CN115960178A

CN115960178A - Expression of human papilloma virus HPV59L1 protein, virus-like particle and preparation method thereof

Info

Publication number: CN115960178A
Application number: CN202211702927.0A
Authority: CN
Inventors: 陈晓; 高文双; 薛俊莲; 刘永江; 张海江; 张瑞霞; 陈丹; 伍树明; 沈迩萃; 张尧; 银飞; 王学红
Original assignee: Beijing Kangleweishi Biological Technology Co ltd
Current assignee: Beijing Kangleweishi Biological Technology Co ltd
Priority date: 2022-12-28
Filing date: 2022-12-28
Publication date: 2023-04-14
Anticipated expiration: 2042-12-28
Also published as: WO2024140162A1; CN115960178B

Abstract

The invention relates to the field of medical biology, in particular to expression of human papilloma virus HPV59 type L1 protein, virus-like particles and a preparation method thereof. Truncating the amino acid sequence of the HPV59 type L1 protein, performing codon optimization on the coding nucleotide sequence of the truncated protein to obtain an optimized coding nucleotide sequence, and finally realizing label-free expression and purification by matching with a label-free expression vector containing a specific SD sequence. The invention makes it possible to obtain higher protein expression amount in prokaryotic expression system, such as colibacillus expression system, and obtain VLP with homogeneous quality.

Description

Expression of human papilloma virus HPV59L1 protein, virus-like particle and preparation method thereof

Technical Field

The invention relates to the field of medical biology, in particular to construction and expression of human papilloma virus HPV59L1 protein VLP (virus-like particle).

Background

Human Papilloma Virus (HPV) is a non-enveloped closed-loop double-stranded DNA virus, belongs to the family of papovaviridae, the sub-family of polyomaviruses, and mainly invades epithelial mucosal tissues of a human body, thereby inducing various benign and malignant hyperplasia lesions. Over 200 different subtypes of HPV have been identified, HPV infection is clearly tissue specific, different types of HPV have different tropisms for skin and mucosa and can induce different papillary lesions, and approximately 30 HPV types are associated with genital tract infections, and more than 20 of them are associated with tumors.

Depending on the malignancy and the quality of the HPV-induced lesions, HPV can be roughly divided into two categories: 1) High risk types (e.g. HPV16, HPV18, HPV31, HPV33, HPV59, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, etc.): the high-risk HPV is closely related to various human tissue malignant tumors, and mainly causes severe atypical hyperplasia and invasive cancer; 2) Low risk types (e.g. HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV72, HPV81, etc.): the low-risk HPV can cause benign proliferative diseases of epidermal cells, such as condyloma acuminatum and flat condyloma. HPV is composed primarily of viral coat and genomic DNA. The genome is about 7900bp long and has 8 virus protein coding genes. Wherein the 6 ORF encoded proteins are expressed early in viral replication, referred to as early proteins; the 2 ORF-encoded proteins are expressed late in viral replication and are referred to as late proteins. The late proteins include major coat protein L1 and minor coat protein L2, and are involved in the formation of the viral coat. The HPV viral coat proteins can self-assemble, and currently in the patent literature, when a yeast expression system or an insect expression system or L1 protein expressed alone in a mammalian cell expression system or L1 protein and L2 protein are co-expressed, they can self-assemble into virus-like particles (VLPs), and after immunization, VLPs produced by an exogenous expression system can induce the production of neutralizing antibodies in vivo, so as to obtain a good immune protection effect. However, when assembling VLPs by direct expression in vivo using eukaryotic expression systems, the properties of VLPs produced are not very uniform, and the cost of eukaryotic expression systems is high, which is not conducive to industrialization.

