WO2024088087A1 - Expression of human papillomavirus (hpv) 68 l1 protein, and virus-like particles and preparation method therefor - Google Patents

Expression of human papillomavirus (hpv) 68 l1 protein, and virus-like particles and preparation method therefor 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
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    • 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
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    • 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

The present invention relates to the field of medical biology, and specifically to expression of a human papillomavirus (HPV) 68 L1 protein, and virus-like particles (VLPs) and a preparation method therefor. The amino acid sequence of the HPV 68 L1 protein of a specific virus strain, especially AAZ39498.1, 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 tag-free expression and purification are achieved by using a tag-free expression vector containing a specific SD sequence. According to the present invention, by means of the improvement, a higher protein expression level can be obtained in a prokaryotic expression system such as an E. coli expression system, and VLPs having more uniform quality are obtained.

Description

人***瘤病毒HPV68 L1蛋白的表达和类病毒样颗粒及其制备方法Expression of human papillomavirus HPV68 L1 protein and virus-like particles and preparation methods thereof 技术领域Technical Field
本发明涉及医药生物领域，具体涉及人***瘤病毒L1蛋白的表达和类病毒样颗粒及其制备方法。更具体涉及人***瘤病毒HPV68 L1蛋白VLP(类病毒样颗粒)的构建及表达。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).
背景技术Background technique
人***瘤病毒(human papillomavirus，HPV)是一种无包膜的闭环双链DNA病毒，属乳多空病毒科多瘤病毒亚科，主要侵犯人体的上皮黏膜组织，进而诱发各种良性及恶性增生病变。目前已鉴定出来的不同亚型HPV超过200种，HPV感染具有明显的组织特异性，不同型别的HPV对于皮肤和黏膜的嗜向性不同，能诱发不同的***状病变，大约有30多种HPV型别与生殖道感染有关，其中有20多种与肿瘤相关。Human papillomavirus (HPV) is a non-enveloped, closed-circular, double-stranded DNA virus belonging to the Polyomavirinae subfamily of the Papovaviridae family. It mainly invades the epithelial mucosal tissues of the human body, thereby inducing various benign and malignant hyperplastic lesions. More than 200 different subtypes of HPV have been identified. 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.
根据HPV诱发病变的良恶性不同，HPV可大致分为两类：1)高危型(如HPV16、HPV18、HPV31、HPV33、HPV35、HPV39、HPV45、HPV51、HPV52、HPV56、HPV58、HPV59、HPV68等)：高危型HPV与人类多种组织恶性肿瘤密切相关，主要引起重度不典型增生和***；2)低危型(如HPV6、HPV11、HPV40、HPV42、HPV43、HPV44、HPV54、HPV72、HPV81等)：低危型HPV可引起表皮细胞良性增殖性性病，如***和***等。HPV主要由病毒外壳和基因组DNA构成。基因组长约7900bp，有8个病毒蛋白编码基因。其中6个ORF编码的蛋白在病毒复制的早期表达，称为早期蛋白；2个ORF编码的蛋白在病毒复制的晚期表达，称为晚期蛋白。晚期蛋白包括主要外壳蛋白L1及次要外壳蛋白L2，并参与病毒外壳的形成。HPV病毒外壳蛋白能够进行自组装，目前在专利文献中，多采用酵母表达***或者昆虫表达***或者在哺乳动物细胞表达***里面单独表达的L1蛋白或将L1蛋白与L2蛋白共表达时均能自组装成病毒样颗粒(virus-like particle,VLP)，利用外源表达体系生产的VLP免疫后能够在体内诱发产生中和抗体，获得良好的免疫保护效果。但是利用真核表达***在体内直接表达组装VLP，产生VLP的性质并不是很均一，并且真核表达***的成本很高，不利于产业化。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. HPV is mainly composed of viral shell and genomic DNA. The genome is about 7900bp long and has 8 viral protein coding genes. Among them, 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. Currently, in patent literature, 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. However, 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.
