity.

Claims (3)

1. A method for producing HPV58 L1 protein, comprising the following steps: a). Produce recombinant Hansenula cells expressing human papillomavirus type 58 L1 (HPV58 L1) protein; b). Cultivating the recombinant Hansenula cells obtained in a) under conditions suitable for expression of said HPV58 L1 protein; and c). recovering and purifying the HPV58 L1 protein from the culture; Wherein a) produces recombinant Hansenula cells expressing human papillomavirus type 58 L1 (HPV58 L1) protein by a method comprising the following steps: a1) constructing expression by inserting the exogenous polynucleotide sequence shown in SEQ ID NO:9 comprising the HPV58L1 protein coding sequence shown in SEQ ID NO:2 operably linked to the MOX promoter and MOX terminator construct; a2) transforming ATCC26012 Hansenula cells with the expression construct obtained in step a1); and a3) Screening the Hansenula cells obtained in step a2) with Zeocin and G418 to obtain recombinant Hansenula cells containing the exogenous polynucleotide with a copy number greater than 60; Wherein the cultivation in b) comprises the following steps b1) and b2): b1) Preparation of fermented seed liquid: Take 50 μl of the recombinant Hansenula cells and insert them into 5ml YPD medium, cultivate them on a shaker at 30°C and 200rpm for 20-24hr, A ₆₀₀ nm2-5, absorb 1ml each and insert them into 2 bottles of 500ml In YPD medium, culture at 30°C, 200rpm shaker for 20-24hr to A ₆₀₀ nm15-20, and use it as fermented seed liquid; b2) Fermentation: 500ml of fermented seed liquid is added to the initial fermentation medium in a 30L fermenter, and the fermentation volume is 15L; the initial stirring speed is 200rpm, the air flow rate is 0.5Nm ³ /hr, and the tank pressure is 0.5bar. The oxygen value is 20-80%; after the initial growth period is maintained for 25 hours, the A ₆₀₀ nm of the bacterial solution reaches 20, the dissolved oxygen begins to rise rapidly, and the feed medium is fed at a flow rate of 100ml/hr; after 6-8 hours of cultivation, the bacteria When the ₆₀₀ nm of solution A reaches 90, stop feeding, and adjust the pH value to 6.0 with ammonia water; after the dissolved oxygen starts to rise, start feeding methanol containing 12ml/L PTM1 to enter the induction expression period. The induction temperature is set at 35°C, and the initial flow of methanol is accelerated. The rate is 50ml/hr, sampling is performed every hour, the concentration of methanol is measured by the methanol electrode, and the methanol concentration is controlled at <5g/L by adjusting the acceleration rate of the methanol flow; after 10 hours of induction, it is put into the tank, and the wet bacteria are collected by centrifugation at 5000rpm for 30min at 4°C , stored at -20°C for later use; Wherein the recovery and purification in c) include the following steps c1) and c2): c1) Take HPV58-L1 expressed wet cells stored at -20°C, add 0.9% normal saline to wash according to the ratio of 10ml/g wet cells; after the cells are washed, add breaking buffer according to 20ml buffer solution/g wet cells Fully dissolve, use high-pressure homogenate to crush, crushing pressure 1500bar, crush 5-8 times in a cycle, and the crushing rate of microscopic examination is >90%; the cell crushing solution is centrifuged at 10000rpm for 30min at 4°C, and the supernatant is collected; c2) The 1 μm filtered supernatant of the crushed bacteria is loaded on the chromatographic medium POROS XS, eluted with a gradient of 0.5M to 1.5M NaCl, and the eluted HPV58 L1 protein is collected; the HPV58 L1 protein after preliminary purification The sample was prepared in the chromatographic medium Macro-Prep ceramic hydroxyapatite, the target protein was bound to the chromatographic medium, and eluted with a concentration gradient of 20-200 mM phosphate, the target protein was separated from impurities, and the eluted HPV58 L1 protein was collected. 2. The method according to claim 1, wherein the Hansenula cells obtained in step a2) are screened with 0.5 mg/ml Zeocin and 16 mg/ml G418 in step a3). 3. The method according to claim 1, wherein said screening in step a3) comprises 1) Spread the Hansenula cells obtained in step a2) on a YPD plate containing 0.5 mg/ml Zeocin, and culture them upside down at 30° C. for 3-7 days; 2) Pick a single colony of the recombinant strain grown on the Zeocin-resistant plate, inoculate it in 5ml of YPD liquid medium containing 0.5mg/ml Zeocin, and culture it on a shaker at 30°C and 200rpm for 24-48 hours until the OD value reaches After 50, transfer to 5ml YPD liquid medium containing 0.5mg/ml Zeocin at a ratio of 1:1000, culture until the OD value reaches 50, then transfer to 5ml containing 0.5mg/ml Zeocin at a ratio of 1:1000 In the YPD liquid culture medium, pass 10 times continuously; 3) Inoculate the recombinant Hansenula expression strain that has been passed 10 times into 5ml of YPD liquid medium without Zeocin resistance, culture it on a shaker at 30°C and 200rpm until the OD value reaches 50, and then use a ratio of 1:1000 Transplant in 5ml YPD liquid medium, pass 5 times continuously in YPD liquid medium without Zeocin resistance; and 4) Spread the obtained product on a YPD plate containing 16 mg/ml G418, culture it upside down at 30° C. for 2-3 days, and pick a single colony for copy number detection.