WO2024088087A1 - Expression de protéine de papillomavirus humain (hpv) 68 l1, particules pseudo-virales et procédé de préparation associé - Google Patents

Expression de protéine de papillomavirus humain (hpv) 68 l1, particules pseudo-virales et procédé de préparation associé Download PDF

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WO2024088087A1
WO2024088087A1 PCT/CN2023/124619 CN2023124619W WO2024088087A1 WO 2024088087 A1 WO2024088087 A1 WO 2024088087A1 CN 2023124619 W CN2023124619 W CN 2023124619W WO 2024088087 A1 WO2024088087 A1 WO 2024088087A1
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protein
truncated
buffer
sequence
hpv68
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Chinese (zh)
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张海江
陈晓
伍树明
刘永江
王学红
薛俊莲
高文双
姜绪林
沈迩萃
银飞
刘玉莹
于泓洋
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北京康乐卫士生物技术股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of medicine and biology, and specifically to the expression of human papillomavirus L1 protein and virus-like particles and preparation methods thereof. More specifically, it relates to the construction and expression of human papillomavirus HPV68 L1 protein VLP (virus-like particle).
  • HPV Human papillomavirus
  • HPV infection has obvious tissue specificity. Different types of HPV have different tropisms for the skin and mucosa, and can induce different papillary lesions. About 30 types of HPV are related to reproductive tract infections, of which more than 20 are related to tumors.
  • HPV can be roughly divided into two categories according to the benign and malignant nature of HPV-induced lesions: 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 malignant tumors of various human tissues, mainly causing severe atypical hyperplasia and invasive cancer; 2) Low-risk types (such as HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV72, HPV81, etc.): Low-risk HPV can cause benign proliferative sexually transmitted diseases of epidermal cells, such as condyloma acuminatum and flat warts.
  • High-risk types such as HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV
  • HPV is mainly composed of viral shell and genomic DNA.
  • the genome is about 7900bp long and has 8 viral protein coding genes.
  • the proteins encoded by 6 ORFs are expressed in the early stage of viral replication and are called early proteins; the proteins encoded by 2 ORFs are expressed in the late stage of viral replication and are called late proteins.
  • Late proteins include the major capsid protein L1 and the minor capsid protein L2, and are involved in the formation of the viral capsid.
  • HPV capsid proteins can self-assemble.
  • yeast expression systems or insect expression systems or L1 proteins expressed alone in mammalian cell expression systems or L1 proteins co-expressed with L2 proteins can self-assemble into virus-like particles (VLPs).
  • VLPs produced by exogenous expression systems can induce neutralizing antibodies in vivo after immunization, and obtain good immune protection effects.
  • the properties of VLPs produced by direct expression and assembly of VLPs in vivo using eukaryotic expression systems are not very uniform, and the cost of eukaryotic expression systems is very high, which is not conducive to industrialization.
  • HPV68 type is only reported in patent CN201310184823.X.
  • This patent uses the Hansen yeast expression system to produce HPV68 L1 protein.
  • the Hansen yeast expression system is a eukaryotic expression system that directly assembles into VLPs in vivo.
  • the patent does not propose whether the qualified protein can be normally expressed in the Escherichia coli prokaryotic expression system, because Escherichia coli
  • the prokaryotic expression system of bacteria does not have the post-translational modification functions of the Hansen yeast expression system, so it is difficult to express HPV68 L1 in the prokaryotic expression system.
  • the inventors expressed the HPV68 L1 protein in a prokaryotic expression system based on the cost of the finished vaccine, and solved the problem of difficulty in expressing the HPV68 L1 protein in a prokaryotic expression system. This was achieved specifically through the following improvements: the amino acid sequence of the HPV68 type L1 protein of a specific strain, especially AAZ39498.1, was truncated, and the codons of the coding nucleotide sequence of the truncated protein were optimized to obtain an optimized coding nucleotide sequence, and finally, a tag-free expression vector containing a specific SD sequence was used to achieve efficient expression and purification.
