CN116041444B - Expression of human papilloma virus HPV39L1 protein, viroid particle and preparation method thereof - Google Patents

Abstract

The invention relates to the field of medical biology, in particular to expression of human papilloma virus HPV39 type L1 protein, viroid particles and a preparation method thereof. Truncating the amino acid sequence of HPV39 type L1 protein, carrying out codon optimization on the truncated protein coding nucleotide sequence to obtain an optimized coding nucleotide sequence, and finally matching with an unlabeled expression vector containing a specific SD sequence to realize unlabeled expression purification. The invention can obtain higher protein expression quantity in a prokaryotic expression system such as an escherichia coli expression system and obtain VLP with more uniform quality through the improvement.

Description

Expression of human papilloma virus HPV39L1 protein, viroid particle and preparation method thereof

Technical Field

The invention relates to the field of medical biology, in particular to construction and expression of human papillomavirus HPV39L1 protein VLP (virus-like particle).

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, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV39, 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, HPV81, 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. The HPV viral coat protein can be self-assembled, and in the patent literature, a yeast expression system or an insect expression system or an L1 protein expressed independently in a mammalian cell expression system or an L1 protein and an L2 protein are co-expressed to form a virus-like particle (VLP), and after immunization with VLP produced by an exogenous expression system, a neutralizing antibody can be induced in vivo, so that a good immune protection effect is obtained. However, the use of eukaryotic expression systems for direct expression of assembled VLPs in vivo is not very uniform in the nature of VLP production and the cost of eukaryotic expression systems is high and is not conducive to industrialization.

At present, aiming at HPV39 type, reported in CN202110981124.2, wherein an HPV39L1 protein is produced by using a Hansenula polymorpha expression system, the Hansenula polymorpha expression system is a eukaryotic expression system, VLP is directly assembled in vivo, and whether a qualified standard protein can be normally expressed in an escherichia coli prokaryotic expression system is not suggested in the patent, and the escherichia coli prokaryotic expression system does not have the functions of post-translational modification and the like of the Hansenula polymorpha expression system, so that the expression of HPV39L1 in the prokaryotic expression system is difficult to achieve. Thus, there is a need to address the problem of difficulties in expressing HPV39L1 protein within prokaryotic expression systems to obtain more uniform VLPs and lower costs for industrial applications.

Disclosure of Invention

The inventor aims at expressing HPV39L1 protein in a prokaryotic expression system based on the cost of vaccine finished products, and solves the problem that the HPV39L1 protein is difficult to express in the prokaryotic expression system. The method is realized by the following improvement: truncating the amino acid sequence of HPV39 type L1 protein, carrying out codon optimization on the truncated protein coding nucleotide sequence to obtain an optimized coding nucleotide sequence, and finally matching with a label-free expression vector containing a specific SD sequence to realize efficient expression and purification.

First, the invention discloses the method for preparing the HPV39L1 protein, which comprises the following steps: 1, and carrying out N/C end truncating treatment on the protein to obtain better protein expression rate. The N-terminal truncation is no more than 10 amino acids, preferably 9 amino acids. The C-terminal truncation is not more than 30 amino acids, preferably 29 amino acids, and the specific truncated amino acids are set forth in SEQ ID NO:2. after N/C terminal truncation treatment, the protein and VLP with higher quality can be expressed on a label-free expression vector.

Wherein, SEQ ID NO:2 is as follows:

1 MVYLPPPSVA KVVNTDDYVT RTGIYYYAGS SRLLTVGHPY FKVGMNGGRK

51 QDIPKVSAYQ YRVFRVTLPD PNKFSIPDAS LYNPETQRLV WACVGVEVGR

101 GQPLGVGISG HPLYNRQDDT ENSPFSSTTN KDSRDNVSVD YKQTQLCIIG

151 CVPAIGEHWG KGKACKPNNV STGDCPPLEL VNTPIEDGDM IDTGYGAMDF

201 GALQETKSEV PLDICQSICK YPDYLQMSAD VYGDSMFFCL RREQLFARHF

251 WNRGGMVGDA IPAQLYIKGT DIRANPGSSV YCPSPSGSMV TSDSQLFNKP

301 YWLHKAQGHN NGICWHNQLF LTVVDTTRST NFTLSTSIES SIPSTYDPSK

351 FKEYTRHVEE YDLQFIFQLC TVTLTTDVMS YIHTMNSSIL DNWNFAVAPP

401 PSASLVDTYR YLQSAAITCQ KDAPAPEKKD PYDGLKFWNV DLREKFSLEL

451 DQFPLGRKFL LQARVRR

secondly, in order to efficiently express HPV39L1 protein using the escherichia coli system, the inventors have identified the sequence according to SEQ ID NO:1, and codon optimization of the nucleotide sequence is performed on an escherichia coli system. The optimization principle comprises the following steps: a) Selecting codons with highest or higher use frequency according to a use frequency table of the escherichia coli genetic code; b) The usual restriction enzyme recognition sites are eliminated. The optimized nucleotide sequence is obtained through the principle and multiple screening, and the optimized nucleotide sequence is shown as SEQ ID NO:3, and further provides expression cassettes, expression vectors and recombinant host cells containing said coding nucleic acids. Preferably, it is E.coli.

Wherein, SEQ ID NO:3 is as follows:

1 ATGGTTTACC TGCCGCCGCC GTCTGTTGCT AAAGTTGTTA ACACCGACGA

51 CTACGTTACC CGTACCGGTA TCTACTACTA CGCTGGTTCT TCTCGTCTGC

101 TGACCGTTGG TCACCCGTAC TTCAAAGTTG GTATGAACGG TGGTCGTAAA

151 CAGGACATCC CGAAAGTTTC TGCTTACCAG TACCGTGTTT TCCGTGTTAC

201 CCTGCCGGAC CCGAACAAAT TCTCTATCCC GGACGCTTCT CTGTACAACC

251 CGGAAACCCA GCGTCTGGTT TGGGCTTGCG TTGGTGTTGA AGTTGGTCGT

301 GGTCAGCCGC TGGGTGTTGG TATCTCTGGT CACCCGCTGT ACAACCGTCA

351 GGACGACACC GAAAACTCTC CGTTCTCTTC TACCACCAAC AAAGACTCTC

401 GTGACAACGT TTCTGTTGAC TACAAACAGA CCCAGCTGTG CATCATCGGT

451 TGCGTTCCGG CTATCGGTGA ACACTGGGGT AAAGGTAAAG CTTGCAAACC

501 GAACAACGTT TCTACCGGTG ACTGCCCGCC GCTGGAACTG GTTAACACCC

551 CGATCGAAGA CGGTGACATG ATCGACACCG GTTACGGTGC TATGGACTTC

601 GGTGCTCTGC AGGAAACCAA ATCTGAAGTT CCGCTGGACA TCTGCCAGTC

651 TATCTGCAAA TACCCGGACT ACCTGCAGAT GTCTGCTGAC GTTTACGGTG

701 ACTCTATGTT CTTCTGCCTG CGTCGTGAAC AGCTGTTCGC TCGTCACTTC

751 TGGAACCGTG GTGGTATGGT TGGTGACGCT ATCCCGGCTC AGCTGTACAT

801 CAAAGGTACC GACATCCGTG CTAACCCGGG TTCTTCTGTT TACTGCCCGT

851 CTCCGTCTGG TTCTATGGTT ACCTCTGACT CTCAGCTGTT CAACAAACCG

901 TACTGGCTGC ACAAAGCTCA GGGTCACAAC AACGGTATCT GCTGGCACAA

951 CCAGCTGTTC CTGACCGTTG TTGACACCAC CCGTTCTACC AACTTCACCC

1001 TGTCTACCTC TATCGAATCT TCTATCCCGT CTACCTACGA CCCGTCTAAA

1051 TTCAAAGAAT ACACCCGTCA CGTTGAAGAA TACGACCTGC AGTTCATCTT

1101 CCAGCTGTGC ACCGTTACCC TGACCACCGA CGTTATGTCT TACATCCACA

1151 CCATGAACTC TTCTATCCTG GACAACTGGA ACTTCGCTGT TGCTCCGCCG

1201 CCGTCTGCTT CTCTGGTTGA CACCTACCGT TACCTGCAGT CTGCTGCTAT

1251 CACCTGCCAG AAAGACGCTC CGGCTCCGGA AAAAAAAGAC CCGTACGACG

1301 GTCTGAAATT CTGGAACGTT GACCTGCGTG AAAAATTCTC TCTGGAACTG

1351 GACCAGTTCC CGCTGGGTCG TAAATTCCTG CTGCAGGCTC GTGTTCGTCG

1401 TTAG

finally, the invention provides a label-free expression vector of a specific SD sequence. For the expression vector, the vector pGEX for expressing the fusion protein is characterized in that the vector is provided with a glutathione S-transferase Gene (GST) of 26kDa, and compared with other fusion vectors, the vector has the characteristics of mild purification condition, simple steps, no addition of denaturing agents and capability of maximally maintaining the spatial conformation and immunogenicity of the purified protein; the GST fusion protein tag has good application value, but the GST fusion protein tag coded by the vector pGEX can increase the potential safety hazard of medicinal protein products. In contrast, the GST tag of the vector is removed, and the SD sequence capable of efficiently expressing the HPV39 type L1 protein is replaced, so that a novel expression vector suitable for the HPV39L1 protein is formed. The SD sequence after the substitution was AGGAGATATA (5 'to 3').

The invention also provides a method for preparing HPV39 type L1 VLP, which is characterized by comprising the following steps: according to the step of HPV39 type L1 protein obtained by the method, the pH and salt concentration of the buffer solution are regulated, so that the HPV39 type L1 protein self-assembles to form 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 pH of the buffer is 4.75-5.25, the salt concentration is 2.0-4.0M, preferably pH4.75, pH5.0, pH5.25; wherein the salt concentration is between 2.0 and 4.0M, preferably 2.0M,2.5M,3.0M,3.5M,4.0M;

the invention can obtain higher protein expression in a prokaryotic, such as escherichia coli expression system and obtain VLPs with more uniform quality through the improvement.

Drawings

FIG. 1 XA90 pKL1-HPV39L1 vial expression electrophoresis detection results. Wherein M is marker; XA90pKL1 negative control; XA90 pKL1-HPV39L1-1 whole bacteria; XA90 pKL1-HPV39L1-1 supernatant; XA90 pKL1-HPV39L1-1 precipitate; 5.HPV18L1;6.XA90 pKL1-HPV39L1-2 whole bacteria; XA90 pKL1-HPV39L1-2 supernatant; XA90 pKL1-HPV39L1-2 precipitate.

FIG. 2 shows the results of detection of XA90 pKL1-HPV39-FL L1 by shake flask expression electrophoresis. Wherein M is marker; XA90pKL1 negative control; XA90 pKL1-HPV39-FL L1 whole bacteria; XA90 pKL1-HPV39-FL L1 supernatant; XA90 pKL1-HPV39-FL L1 precipitate; XA90 pKL1-HPV39-FL L1-2 whole bacteria; XA90 pKL1-HPV39-FL L1-2 supernatant; XA90 pKL1-HPV39-FL L1-2 precipitate.

FIG. 3 shows the results of detection of XA90pKL1-HPV 39-N9L 1 expression in shake flasks. Wherein M is marker; XA90pKL1 negative control; XA90 pKL1-HPV39L1N9-1 whole bacteria; XA90 pKL1-HPV39L1N9-1 supernatant; XA90 pKL1-HPV39L1N9-1 precipitate; XA90 pKL1-HPV39L1N9-2 whole bacteria; XA90 pKL1-HPV39L1N9-2 supernatant; XA90 pKL1-HPV39L1N9-2 precipitate; XA90pKL1 negative control.

FIG. 4 HPV 39-N9L 1 pentamer electrophoresis detection results. Wherein M is marker; HPV 39-N9L 1 pentamer.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Embodiment one: construction of a Label-free expression vector containing specific SD sequences

1. NdeI cleavage site was introduced into pGEX-6P-2 plasmid by mutation PCR:

the PCR primer names and sequences were as follows:

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 Xphusion HF buffer 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, pGEX-6P-2 (20-fold dilution) 5. Mu.L, phusion HF Enzyme 0.5.5. Mu.L.