目前针对HPV68型仅在专利CN201310184823.X中报道，此专利用的是汉逊酵母表达***产生HPV68 L1蛋白，汉逊酵母表达***是真核表达***，在体内直接组装成VLP，该专利中并没有提出在大肠杆菌原核表达***里是否可以正常表达合格标准的蛋白，因为大肠杆 菌原核表达***并不具备汉逊酵母表达***翻译后修饰等功能，所以在原核表达***里表达HPV68 L1是有一定难度的。Currently, the 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.
因此，需要研究解决在原核表达***里面表达HPV68 L1蛋白困难的问题，来获得更均一的VLP以及在产业应用上有更低的成本。Therefore, it is necessary to study and solve the problem of difficulty in expressing HPV68 L1 protein in prokaryotic expression systems to obtain more uniform VLPs and lower costs for industrial applications.
发明内容Summary of the invention
本发明人针对基于疫苗成品成本的考虑，在原核表达***里面表达HPV68 L1蛋白，并解决了在原核表达***里面表达HPV68L1蛋白困难的问题。具体通过以下改进实现：对特定毒株尤其是AAZ39498.1的HPV68型L1蛋白的氨基酸序列进行截短，并针对该截短后的蛋白的编码核苷酸序列进行密码子优化得到优化的编码核苷酸序列，最后配合含有特定SD序列的无标签表达载体实现高效表达和纯化。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.
首先，申请人通过很多次试验发现，并不是所有毒株的HPV68型L1蛋白能够在大肠杆菌***表达并组装成质量合格的VLP，例如毒株ACX32384.1等。因此本发明经过序列比对筛选改造，找到了与ACX32384.1毒株有29个氨基酸差异的毒株AAZ39498.1序列做表达，比对结果见图3，具体的毒株AAZ39498.1的HPV68型L1蛋白(野生型)序列见SEQ ID NO.1。本发明将SEQ ID NO.1的氨基酸序列做N/C端截短处理，为了获得更好的蛋白表达率。N端截短不多于10个氨基酸，优选4个氨基酸。C端截短不多于30个氨基酸，优选28个氨基酸，具体的截短后的氨基酸见SEQ ID NO.2。N/C端截短处理，能够使得蛋白性质从偏碱性到偏酸性的转换，在无标签表达载体上表达并且获得更高质量的蛋白及VLP。First, the applicant discovered through many experiments that not all strains of HPV68 L1 proteins can be expressed in the E. coli system and assembled into qualified VLPs, such as strain ACX32384.1. Therefore, 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.
因此，本发明首先提供一种截短的HPV68型L1蛋白，其是在野生型HPV68型L1蛋白的基础上，在其N端截短不多于10个氨基酸，优选4个氨基酸，且在其C端截短不多于30个氨基酸，优选28个氨基酸。优选地，野生型HPV68型L1蛋白的氨基酸序列如SEQ ID NO.1所示。更优选地，截短的HPV68型L1蛋白的氨基酸序列如SEQ ID NO.2所示。Therefore, 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. Preferably, 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.
其次，为了利用大肠杆菌***高效的表达HPV68L1蛋白，本发明人根据SEQ ID NO.2所示的氨基酸序列，针对大肠杆菌***进行核苷酸序列的密码子优化。优化原则包括：a)按照大肠杆菌遗传密码使用频率表选用使用频率最高或较高的密码子；b)消除常用的限制性内切酶识别位点。通过上述原则经过优化的核苷酸序列并进行多次筛选，获得了优化后的核苷酸序列如SEQ ID NO.3所示。Secondly, in order to efficiently express HPV68L1 protein using the E. coli system, 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.
由此本发明提供一种截短的HPV68型L1蛋白的编码核酸。优选地，其经过密码子优化的核酸。更优选地，其核苷酸序列如SEQ ID NO.3所示。也进一步提供含有所述编码核酸的表达盒，表达载体和重组宿主细胞。优选地，其是大肠杆菌。 Thus, the present invention provides a nucleic acid encoding a truncated HPV68 type L1 protein. Preferably, it is a codon-optimized nucleic acid. More preferably, its nucleotide sequence is shown in SEQ ID NO.3. Also provided are expression cassettes, expression vectors and recombinant host cells containing the encoding nucleic acid. Preferably, it is Escherichia coli.