  • the present invention through sequence alignment screening and modification, found the strain AAZ39498.1 sequence with 29 amino acid differences from the ACX32384.1 strain for expression.
  • the alignment results are shown in Figure 3.
  • the specific HPV68 L1 protein (wild type) sequence of strain AAZ39498.1 is shown in SEQ ID NO.1.
  • the present invention performs N/C-terminal truncation on the amino acid sequence of SEQ ID NO.1 in order to obtain a better protein expression rate.
  • the N-terminal truncation is not more than 10 amino acids, preferably 4 amino acids.
  • the C-terminal truncation is not more than 30 amino acids, preferably 28 amino acids.
  • the specific amino acids after truncation are shown in SEQ ID NO.2.
  • N/C-terminal truncation can convert the protein properties from alkaline to acidic, express it on a tag-free expression vector and obtain higher quality proteins and VLPs.
  • the present invention first provides a truncated HPV68 L1 protein, which is based on the wild-type HPV68 L1 protein, with no more than 10 amino acids, preferably 4 amino acids, truncated at its N-terminus, and no more than 30 amino acids, preferably 28 amino acids, truncated at its C-terminus.
  • the amino acid sequence of the wild-type HPV68 L1 protein is shown in SEQ ID NO.1. More preferably, the amino acid sequence of the truncated HPV68 L1 protein is shown in SEQ ID NO.2.
  • the inventors optimized the codons of the nucleotide sequence for the E. coli system based on the amino acid sequence shown in SEQ ID NO.2.
  • the optimization principles include: a) selecting the codons with the highest or higher usage frequency according to the E. coli genetic code usage frequency table; b) eliminating the commonly used restriction endonuclease recognition sites.
  • the nucleotide sequence optimized by the above principles was screened multiple times to obtain the optimized nucleotide sequence shown in SEQ ID NO.3.
  • the present invention provides a nucleic acid encoding a truncated HPV68 type L1 protein.
  • it is a codon-optimized nucleic acid. More preferably, its nucleotide sequence is shown in SEQ ID NO.3.
  • expression cassettes, expression vectors and recombinant host cells containing the encoding nucleic acid Preferably, it is Escherichia coli.
  • the present invention also studies the use of a specific SD sequence.
  • the plasmid expression vectors of Escherichia coli mainly include two types of expression vectors for expressing fusion proteins and expression vectors for non-fusion proteins. If protein drugs or vaccine products are expressed using fusion proteins, the introduction of new exogenous proteins or polypeptides (residues of tag proteins or cutting enzymes) may increase the risk of drug safety.
  • the SD sequence (Shine-Dalgarno sequence) was first discovered by Shine and Dalgarno in 1974. There are ribosome binding sites on mRNA, which are the start codon AUG and a sequence consisting of 3 to 9 bp located 3 to 10 bp upstream of AUG.
  • This sequence is rich in purine nucleotides and is complementary to the pyrimidine-rich sequence at the 3' end of 16S rRNA. It is the recognition and binding site of ribosomal RNA. Since then, people have named this sequence the Shine-Dalgarno sequence, or SD sequence for short. Different SD sequences and their distances from the start codon AUG are important factors that affect mRNA transcription and translation into proteins. The binding of certain proteins to the SD sequence will also affect the binding of mRNA to the ribosome, thereby affecting the translation of the protein, that is, affecting the expression level of the recombinant exogenous factor.
  • the present invention finally determined that the SD sequence used for the recombinant expression of the HPV68 type L1 protein of the present invention is: 5'-AGGAGGAATTA-3' (the reverse sequence is 3'-TAATTCCTCCT-5').
  • the present invention provides a tag-free expression vector containing the SD sequence and a nucleic acid molecule containing a truncated HPV68 type L1 protein, and further provides an expression cassette, an expression vector and a recombinant host cell containing the nucleic acid molecule, more specifically, Escherichia coli.
  • the characteristic of the vector pGEX expressing fusion protein is that a 26kDa glutathione S-transferase gene (GST) is connected to the vector.