At present, the HPV59 is reported in CN202110442661.X, a Hansenula polymorpha expression system is adopted to generate HPV59L1 protein, the Hansenula polymorpha expression system is a eukaryotic expression system and is directly assembled into VLP in vivo, whether the protein of a qualified standard can be normally expressed in an escherichia coli prokaryotic expression system is not provided in the patent, and the expression of HPV59L1 in the prokaryotic expression system has certain difficulty because the escherichia coli prokaryotic expression system does not have the functions of post-translational modification and the like of the Hansenula polymorpha expression system. Therefore, research is needed to solve the problem of difficulty in expressing HPV59L1 protein in prokaryotic expression systems to obtain more uniform VLPs and lower cost for industrial applications.

Disclosure of Invention

The inventor aims at the consideration of the cost of vaccine finished products, expresses the HPV59L1 protein in a prokaryotic expression system, and solves the problem of difficulty in expressing the HPV59L1 protein in the prokaryotic expression system. The method is realized by the following improvements: the amino acid sequence of the HPV59 type L1 protein is truncated, codon optimization is carried out on the coding nucleotide sequence of the truncated protein to obtain an optimized coding nucleotide sequence, and finally high-efficiency expression and purification are realized by matching with a tag-free expression vector containing a specific SD sequence.

Firstly, the amino acid sequence (SEQ ID NO: 1) of the HPV59L1 protein is subjected to N/C end truncation treatment so as to obtain a better protein expression rate. The N end is truncated by 4 amino acids, and the C end is truncated by 31 amino acids, and the specific truncated amino acids are shown in SEQ ID NO:2. after the N/C end truncation treatment, the protein and the VLP can be expressed on a label-free expression vector and higher quality can be obtained.

Wherein, the sequence shown in SEQ ID NO.2 is as follows:

1 MSSDNKVYLP PPSVAKVVST DEYVTRTSIF YHAGSSRLLT VGHPYFKVPK

51 GGNGRQDVPK VSAYQYRVFR VKLPDPNKFG LPDNTVYDPN SQRLVWACVG

101 VEIGRGQPLG VGLSGHPLYN KLDDTENSHV ASAVDTKDTR DNVSVDYKQT

151 QLCIIGCVPA IGEHWTKGTA CKPTTVVQGD CPPLELINTP IEDGDMVDTG

201 YGAMDFKLLQ DNKSEVPLDI CQSICKYPDY LQMSADAYGD SMFFCLRREQ

251 VFARHFWNRS GTMGDQLPES LYIKGTDIRA NPGSYLYSPS PSGSVVTSDS

301 QLFNKPYWLH KAQGLNNGIC WHNQLFLTVV DTTRSTNLSV CASTTSSIPN

351 VYTPTSFKEY ARHVEEFDLQ FIFQLCKITL TTEVMSYIHN MNTTILEDWN

401 FGVTPPPTAS LVDTYRFVQS AAVTCQKDTA PPVKQDPYDK LKFWPVDLKE

451 RFSADLDQFP LGRKFLLQLG ARP

secondly, in order to express HPV59L1 protein efficiently using the e.coli system, the inventors have expressed the HPV59L1 protein according to SEQ ID NO:2, and carrying out codon optimization of the nucleotide sequence aiming at an escherichia coli system. The optimization principle comprises the following steps: a) Selecting codons with highest or higher use frequency according to the Escherichia coli genetic code use frequency table; b) The commonly used restriction enzyme recognition sites were eliminated. The optimized nucleotide sequence is obtained by the principle and is screened for many times, and the optimized nucleotide sequence is shown as SEQ ID NO:3, further provides an expression cassette, an expression vector and a recombinant host cell containing the encoding nucleic acid. Preferably, it is Escherichia coli.