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

The PCR product was digested with DpnI and then transformed intoE. coli In DH 5. Alpha. The monoclonal colonies were obtained after overnight culture. And (3) performing amplification culture on the monoclonal colony, sequencing a vector sequence in the monoclonal colony by a professional gene sequencing company, selecting a clone with a correct sequencing result, and performing cloning amplification and plasmid extraction on the clone to obtain a vector successfully introduced into NdeI restriction enzyme sites.

2. Designing mutation PCR primer for replacing SD sequence, and replacing SD sequence of original carrier by PCR method

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 buffer 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, 5. Mu.L of plasmid obtained in the previous step, and 0.5. Mu.L of Phusion HF enzyme.

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

The PCR product was transformed into a template DNA digested with DpnIE.coliIn DH 5. Alpha. The monoclonal colonies were obtained after overnight culture. Amplifying and culturing the monoclonal colony, sequencing the carrier sequence by special gene sequencing company, selecting the clone with correct sequencing result, amplifying and extracting the cloneTaking the plasmid to obtain the vector with the SD sequence replaced successfully. The SD sequence after substitution was AGGAGATATA (5 'to 3').

3. Vector was digested with NdeI and BamHI to remove 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 obtained vector, ndeI 2. Mu.l, bamHI 2. Mu.l.

Enzyme cutting at 37 ℃ for 2 hours; 0.8% agarose gel electrophoresis, 120V,1h; and (3) cutting gel to obtain a carrier fragment with GST genes removed, corresponding to the electrophoresis band, and preserving at 4 ℃.

The carrier fragment was recovered by using an agarose gel recovery kit, and 3. Mu.l of the obtained carrier fragment was subjected to electrophoresis detection and recovery. Then the double enzyme cutting product is used for filling the sticky end by DNA polymerase I, and the reaction system is as follows: 10×T4 DNA ligase buffer 2.5 μl, ddH2O1.8 μl, gel recovered cleaved vector fragment 20 μl,10mM dNTP0.2 μl, DNA polymerase I0.5 μl, reaction at 25deg.C for 15min, EDTA (final EDTA concentration of 10 mM) added and heating at 75deg.C for 20min to terminate the reaction.

And (3) carrying out religation cyclization on the carrier subjected to enzyme digestion and end-complemented, wherein a connection system is as follows: 2. Mu.l of T4 DNA ligase buffer, 16. Mu.l of linear blunt end vector fragment, 2. Mu.l of T4 DNA ligase. The connection is carried out at 16℃for 4h.

The ligation product is digested and then converted intoE.coliIn DH 5. Alpha. The monoclonal colonies were obtained after overnight culture. And (3) performing amplification culture on the monoclonal colony, sequencing a vector sequence in the monoclonal colony by a professional gene sequencing company, selecting a clone with a correct sequencing result, performing cloning amplification and plasmid extraction on the clone, and obtaining a vector for successfully replacing the SD sequence and removing the GST gene.

PCR amplified plasmid, reintroduced NdeI and BamHI cleavage 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 O30.5μL，10mM dNTP 2μL，6PNE-SDm-noG-F1. Mu.L, 6 PNE-SDm-noG-R1. Mu.L, template plasmid 5. Mu.L, phusion HF enzyme 0.5. Mu.L.

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

The PCR product was transformed into a template DNA digested with DpnIE.coliIn DH 5. Alpha. The monoclonal colonies were obtained after overnight culture. The monoclonal colony is subjected to amplification culture, then the vector sequence in the monoclonal colony is sequenced by a professional gene sequencing company, clones with correct sequencing results are selected, then the clones are amplified and plasmids are extracted from the clones, the SD sequence is successfully replaced, GST genes are removed, and NdeI and BamHI vectors are reintroduced. Thus, the vector pKL1 was constructed.