  • GST glutathione S-transferase gene
  • the present invention removes the GST tag of the vector and replaces the SD sequence that can efficiently express the HPV68 type L1 protein to form a new expression vector suitable for the HPV68 L1 protein.
  • the present invention also provides a method for expressing the truncated HPV68 type L1 protein, which cultured the recombinant host cell as described above to produce the truncated HPV68 type L1 protein, and optionally, included a purification step; preferably, the purification step is: taking the bacterial cells of the recombinant host cell and fully resuspending them with a bacterial lysis buffer, and then high-pressure crushing the bacterial cells with a high-pressure homogenizer, and centrifuging and collecting the supernatant; the supernatant is further precipitated by ammonium sulfate, and the final saturation of ammonium sulfate is 30%, and the precipitate is redissolved and the supernatant is collected again by centrifugation to obtain a crude pure liquid;
  • the crude pure liquid was first loaded for Superdex200 molecular sieve chromatography, and the fractions containing the target protein L1 were collected according to its peak position;
  • the present invention also provides a method for preparing truncated HPV68 type L1 protein VLP, comprising the following steps: The method comprises the steps of obtaining the truncated HPV68 type L1 protein by adjusting the pH and salt concentration of the buffer in which the truncated HPV68 type L1 protein is located so as to enable the truncated HPV68 type L1 protein to self-assemble to form a truncated HPV68 type L1 protein VLP.
  • the buffer includes but is not limited to Tris buffer, phosphate buffer, acetate buffer, HEPES buffer, MOPS buffer, citrate buffer, histidine buffer, borate buffer, preferably phosphate buffer;
  • the pH of the buffer solution is between 5 and 5.75, and the salt concentration is between 1.5 and 3.0 M, preferably pH 5, pH 5.25, pH 5.5, and pH 5.75; wherein the salt concentration is between 1.5 M and 3.0 M, preferably 1.5 M, 2.0 M, 2.5 M, and 3.0 M;
  • the method further comprises the step of purifying the obtained truncated HPV68 type L1 protein VLP.
  • the present invention through the above-mentioned improvements, can obtain a higher protein expression amount in a prokaryotic expression system such as Escherichia coli, and obtain VLPs with more uniform quality.
  • Figure 1 XA90 pKL1-HPV68L1 expression electrophoresis test results in small shake flasks (SD not modified).
  • M marker; 1. XA90 pKL1 negative control; 2. XA90 pKL1-HPV68L1-1 whole bacteria; 3. XA90 pKL1-HPV68L1-1 supernatant; 4. XA90 pKL1-HPV68L1-1 precipitate; 5. HPV68L1-VLP; 6. XA90 pKL1-HPV68L1-2 whole bacteria; 7. XA90 pKL1-HPV68L1-2 supernatant; 8. XA90 pKL1-HPV68L1-2 precipitate.
  • Figure 2 XA90 pKL30-HPV68L1 small shake flask expression electrophoresis detection results (SD transformation).
  • M marker; 1. XA90 pKL30 negative control; 2. XA90 pKL30-HPV68L1-1 uninduced negative control; 3. XA90 pKL30-HPV68L1-1 whole bacteria; 4. XA90 pKL30-HPV68L1-1 supernatant; 5. XA90 pKL30-HPV68L1-1 precipitate; 6. XA90 pKL30-HPV68L1-2 whole bacteria; 7. XA90 pKL30-HPV68L1-2 supernatant; 8. XA90 pKL30-HPV68L1-2 precipitate.
  • Fig. 4 shows the expression results of the improved truncated HPV68L1 sequence (HPV68L1a) of the present invention.
  • M marker; 1. XA90 pKL30 negative control; 2. XA90 pKL30-HPV68L1a-1 uninduced negative control; 3. XA90 pKL30-HPV68L1a-1 whole bacteria; 4. XA90 pKL30-HPV68L1a-1 supernatant; 5. XA90 pKL30-HPV68L1a-1 precipitate; 6. XA90 pKL30-HPV68L1a-2 whole bacteria; 7. XA90 pKL30-HPV68L1a-2 supernatant; 8. XA90 pKL30-HPV68L1a-2 precipitate.