Wherein, the sequence shown in SEQ ID NO.3 is as follows:

1 ATGTCTTCTG ACAACAAAGT TTACCTGCCG CCGCCGTCTG TTGCTAAAGT

51 TGTTTCTACC GACGAATACG TTACCCGTAC CTCTATCTTC TACCACGCTG

101 GTTCTTCTCG TCTGCTGACC GTTGGTCACC CGTACTTCAA AGTTCCGAAA

151 GGTGGTAACG GTCGTCAGGA CGTTCCGAAA GTTTCTGCTT ACCAGTACCG

201 TGTTTTCCGT GTTAAACTGC CGGACCCGAA CAAATTCGGT CTGCCGGACA

251 ACACCGTTTA CGACCCGAAC TCTCAGCGTC TGGTTTGGGC TTGCGTTGGT

301 GTTGAAATCG GTCGTGGTCA GCCGCTGGGT GTTGGTCTGT CTGGTCACCC

351 GCTGTACAAC AAACTGGACG ACACCGAAAA CTCTCACGTT GCTTCTGCTG

401 TTGACACCAA AGACACCCGT GACAACGTTT CTGTTGACTA CAAACAGACC

451 CAGCTGTGCA TCATCGGTTG CGTTCCGGCT ATCGGTGAAC ACTGGACCAA

501 AGGTACCGCT TGCAAACCGA CCACCGTTGT TCAGGGTGAC TGCCCGCCGC

551 TGGAACTGAT CAACACCCCG ATCGAAGACG GTGACATGGT TGACACCGGT

601 TACGGTGCTA TGGACTTCAA ACTGCTGCAG GACAACAAAT CTGAAGTTCC

651 GCTGGACATC TGCCAGTCTA TCTGCAAATA CCCGGACTAC CTGCAGATGT

701 CTGCTGACGC TTACGGTGAC TCTATGTTCT TCTGCCTGCG TCGTGAACAG

751 GTTTTCGCTC GTCACTTCTG GAACCGTTCT GGTACCATGG GTGACCAGCT

801 GCCGGAATCT CTGTACATCA AAGGTACCGA CATCCGTGCT AACCCGGGTT

851 CTTACCTGTA CTCTCCGTCT CCGTCTGGTT CTGTTGTTAC CTCTGACTCT

901 CAGCTGTTCA ACAAACCGTA CTGGCTGCAC AAAGCTCAGG GTCTGAACAA

951 CGGTATCTGC TGGCACAACC AGCTGTTCCT GACCGTTGTT GACACCACCC

1001 GTTCTACCAA CCTGTCTGTT TGCGCTTCTA CCACCTCTTC TATCCCGAAC

1051 GTTTACACCC CGACCTCTTT CAAAGAATAC GCTCGTCACG TTGAAGAATT

1101 CGACCTGCAG TTCATCTTCC AGCTGTGCAA AATCACCCTG ACCACCGAAG

1151 TTATGTCTTA CATCCACAAC ATGAACACCA CCATCCTGGA AGACTGGAAC

1201 TTCGGTGTTA CCCCGCCGCC GACCGCTTCT CTGGTTGACA CCTACCGTTT

1251 CGTTCAGTCT GCTGCTGTTA CCTGCCAGAA AGACACCGCT CCGCCGGTTA

1301 AACAGGACCC GTACGACAAA CTGAAATTCT GGCCGGTTGA CCTGAAAGAA

1351 CGTTTCTCTG CTGACCTGGA CCAGTTCCCG CTGGGTCGTA AATTCCTGCT

1401 GCAGCTGGGT GCTCGTCCGT AA

finally, the invention provides tag-free expression vectors of specific SD sequences. As for an expression vector, the pGEX vector for expressing the fusion protein is characterized in that a glutathione S-transferase Gene (GST) with 26kDa is arranged on the vector, compared with other fusion vectors, the pGEX vector has the characteristics of mild purification conditions, simple steps, no denaturant addition and capability of keeping the spatial conformation and immunogenicity of the purified protein to the maximum extent; the GST fusion protein tag coded by the vector pGEX has a good application value, but the potential safety hazard of a medicinal protein product can be increased by the GST fusion protein tag coded by the vector pGEX. In contrast, the present invention removes the GST tag of the vector and replaces the SD sequence capable of efficiently expressing the HPV59 type L1 protein, thereby forming a novel expression vector suitable for the HPV59 type L1 protein. The shifted SD sequence was AGGAGGAATTA (5 'to 3').