Embodiment two: construction of expression vector containing codon optimized HPV39L1 Gene

Three truncated forms of human papillomavirus type 39 coat protein L1 (HPV 39L1, the full-length amino acid sequence of which is shown as SEQ ID NO: 1) are respectively N-terminal truncated by 4 amino acids and C-terminal truncated by 29 amino acids; the full length of the N end and the C end are truncated by 29 amino acids; and N-terminal truncated by 9 amino acids, C-terminal truncated by 29 amino acids (the amino acid sequence is shown as SEQ ID NO:2, the coding nucleotide sequence after optimizing the codon is shown as SEQ ID NO: 3), and the truncated coding nucleotide sequence is synthesized artificially.

The DNA fragment of HPV39L1 is amplified by PCR, the L1 gene PCR fragment containing NdeI and Xho1 cleavage sites and the recombinant vector are respectively subjected to NdeI/Xho1 double-enzyme digestion, and then the recovered gene fragment and pKL1 containing corresponding cohesive ends are subjected to ligation reaction by using T4 DNA ligase, and the temperature is between 16 ℃ and 10 and 15 h. The connection system is as follows: 6. Mu.l of pKL1 vector fragment, 2. Mu.l of the aforementioned HPV39L1 gene fragment, 1. Mu.l of T4 DNA ligase and 1. Mu.l of T4 DNA ligase buffer. Conversion of ligation products to ligation products after ligation reactionsE. coli Screening of recombinants was performed in DH 5. Alpha. Amplifying the selected monoclonal colony, extracting plasmid, and sequencing to obtain recombinant expression vector pKL1-HPV39L1 (the three truncated forms include pKL1-HPV39L1Is N-end truncated by 4 amino acids, C-end truncated by 29 amino acids; pKL1-HPV39-FL L1 is N-terminally full length, C-terminally truncated by 29 amino acids, pKL1-HPV 39-N9L 1 is N-terminally truncated by 9 amino acids, C-terminally truncated by 29 amino acids).

Embodiment III: expression of HPV39L1 protein

Three recombinant vectors pKL1-HPV39L1 with correct sequencing results in the second embodiment are transformed into an escherichia coli XA90 host cell, and are used as engineering bacteria for expressing recombinant proteins to express HPV L1 proteins. 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 inoculum size of 0.5%, culturing at 30 ℃ for 7h at 220rpm, adding IPTG with the final concentration of 0.2mM, performing induction culture at 30 ℃ for 16h at 220rpm, ending fermentation, and centrifugally collecting bacterial bodies for expression amount detection and purification experiments. From the results of SDS-PAGE (FIGS. 1 and 2), the expression vector pKL1-HPV39L1 (N-terminally truncated by 4 amino acids and C-terminally truncated by 29 amino acids) and pKL1-HPV39-FL L1 (N-terminally full length and C-terminally truncated by 29 amino acids) were both expressed, and the results of the shake flask expression test showed that it was not able to efficiently express the HPV39L1 protein of interest. As can be seen from FIG. 3, only XA90pKL1-HPV 39-N9L 1 (9 amino acids N-terminally truncated and 29 amino acids C-terminally truncated) was able to efficiently express the target protein, and although some of the target proteins formed inclusion bodies (in the pellet), there were also more target proteins in the bacterial supernatant. Therefore, different experimental results are obtained by carrying out different optimization on the N end and the C end, the N end is truncated by nine amino acids, so that unexpected technical effects can be brought, and the expression quantity of target protein unit thalli is about 0.2mg/g wet thalli.

Embodiment four: HPV39L1 protein VLP purification and assembly

Taking a proper amount of thalli according to the mass volume ratio of 1:10, fully re-suspending the bacterial cells by using a bacteria breaking buffer (20 mM PB,20 mM DTT,pH8.0), and then crushing the bacterial cells by using a high-pressure homogenizer under the following conditions: 800 bar,3 times. The cell disruption solution was then centrifuged at high speed (4 ℃,12000 rpm,60 min) to collect the supernatant. The supernatant was further precipitated by ammonium sulfate with a saturation of 30%, and the precipitate was collected by centrifugation (4 ℃,12000 rpm,60 min) at a mass to volume ratio of 1:10, and re-centrifuging (at 4 ℃ C., 12000 rpm,60 min) after fully re-dissolving with a re-dissolving buffer (20 mM PB,20 mM DTT,pH8.0), and collecting the supernatant to obtain a crude pure solution. The crude pure solution is firstly loaded to carry out Superdex200 molecular sieve chromatography, and the molecular sieve buffer solution (20 mM PB,20 mM DTT,pH8.0) collects the components of the L1 target protein according to the peak position. And then loading the molecular sieve collected sample 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,pH8.0), and linearly eluting and collecting components of the L1 target protein by 10 column volumes through 0-20% high-salt buffer solution, wherein the components are the purified L1 protein. The mass of the L1 pentamer was determined by Dynamic Light Scattering (DLS). The SDS gel electrophoresis of pentamer is shown in FIG. 4. It can be seen that the pentamer formed in the target protein region is single, the purity is high, and the expression amount is high. Finally, the pH and salt concentration of the buffer solution in which the L1 protein is positioned are adjusted to self-assemble to form VLPs, and the preparation of the VLPs is completed. Finally, the quality of VLPs was determined by DLS.