  • FIG. 6 Protein expression test results of different SD sequences.
  • M marker; 1. Control XA90 pKL1; 2. Uninduced XA90 pBSDm-68L1; 3. Induced whole bacteria XA90 pBSDm-68L1; 4. Induced supernatant XA90 pBSDm-68L1; 5. Induced precipitate XA90 pBSDm-68L1; 6. Uninduced XA90 pT1SDm-68L1; 7. Induced whole bacteria XA90 pT1SDm-68L1; 8. Induced supernatant XA90 pT1SDm-68L1; 9.
  • the position indicated by the arrow in the above figure is the theoretical position of the target protein.
  • Example 1 Construction of an untagged HPV68L1 expression vector containing a specific SD sequence
  • the inherent SD of the pGEX-6P-1 plasmid vector cannot effectively express exogenous genes such as HPV polytype L1 in a soluble manner in a non-fusion manner. Even if there is expression, the expression level is very low. Therefore, for the HPV68 L1 protein of strain ACX32384.1 (its amino acid sequence is shown in Figure 3, and the nucleotide sequence is shown in GenBank: GQ472851.1, 5515-7032bp), a cluster of new non-fusion SD sequence expression vectors was constructed by modifying the SD sequence based on the pGEX-6P-1 plasmid vector.
  • the modified SD sequence is: 5'-AGGAGGAATTA-3' (the reverse sequence is 3'-TAATTCCTCCT-5').
  • PCR primers The names and sequences of PCR primers are as follows:
  • the PCR reaction system was as follows: 5 ⁇ phusion HF buffer 10 ⁇ L, ddH 2 O 3 0.5 ⁇ L, 10 mM dNTP 2 ⁇ L, 6PNE-SDm-F 1 ⁇ L, 6PNE-SDm-R 1 ⁇ L, pGEX-6P-2 (diluted 20 times) 5 ⁇ L, and Phusion HF Enzyme 0.5 ⁇ L.
  • the PCR product was digested with DpnI and transformed into the host bacteria DH5 ⁇ . After overnight culture, a monoclonal colony was obtained. The monoclonal colony was expanded and cultured, and then the vector sequence was sequenced by a professional gene sequencing company. The clone with the correct sequencing result was selected, and then the clone was expanded and the plasmid was extracted from it to obtain the vector with the NdeI restriction site successfully introduced.
  • Primer information is as follows:
  • the PCR reaction system was as follows: 5 ⁇ phusion HF buffer 10 ⁇ L, ddH 2 O 3 0.5 ⁇ L, 10 mM dNTP 2 ⁇ L, SDm-F 1 ⁇ L, SDm-R 1 ⁇ L, plasmid obtained in step 1 5 ⁇ L, Phusion HF Enzyme 0.5 ⁇ L.
  • the template DNA was transformed into E. coli DH5 ⁇ and cultured overnight to obtain a monoclonal colony.
  • the monoclonal colony was expanded and then sequenced by a professional gene sequencing company. The clone with the correct sequencing result was selected, and then the clone was expanded and the plasmid was extracted from it to obtain the vector that successfully replaced the SD sequence.
  • the enzyme digestion system is as follows: Cutsmart buffer 3 ⁇ l, ddH 2 O 3 ⁇ l, 1.2 obtained vector 20 ⁇ l, NdeI 2 ⁇ l, BamHI 2 ⁇ l. 37°C digestion for 2h; 0.8% agarose gel electrophoresis, 120V, 1h; gel cutting to obtain the electrophoresis band corresponding to the vector fragment after removing the GST gene, and store at 4°C. Use agarose gel recovery kit to recover the vector fragment, and take 3 ⁇ l of the obtained vector fragment for electrophoresis to detect the recovery result. Then the double enzyme digestion product is filled with DNA polymerase I to fill the sticky ends.