The present invention also provides a method for preparing HPV type 59L1 VLPs, comprising the steps of: adjusting the pH and salt concentration of the buffer solution of the HPV59 type L1 protein obtained by the method to enable the protein to self-assemble into VLPs.

Preferably, the buffer includes, but is not limited to, tris buffer, phosphate buffer, acetate buffer, HEPES buffer, MOPS buffer, citric acid buffer, histidine buffer, boric acid buffer, preferably phosphate buffer;

the buffer has a pH of 5-5.75 and a salt concentration of 1.5-3.0M, preferably pH5, pH5.25, pH5.5, pH5.75; wherein the salt concentration is between 1.5M and 3.0M, preferably 1.5M,2.0M,2.5M,3.0M.

The invention makes it possible to obtain higher protein expression levels in prokaryotic, e.g., E.coli, expression systems and to obtain VLPs of more uniform quality through the above improvements.

Drawings

FIG. 1 XA90 pKL1-HPV59L1a small shake flask expression electrophoresis detection result (SD is not modified, namely SD sequence is AGGATATACAT). Wherein M is marker; XA90pKL1 negative control; 2.XA90 pKL1-HPV59L1-1 whole strain; supernatant of XA90 pKL1-HPV59L 1-1; XA90 pKL1-HPV59L1-1 precipitate; 5.HPV18L1, 6.XA90 pKL1-HPV59L1-2 whole bacteria; XA90 pKL1-HPV59L1-2 supernatant; XA90 pKL1-HPV59L1-2 precipitate. XA90 is a host bacterium, pKL1 is a vector with the SD sequence AGGATATACAT.

FIG. 2 protein expression assay results for different SD sequences. Wherein M is marker;1. non-induced XA90 pBSDm-59L1;2. inducing a whole bacterium XA90 pBSDm-59L1;3. induction of supernatant XA90 pBSDm-59L1;4. induction precipitation XA90 pBSDm-59L1. Non-induction XA90 pT1SDm-59L1;6. inducing a whole bacterium XA90 pT1SDm-59L1;7. induction of supernatant XA90 pT1SDm-59L1;8. induction precipitation XA90 pT2SDm-59l 1. No induction of XA90 pT2SDm-59L1;10. inducing whole bacteria XA90 pT2SDm-59L1;11. inducing supernatant XA90 pT2SDm-59L1;12. induction precipitation XA90 pT2 SDm-59l1. Control XA90 pKL1.

FIG. 3 shows the result M of electrophoretic detection of pentamer HPV59L1: marker;1 HPV59L1 pentamer.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The first embodiment is as follows: construction of tag-free expression vectors containing specific SD sequences

1. NdeI restriction sites were introduced into pGEX-6P-2 plasmid by mutation PCR:

the PCR primer names and sequences are as follows:

a forward primer: 6p1-NdeImut-F (5 'to 3'):

ATTTCA CACAGG AAACAG TACATA TGTCCC CTATAC TAGGTT ATTGGA AAATTA AG；

reverse primer: 6p1-NdeImut-R sequence (5 'to 3'):

ATAACC TAGTAT AGGGGA CATATG TACTGT TTCCTG TGTGAA ATTGTT ATCC。

the PCR reaction system is as follows: 5 XPisuion HF buffer 10 uL, ddH ₂ O30.5. Mu.L, 10mM dNTP 2. Mu.L, 6 PNE-SDm-F1. Mu.L, 6 PNE-SDm-R1. Mu.L, pGEX-6P-2 (diluted 20-fold) 5. Mu.L, phusion HF enzyme 0.5. Mu.L.

Setting a PCR reaction program: 3min at 95 ℃; 1min at 95 ℃, 1min at 55 ℃ and 10 min at 72 ℃; circulating for 20 times; 72 ℃ for 15 min.