TABLE 1 DLS detection results before and after HPV 39-N9L 1 protein Assembly

The purification experiment result shows that the pentamer state of HPV 39-N9L 1 is good (PdI is less than or equal to 0.1), and VLP with good state can be effectively assembled and formed (the particle size is less than or equal to 45 nm and less than or equal to 75 nm, and the PdI is less than or equal to 0.1)

Example five, protein Long-term stability experiment

The long-term stability data of HPV39L1 protein VLP prepared in example four was examined at-70 ℃ and the results were as follows.

"-" indicates no such provision or no test was performed on the item.

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

Claims (19)

1. A truncated HPV39 type L1 protein is characterized in that the amino acid sequence is shown in SEQ ID NO. 2.

2. A nucleic acid encoding the truncated HPV type 39L1 protein of claim 1.

3. The nucleic acid of claim 2, wherein the nucleotide sequence is set forth in SEQ ID No. 3.

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

5. A nucleic acid consisting of an SD sequence and a nucleic acid according to claim 2 or 3, the nucleotide sequence of said SD sequence being 5 '-AGGAGATATA-3'.

6. An expression cassette comprising the nucleic acid of claim 5.

7. An expression vector comprising the expression cassette of claim 6.

8. The expression vector of claim 7, which is a prokaryotic expression vector.

9. The expression vector of claim 8, wherein the GST tag sequence is deleted based on the vector pGEX, and the coding nucleic acid of claim 5 is integrated.

10. A recombinant host cell comprising the expression vector of claim 7 or 8 or 9.

11. The recombinant host cell of claim 10, which is a prokaryotic cell.

12. The recombinant host cell of claim 11, which is e.

13. A method of expressing a truncated HPV type 39L1 protein of claim 1, wherein the recombinant host cell of claim 10 or 11 or 12 is cultured to produce HPV type 39L1 protein.

14. The method of claim 13, further comprising a purification step.

15. The method of claim 14, wherein the purifying step is: taking thalli of the recombinant host cells, fully re-suspending the thalli by using a bacteria breaking buffer solution, then crushing the thalli at high pressure by using a high-pressure homogenizer, and centrifugally collecting supernatant; the supernatant is further precipitated by ammonium sulfate, the final saturation of ammonium sulfate is 30%, and the supernatant is centrifugally collected again after precipitation and redissolution to obtain crude pure liquid;

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

and then loading a molecular sieve collected sample to perform Source15Q anion exchange chromatography, and collecting components of the L1 target protein by NaCl linear elution to obtain HPV39 type L1 protein.

16. A method of making HPV type 39L1 VLPs, comprising the steps of: the process of claim 14 or 15, wherein the buffer is adjusted to a pH of 4.75-5.25 and a salt concentration of 2.0-4.0M to self-assemble HPV39 type L1 VLPs.

17. The method of claim 16, wherein the buffer is selected from Tris buffer, phosphate buffer, acetate buffer, HEPES buffer, MOPS buffer, citrate buffer, histidine buffer, or borate buffer.

18. The method of claim 17, wherein the buffer has a pH of 4.75, pH5.0, pH5.25; the salt concentration was 2.0m,2.5m,3.0m,3.5m,4.0m.

19. The method of claim 18, further comprising the step of purifying the resulting HPV type 39L1 VLP.