  • the reaction system is as follows: 10 ⁇ T 4 DNA Ligase buffer 2.5 ⁇ l, ddH 2 O 1.8 ⁇ l, gel-recovered enzyme digestion vector fragment 20 ⁇ l, 10mM dNTP 0.2 ⁇ l, DNA polymerase I 0.5 ⁇ l, 25°C reaction for 15min, add EDTA (EDTA final concentration is 10mM) and heat at 75°C for 20min to terminate the reaction.
  • the vector that was digested and end-filled was re-ligated and circularized.
  • the ligation system was as follows: T4 DNA Ligase buffer 2 ⁇ l, linear blunt-end vector fragment 16 ⁇ l, T4 DNA ligase 2 ⁇ l. Ligation was carried out at 16°C for 4h.
  • the ligation product was digested and transformed into E.coliDH5 ⁇ , and monoclonal colonies were obtained after overnight culture. The monoclonal colonies were expanded and cultured, and then the vector sequences were sequenced by a professional gene sequencing company. The clones with correct sequencing results were selected, and then the clones were expanded and plasmids were extracted from them to obtain vectors that successfully replaced the SD sequence and removed the GST gene.
  • PCR primers are as follows:
  • 6PNE-SDm-noG-F (5′to3′): CAGGAGATATACATATGGGATCCCCGGAATTCCCG;
  • the PCR reaction system was as follows: 5 ⁇ phusion HF buffer 10 ⁇ L, ddH 2 O 30.5 ⁇ L, 10 mM dNTP 2 ⁇ L, 6PNE-SDm-noG-F 1 ⁇ L, 6PNE-SDm-noG-R 1 ⁇ L, template plasmid 5 ⁇ L, and Phusion HF Enzyme 0.5 ⁇ L.
  • the PCR reaction program was set as follows: 95°C for 3 min; 95°C for 1 min, 55°C for 1 min, 72°C for 10 min; 20 cycles; 72°C for 15 min.
  • the PCR product was digested with DpnI and then transformed into E. coli DH5 ⁇ . After overnight culture, a single clone was obtained. Colonies. The monoclonal colonies were expanded and cultured, and then the vector sequences were sequenced by a professional gene sequencing company. The clones with correct sequencing results were selected, and then the clones were expanded and plasmids were extracted from them to obtain a vector (pKL30) that successfully replaced the SD sequence, removed the GST gene, and reintroduced NdeI and BamHI.
  • the vector obtained in the previous step and the HPV68L1 protein gene of the artificially synthesized strain ACX32384.1 were double-digested with NdeI and Xho1 restriction endonucleases, respectively, and then recovered.
  • the recovered vector fragment and gene fragment were ligated with T4 DNA ligase at 16°C for 10 to 15 hours to obtain an untagged HPV68L1 expression vector (pKL30-HPV68L1) containing a specific SD sequence.
  • the SD sequence (5'-AGGAGGAATTA-3') used in the present invention can effectively express the HPV68L1 protein of strain ACX32384.1 in the E. coli XA90 host cell, but the soluble expression amount is low, as shown in Figure 2.
  • the target protein cannot be expressed using the original SD sequence (5'-AGGAGATATA-3') of the vector, as shown in Figure 1.
  • the electrophoresis results of SDS-PAGE show that the expression of the target protein of the vector without SD sequence improvement is significantly worse than that of the SD sequence (5'-AGGAGGAATTA-3') vector with sequence improvement.
  • XA90 is the transformed host bacteria.
  • pKL30 is the vector in Example 1 from which the GST gene is removed and transformed with the SD sequence (pBSDm: 5'-AGGAGGAATTA-3').
  • pKL1 is the vector from which the GST gene is removed but not transformed with the SD sequence.
  • HPV68L1 refers to the HPV68L1 protein sequence of strain ACX32384.1.