最后，本发明还研究采用了特定SD序列。大肠杆菌的质粒表达载体主要包括表达融合蛋白的表达载体和非融合蛋白的表达载体两类。蛋白药物类或疫苗类产品如果应用融合蛋白表达，由于引入新的外源蛋白或多肽(标签蛋白或切割酶的残留)，可能增加药物安全性风险。SD序列(Shine-Dalgarno sequence)是1974年Shine和Dalgarno首先发现，在mRNA上有核糖体的结合位点，它们是起始密码子AUG和一段位于AUG上游3～10bp处的由3～9bp组成的序列。这段序列富含嘌呤核苷酸，刚好与16S rRNA 3'末端的富含嘧啶的序列互补，是核糖体RNA的识别与结合位点。此后人们将此序列命名为Shine-Dalgarno序列，简称SD序列。不同的SD序列及其与起始密码子AUG之间的距离是影响mRNA转录、翻译成蛋白的重要因素之一，某些蛋白质与SD序列结合也会影响mRNA与核糖体的结合，从而影响蛋白质的翻译,也就是影响重组外源因的表达水平。本发明经过研究和优化，最终确定用于本发明HPV68型L1蛋白重组表达的SD序列为：5'-AGGAGGAATTA-3'(反向序列为3'-TAATTCCTCCT-5')。Finally, 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. After research and optimization, 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').
在此基础上，本发明提供含所述SD序列的无标签表达载体以及含有截短的HPV68型L1蛋白的核酸分子。也进一步提供含有所述核酸分子的表达盒，表达载体和重组宿主细胞，更具体地，其是大肠杆菌。On this basis, 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.
对于表达载体而言，表达融合蛋白的载体pGEX的特点是在载体上接上了一种26kDa的谷胱甘肽S-转移酶基因(GST)，与其它的融合载体相比，它具有纯化条件温和、步骤简单、无变性剂加入，以及纯化后蛋白能最大限度保持其空间构象和免疫原性的特点；具有较好的应用价值，但载体pGEX编码的GST融合蛋白标签可能会增加药用蛋白产品的安全性隐患。对此，本发明将该载体的GST标签去除，并且替换能够高效表达HPV68型L1蛋白的SD序列，而形成新的适合HPV68 L1蛋白的表达载体。For expression vectors, the characteristic of the vector pGEX expressing fusion protein is that a 26kDa glutathione S-transferase gene (GST) is connected to the vector. Compared with other fusion vectors, it has mild purification conditions, simple steps, no denaturant addition, and the purified protein can maintain its spatial conformation and immunogenicity to the maximum extent; it has good application value, but the GST fusion protein tag encoded by the vector pGEX may increase the safety risks of pharmaceutical protein products. In this regard, 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.
本发明最后还提供一种表达所述截短的HPV68型L1蛋白的方法，其培养如所述的重组宿主细胞以产生截短的HPV68型L1蛋白，任选地，包括纯化步骤；优选地所述纯化步骤为：取所述的重组宿主细胞的菌体用破菌缓冲液充分重悬，然后用高压均质机对菌体进行高压破碎，离心收集上清；上清进一步通过硫酸铵沉淀，硫酸铵的最终饱和度为30％，沉淀复溶后再次离心收集上清获得粗纯液；Finally, 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;
粗纯液先上样进行Superdex200分子筛层析，根据L1目的蛋白的出峰位置收集其所在组分；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;
接着将分子筛收集样品上样进行Source15Q阴离子交换层析，通过NaCl线性洗脱收集L1目的蛋白所在组分，得到截短的HPV68型L1蛋白。Then, the sample collected by the molecular sieve was loaded onto Source15Q anion exchange chromatography, and the fraction containing the L1 target protein was collected by NaCl linear elution to obtain a truncated HPV68 type L1 protein.
本发明还提供一种制备截短的HPV68型L1蛋白VLP的方法，包括如下步骤：根据所述 的方法得到的截短的HPV68型L1蛋白的步骤，调节其所在缓冲液的pH和盐浓度，使其自组装形成截短的HPV68型L1蛋白VLP。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.