The PCR product was digested with DpnI and transformed into E.coli DH 5. Alpha. And cultured overnight to obtain a single colony. And carrying out amplification culture on the single clone colony, sequencing the vector sequence in the single clone colony by a professional gene sequencing company, selecting a clone with a correct sequencing result, carrying out amplification on the clone, and extracting a plasmid from the clone to obtain the vector with the NdeI restriction enzyme site successfully introduced.

2. Designing a mutation PCR primer for replacing the SD sequence, and then replacing the SD sequence of the original vector by a PCR method

The primer information is as follows:

6PNE-SDm-F(5'to3')：CAATTTCACACAGGAGATATACATATGTCCCCTATACTAGG

6PNE-SDm-R(5'to3')：GTATAGGGGACATATGTATATCTCCTGTGTGAAATTGTTATCC

the PCR reaction system is as follows: 5 XPhusion HF bufferWash 10. Mu.L, ddH ₂ O30.5. Mu.L, 10mM dNTP 2. Mu.L, 6 PNE-SDm-F1. Mu.L, 6 PNE-SDm-R1. Mu.L, plasmid 5. Mu.L obtained in the 1.1 step, phusion HF Enzyme 0.5. Mu.L.

Setting a PCR reaction program: 3min at 95 ℃;95 deg.C for 1min,55 deg.C for 1min, and 72 deg.C for 10 min; circulating for 20 times; 72 ℃ for 15 min.

The PCR product was transformed into DNA by digesting the template DNA with DpnIE.coliIn DH 5. Alpha. A monoclonal colony was obtained after overnight culture. And carrying out amplification culture on the monoclonal colony, sequencing the vector sequence in the colony by a professional gene sequencing company, selecting a clone with a correct sequencing result, carrying out amplification on the clone, extracting a plasmid from the clone, and obtaining the vector for successfully replacing the SD sequence. The SD sequence after substitution is AGGAGATATA (5 'to 3').

3. NdeI and BamHI double enzyme digestion removal of GST gene

The enzyme digestion system is as follows: cutsmart buffer 3. Mu.l, ddH ₂ O3. Mu.l, 1.2. Mu.l of the vector obtained, ndeI 2. Mu.l, bamHI 2. Mu.l.

Carrying out enzyme digestion for 2h at 37 ℃;0.8% agarose gel electrophoresis, 120V,1h; and cutting the gel to obtain a corresponding electrophoresis band of the vector fragment with the GST gene removed, and storing at 4 ℃.

And (3) recovering the carrier fragment by using an agarose gel recovery kit, and detecting and recovering the result by taking 3 mu l of the obtained carrier fragment through electrophoresis. Then, the double-restriction enzyme products are filled with DNA polymerase I to fill the cohesive ends, and the reaction system is as follows: 10 XT 4 DNA ligase buffer 2.5. Mu.l, ddH ₂ O1.8. Mu.l, gel-recovered cleavage vector fragment 20. Mu.l, 10mM dNTP0.2. Mu.l, DNA polymerase I0.5. Mu.l, reaction at 25 ℃ for 15min, addition of EDTA (EDTA to a final concentration of 10 mM) and heating at 75 ℃ for 20min to terminate the reaction.

Carrying out reconnection cyclization on the vector subjected to enzyme digestion and with the complete tail end, wherein a connection system is as follows: 2. Mu.l of T4 DNA ligase buffer, 16. Mu.l of linear blunt-ended vector fragment, 2. Mu.l of T4 DNA ligase. Ligation was carried out at 16 ℃ for 4h.

Conversion of ligation product toE.coliIn DH 5. Alpha. A monoclonal colony was obtained after overnight culture. Amplifying and culturing the monoclonal colony, sequencing the vector sequence by a professional gene sequencing company,selecting the clone with correct sequencing result, then expanding the clone and extracting plasmid from the clone to obtain the vector for successfully replacing SD sequence and removing GST gene.