  • Example 2 Screening of HPV68 L1 proteins from different strains and expression of truncated HPV68 L1 proteins
  • the applicant has found through many experiments that not all strains of HPV68 L1 protein can be expressed in the E. coli system and assembled into VLPs of good quality, such as strain ACX32384.1. Therefore, the present invention has found the strain AAZ39498.1 sequence with 29 amino acid differences from the ACX32384.1 strain for expression through sequence comparison screening and modification. The comparison result is shown in Figure 3; the amino acids of the HPV68 L1 protein of strain AAZ39498.1 are shown in SEQ ID NO.1.
  • the present invention performs N/C-terminal truncation on the amino acid sequence of the HPV68 L1 protein (SEQ ID NO.1) of the strain AAZ39498.1, truncating 4 amino acids at the N-terminus and 28 amino acids at the C-terminus, and the specific truncated amino acids are shown in SEQ ID NO.2.
  • the expression results of the HPV68 L1 protein of the strain ACX32384.1 are shown in Figure 2
  • the expression results of the truncated HPV68 L1 protein (SEQ ID NO.2, marked as HPV68L1a) of the strain AAZ39498.1 are shown in Figure 4. Comparing Figures 2 and 4, it can be seen that the expression level of the truncated HPV68 L1 of the optimized strain AAZ39498.1 is higher than that of the strain ACX32384.1.
  • Example 3 Construction of an expression vector containing a truncated HPV68 L1 protein of the codon-optimized strain AAZ39498.1
  • the gene of the truncated HPV68L1 protein (HPV68L1a) of the codon-optimized strain AAZ39498.1 was artificially synthesized (the sequence is shown in SEQ ID No. 3).
  • the L1 gene and the pKL30 vector were double-digested with NdeI and Xho1 restriction endonucleases, respectively, and then the recovered L1 gene fragment was ligated with the pKL30 vector fragment using T4 DNA ligase at 16°C for 10 to 15 hours.
  • the ligation system is as follows: 6 ⁇ l of pKL30 vector fragment, 2 ⁇ l of L1 gene fragment, 1 ⁇ l of T4 DNA ligase, and 1 ⁇ l of T4 DNA Ligase buffer. After the ligation reaction, the ligation product was transformed into E. coli DH5 ⁇ for recombinant screening. The screened monoclonal colonies were expanded and the plasmids were extracted, and then sequenced and verified to obtain the recombinant expression vector pKL30-HPV68L1a.
  • Example 4 Expression of truncated HPV68 L1 protein of strain AAZ39498.1
  • the pKL30-HPV68L1a with the correct sequencing result in Example 3 was transformed into the E. coli XA90 host cell, and used as an engineered bacterium to express the truncated HPV68L1 (HPV68L1a) protein for expressing the recombinant protein.
  • the expression process is as follows: XA90 pKL30-HPV68L1a was inoculated into LB medium (Amp+) at an inoculation rate of 0.05%, and cultured at 37°C, 220rpm for 16h for activation.
  • the activated bacterial solution was inoculated into 2YT medium at an inoculation rate of 0.5%, and IPTG was added at a final concentration of 0.2mM after culture at 30°C, 220rpm for 7h.
  • the expression was induced at 30°C, 220rpm for 16h, and the fermentation was terminated.
  • the bacterial cells were collected by centrifugation for expression detection and purification experiments.
  • the SD sequence 5'-AGGAGGAATTA-3'
  • codon optimization and amino acid truncation the soluble expression level of the protein without modification and optimization was low, and most of the expression was in the precipitate.
  • the target protein could not be purified and recovered, which was equivalent to expression failure.
  • the protein expression level was significantly improved, and in the supernatant, it was conducive to purification and recovery, and the expression of human papillomavirus L1 antigen protein could be achieved from scratch.
  • a lysis buffer (20 mM Pb, 20 mM DTT, pH 8.0) at a mass volume ratio of 1:10. Then use a high-pressure homogenizer to rupture the cells under high pressure at 800 bar for 3 times. The cell rupture solution is then centrifuged at high speed (4°C, 12000 rpm, 60 min) to collect the supernatant.
  • the supernatant is further purified by 30% saturation sulfur Ammonium acid precipitation, centrifugation (4 °C, 12000rpm, 60min) to collect the precipitate, the precipitate was fully re-dissolved with re-dissolution buffer (20mM PB, 20mM DTT, pH8.0) at a mass volume ratio of 1:10, and then centrifuged again (4 °C, 12000rpm, 60min) to collect the supernatant to obtain a crude pure solution.
  • the crude pure solution was first loaded for Superdex200 molecular sieve chromatography, molecular sieve buffer (20mM PB, 20mM DTT, pH8.0), and the components where the L1 target protein was located were collected according to its peak position.
  • the sample collected by molecular sieve was loaded for Source15Q anion exchange chromatography (SQ low salt buffer: 5mM PB, 10mM DTT, pH8.0, SQ high salt buffer: 5mM PB, 1M NaCl, 10mM DTT, pH8.0), and the fraction containing the truncated L1 target protein was collected by linear elution with 0-20% high salt buffer and 10 column volumes. This fraction is the truncated L1 protein after purification. Finally, the pH and salt concentration of the buffer containing the truncated L1 protein were adjusted to form VLPs by self-assembly, and the preparation of truncated HPV68L1-VLPs was completed. Finally, the quality of VLPs was determined by dynamic light scattering (DLS).
  • DLS dynamic light scattering
  • the improvement of the present invention is to study the truncation of a small number of amino acid sequences at the N-terminus or C-terminus to increase the expression amount, and further through codon optimization and SD sequence modification, to achieve efficient expression of HPV68L1 protein.
  • the present invention only involves the two ends of the HPV68 type L1 protein (SEQ ID NO.1) of strain AAZ39498.1, and does not involve the core region of its immunogenicity, that is, the truncation does not affect its immunogenicity; and it is verified by the following experiments:
  • the truncated HPV68L1-VLP protein stock solution obtained in Example 5 above was adsorbed with aluminum hydroxide adjuvant to prepare a vaccine and stored at 4°C for use. Take the VLP protein vaccine and inject 0.1 ml into BAB/c mice intramuscularly, with 10 mice in each group. The mice were boosted once every 4 weeks, for a total of 2 immunizations. Four weeks after the second immunization, the pseudovirus cell neutralization test method (since the HPV virus cannot be artificially cultured in large quantities in vitro, the pseudovirus method is a standard method for evaluating the efficacy of HPV vaccines.
  • the pseudovirus used simulates the structure of the HPV virus to the maximum extent, and the L1 and L2 proteins of the wild-type HPV virus are assembled into pseudovirus particles, in which the L1 and L2 proteins are both unmodified full-length sequences) was used to measure the neutralizing antibody titer against HPV type 68 in the serum of immunized mice. The results are shown in Table 2.

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Abstract

La présente invention relève du domaine de la biologie médicale, et concerne en particulier l'expression d'une protéine de papillomavirus humain (HPV) 68 L1, des particules pseudo-virales (VLP) et un procédé de préparation associé. La séquence d'acides aminés de la protéine HPV 68 L1 d'une souche virale spécifique, en particulier AAZ39498.1, est tronquée, l'optimisation des codons est effectuée sur la séquence nucléotidique codante de la protéine tronquée pour obtenir une séquence nucléotidique codante optimisée, et enfin l'expression et la purification sans marqueur sont obtenues à l'aide d'un vecteur d'expression sans marqueur contenant une séquence SD spécifique. Selon la présente invention, une amélioration permet d'obtenir un niveau d'expression de protéine plus élevé dans un système d'expression procaryote tel qu'un système d'expression d'E. coli, et des VLP ayant une qualité plus uniforme sont obtenues.
PCT/CN2023/124619 2022-10-28 2023-10-13 Expression de protéine de papillomavirus humain (hpv) 68 l1, particules pseudo-virales et procédé de préparation associé WO2024088087A1 (fr)

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