4.PCR amplification of the plasmid, reintroduction of NdeI and BamHI sites

The PCR primers were as follows:

6PNE-SDm-noG-F (5'to3')：CAGGAGATATACATATGGGATCCCCGGAATTCCCG

6PNE-SDm-noG-R (5'to3')：GAATTCCGGGGATCCCATATGTATATCTCCTGTGTG

the PCR reaction system is as follows: 5 XPhusion HF buffer 10. Mu.L, ddH ₂ O30.5. Mu.L, 10mM dNTP 2. Mu.L, 6 PNE-SDm-noG-F1. Mu.L, 6 PNE-SDm-noG-R1. Mu.L, template plasmid 5. Mu.L, phusion HF enzyme 0.5. Mu.L.

Setting a PCR reaction program: 3min at 95 ℃;95 ℃ for 1min,55 ℃ for 1min,72 ℃ for 10 min; circulating for 20 times; 72 ℃ for 15 min.

The PCR product was transformed into DNA by digesting the template DNA with DpnIE.coliIn DH 5. Alpha. A monoclonal colony was obtained after overnight culture. And carrying out amplification culture on the monoclonal colony, sequencing the vector sequence in the colony by a professional gene sequencing company, selecting a clone with a correct sequencing result, carrying out amplification on the clone, extracting a plasmid from the clone, obtaining a vector which successfully replaces an SD sequence, removing a GST gene, and reintroducing NdeI and BamHI enzyme cutting sites.

Through the improvement of different SD sequences (three SD sequences are screened, namely AGGAGGAATTTA, SD sequence 1, AGAGAGGTATATA, SD sequence 3.

Electrophoresis results by SDS-PAGE showed that the expression of the target protein in the vector without the sequence-modified SD sequence (AGGAGATATACAT) was significantly inferior to that in the vector with the sequence-modified SD sequence (AGGAGGAATTA) (as shown in FIG. 2). XA90 is a transformed host bacterium. As in FIG. 2, XA90pKL1 is a negative control. Compared with a negative control, an obvious expression band (indicated by an arrow) appears in a position of a XA90 pBSDm-59L1 (namely an expression vector with an improved SD sequence) test sample lane corresponding to the theoretical molecular weight of the target protein (about 52.89 KDa), and the unit thallus expression quantity of the target protein is about 0.3mg/g wet thallus. While the XA90 pT1SDm-59L1 (SD sequence is AGGAATAA) and XA90 pT2SDm-59L1 (SD sequence is AGAGGTATATA) test sample lanes show no expression band corresponding to the theoretical molecular weight of the target protein. The specific gene sequence information for the 3 SD sequences in the figure is as follows: SD sequence 1: AGGAGGAATTA, SD sequence 2: AGGAATAA, SD sequence 3: AGAGGTATATA.

The second embodiment: construction of expression vector containing codon-optimized HPV59L1 Gene

The wild type full-length amino acid sequence of human papilloma virus 59 type coat protein L1 (HPV 59L 1) is shown as SEQ ID NO:1 is shown. Truncating the gene fragment, namely truncating 4 amino acids at the N terminal and 31 amino acids at the C terminal, carrying out codon optimization on the coded nucleotide sequence, artificially synthesizing, specifically referring to SEQ ID No.3, carrying out PCR amplification on a DNA fragment of HPV59L1, carrying out NdeI/Xho1 double enzyme digestion on an L1 gene PCR fragment containing NdeI and Xho1 enzyme digestion sites and a recombinant vector respectively, and then carrying out ligation reaction on the recovered gene fragment and pKL1 (NdeI/Xho 1 double enzyme digestion and glue recovery) containing the corresponding cohesive ends by utilizing T4 DNA ligase at 16 ℃ of 10-15 h.

The linking system is as follows: pKL1 vector fragment 6. Mu.l, HPV59L1 gene fragment 2. Mu.l, T4 DNA ligase 1. Mu.l, T4 DNA ligase buffer 1. Mu.l. Conversion of ligation products toE. coli Selection of recombinants was performed in DH 5. Alpha. And carrying out amplification culture on the screened monoclonal colonies, extracting plasmids, and carrying out sequencing verification to obtain a recombinant expression vector pKL1-HPV59L1.

Example three: expression of HPV59L1 protein

And (3) transforming the recombinant vector-HPV 59L1 with correct sequencing result in the second embodiment into an Escherichia coli XA90 host cell, and expressing the HPV L1 protein as an engineering bacterium for expressing recombinant protein. 0.05% of the inoculum size was inoculated into LB medium (Amp +) and cultured at 37 ℃ and 220rpm for 16 hours for activation. Inoculating the activated bacterial liquid into a 2YT culture medium according to the inoculation amount of 0.5%, culturing at 30 ℃ and 220rpm for 7h, adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 0.2mM, performing induced culture at 30 ℃ and 220rpm for 16h, ending fermentation, and centrifugally collecting thalli for expression amount detection and purification experiments. By combining modification of the SD sequence with codon optimization and screening of three SD sequences, the protein expression vector which is not modified and optimized does not have obvious soluble expression, and the purification and recovery of the target protein are unsuccessful (namely, the target protein cannot be purified and obtained), which is equivalent to expression failure; the protein expression quantity is obviously improved after the SD sequence modification and optimization, and the purification and recovery of the antigen protein are facilitated in the supernatant, so that the soluble expression quantity of the human papilloma virus L1 antigen protein can be realized from nothing to nothing.

From the results of SDS-PAGE, the expression level of the target protein was very low in the vector which had not been modified with the sequence and SD sequence. The protein after the technical scheme improvement is soluble and highly expressed (figure 2, lanes 2 and 3), and the molecular weight of the HPV59L1 protein is 52.89 KDa.

Example four: HPV59L1 protein VLP purification and assembly

Taking a proper amount of thalli according to a mass-volume ratio of 1:10 (10) in a cell disruption buffer (20 mM PB,20 mM DTT, pH 8.0), and then crushing the cells under high pressure by a high pressure homogenizer under the following conditions: 800 bar,3 times. The disrupted cell solution was then subjected to high-speed centrifugation (4 ℃,12000 rpm,60 min) to collect the supernatant. The supernatant was further precipitated by ammonium sulfate at a saturation of 30%, and the precipitate was collected by centrifugation (4 ℃,12000 rpm,60 min) at a mass/volume ratio of 1:10 was sufficiently reconstituted with a reconstitution buffer (20 mM PB,20 mM DTT, pH 8.0) and the supernatant was collected by centrifugation (4 ℃,12000 rpm,60 min) to obtain a crude pure solution. The crude pure solution is loaded to Superdex200 molecular sieve chromatography, and the components of the crude pure solution are collected according to the peak position of the L1 target protein in a molecular sieve buffer solution (20 mM PB,20 mM DTT, pH8.0). Then, the molecular sieve collected sample is loaded to perform Source15Q anion exchange chromatography (SQ low salt buffer solution: 5 mM PB,10 mM DTT, pH8.0, SQ high salt buffer solution: 5 mM PB, 1M NaCl,10 mM DTT, pH 8.0), and a component of the L1 target protein is collected through linear elution of 0-20% high salt buffer solution and 10 column volumes, wherein the component is the purified L1 protein. The mass of the L1 pentamer was determined by Dynamic Light Scattering (DLS) method. Finally, the pH and salt concentration of the buffer in which the L1 protein is present are adjusted until it self-assembles to form VLPs, by which time the preparation of VLPs is complete. Finally the quality of VLPs was determined by DLS.

TABLE 1 DLS detection results before and after HPV 59-N9L 1 protein assembly

As is apparent from the results of the purification experiments shown in Table 1 above, when the pH of the assembly buffer is 5-5.75, preferably 5, 5.25, 5.5, 5.75, and the salt concentration is 1.5M-3.0M, preferably 1.5M,2.0M,2.5M,3.0M, the pentamer state of the novel HPV59L1 is good (PdI. Ltoreq.0.1), and VLPs in good state can be efficiently assembled (45 nm. Ltoreq. Particle size. Ltoreq.75 nm, pdI. Ltoreq.0.1), the result of electrophoretic detection of the HPV59L1 pentamer obtained by purification is shown in FIG. 3.

Example five Long-term stability of proteins

The HPV59L1 protein VLP prepared in example 4 was taken and subjected to investigation of long-term stability data at-70 ℃ and the results are as follows.

"-" indicates that there is no such provision or that the item has not been tested.

It can be seen that, after long-term investigation for 9 months, the appearance, pH value, VLP average particle size and dispersion coefficient, purity, in vitro potency, etc. of the antigen protein are not obviously changed, and the antigen protein is quite stable.

Claims

1. A truncated HPV type 59L1 protein which is truncated by 4 amino acids at its N-terminus and 31 amino acids at its C-terminus, based on a wild-type HPV type 59L1 protein; preferably, the amino acid sequence of the truncated HPV59 type L1 protein is shown in SEQ ID NO. 2.

2. Nucleic acid encoding a truncated HPV type 59L1 protein of claim 1; preferably, it is a codon optimized nucleic acid; more preferably, the nucleotide sequence is shown in SEQ ID NO. 3.

3. Nucleic acid comprising an SD sequence and a nucleotide sequence encoding a truncated HPV type 59L1 protein according to claim 2, preferably the SD sequence has a nucleotide sequence of 5 '-AGGAGGAATTA-3'.

4. An expression cassette or expression vector comprising the coding nucleic acid of claim 2 or 3.

5. The expression cassette or expression vector according to claim 4, which is a prokaryotic expression vector, more preferably a nucleic acid molecule of truncated HPV type 59L1 protein with the GST tag sequence removed and the SD sequence integrated on the basis of the vector pGEX.

6. A recombinant host cell comprising an expression cassette or expression vector encoding the nucleic acid of claim 2 or 3.

7. The recombinant host cell according to claim 6, characterized in that it is a prokaryotic cell, preferably E.coli.

8. A method of expressing a truncated HPV59 type L1 protein according to claim 1, comprising culturing a recombinant host cell according to claim 6 or 7 to produce an HPV59 type L1 protein, optionally comprising a purification step, preferably said purification step being: taking thalli of the recombinant host cell, fully suspending the thalli by using a bacterium breaking buffer solution, then carrying out high-pressure crushing on the thalli by using a high-pressure homogenizer, and centrifugally collecting a supernatant; precipitating the supernatant with ammonium sulfate to obtain a final saturation of 30%, re-dissolving the precipitate, centrifuging again, and collecting the supernatant to obtain a crude pure solution;

firstly, loading the crude pure solution to perform Superdex200 molecular sieve chromatography, and collecting the components of the crude pure solution according to the peak position of the L1 target protein;

and then, carrying out Source15Q anion exchange chromatography on the molecular sieve collected sample, and collecting the component of the L1 target protein through NaCl linear elution to obtain the HPV59 type L1 protein.

9. A method of expressing a truncated HPV type 59L1 protein according to claim 1, comprising the steps of: the step of adjusting the pH and salt concentration of the buffer in which the HPV type 59L1 protein is obtained according to the method of claim 8, such that it self-assembles into VLPs.

10. The method of claim 9, wherein the buffer includes, but is not limited to, tris buffer, phosphate buffer, acetate buffer, HEPES buffer, MOPS buffer, citric acid buffer, histidine buffer, boric acid buffer, preferably phosphate buffer;

the buffer has a pH of 5-5.75 and a salt concentration of 1.5-3.0M, preferably pH5, pH5.25, pH5.5, pH5.75; wherein the salt concentration is between 1.5M and 3.0M, preferably 1.5M,2.0M,2.5M,3.0M;

optionally, a step of purifying the resulting HPV59L1 pentamer is also included.