优选地，所述缓冲液包括但不限于Tris缓冲液，磷酸盐缓冲液，醋酸缓冲液，HEPES缓冲液，MOPS缓冲液，枸橼酸缓冲液、组氨酸缓冲液，硼酸缓冲液，优选磷酸盐缓冲液；Preferably, 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;
缓冲液的pH在5-5.75，盐浓度在1.5-3.0M之间，优选pH5，pH5.25，pH5.5，pH5.75；其中的盐浓度在1.5M-3.0M之间，优选1.5M，2.0M，2.5M，3.0M；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;
任选地，还包括纯化所得截短的HPV68型L1蛋白VLP的步骤。Optionally, the method further comprises the step of purifying the obtained truncated HPV68 type L1 protein VLP.
本发明通过上述改进使得在原核例如大肠杆菌表达***中可以获得更高的蛋白表达量，且得到质量更均一的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.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1 XA90 pKL1-HPV68L1小摇瓶表达电泳检测结果(SD未改造)。其中，M:marker；1.XA90pKL1阴性对照；2.XA90 pKL1-HPV68L1-1全菌；3.XA90 pKL1-HPV68L1-1上清；4.XA90 pKL1-HPV68L1-1沉淀；5.HPV68L1-VLP；6.XA90 pKL1-HPV68L1-2全菌；7.XA90 pKL1-HPV68L1-2上清；8.XA90 pKL1-HPV68L1-2沉淀。Figure 1 XA90 pKL1-HPV68L1 expression electrophoresis test results in small shake flasks (SD not modified). Among them, 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.
图2 XA90 pKL30-HPV68L1小摇瓶表达电泳检测结果(SD改造)。其中，M:marker；1.XA90 pKL30阴性对照；2.XA90 pKL30-HPV68L1-1未诱导阴性对照；3.XA90pKL30-HPV68L1-1全菌；4.XA90 pKL30-HPV68L1-1上清；5.XA90 pKL30-HPV68L1-1沉淀；6.XA90 pKL30-HPV68L1-2全菌；7.XA90 pKL30-HPV68L1-2上清；8.XA90 pKL30-HPV68L1-2沉淀。Figure 2 XA90 pKL30-HPV68L1 small shake flask expression electrophoresis detection results (SD transformation). Among them, 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.
图3毒株ACX32384.1的HPV68 L1序列和毒株AAZ39498.1的HPV68 L1序列(SEQ ID NO.1)的比对。Figure 3 Alignment of the HPV68 L1 sequence of strain ACX32384.1 and the HPV68 L1 sequence of strain AAZ39498.1 (SEQ ID NO.1).
图4本发明改进后的截短的HPV68L1序列(HPV68L1a)表达结果。其中，M:marker；1.XA90 pKL30阴性对照；2.XA90 pKL30-HPV68L1a-1未诱导阴性对照；3.XA90 pKL30-HPV68L1a-1全菌；4.XA90 pKL30-HPV68L1a-1上清；5.XA90 pKL30-HPV68L1a-1沉淀；6.XA90 pKL30-HPV68L1a-2全菌；7.XA90 pKL30-HPV68L1a-2上清；8.XA90 pKL30-HPV68L1a-2沉淀。Fig. 4 shows the expression results of the improved truncated HPV68L1 sequence (HPV68L1a) of the present invention. Wherein, 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.
图5纯化所得HPV68L1a五聚体电泳检测结果。其中，M:marker；1.HPV68L1a五聚体。Figure 5. Electrophoresis detection results of purified HPV68L1a pentamer. Wherein, M: marker; 1. HPV68L1a pentamer.
图6不同SD序列的蛋白表达检测结果。其中，M:marker；1.对照XA90 pKL1；2.未诱导XA90 pBSDm-68L1；3.诱导全菌XA90 pBSDm-68L1；4.诱导上清XA90 pBSDm-68L1；5.诱导沉淀XA90 pBSDm-68L1；6.未诱导XA90 pT1SDm-68L1；7.诱导全菌XA90 pT1SDm-68L1； 8.诱导上清XA90 pT1SDm-68L1；9.诱导沉淀XA90 pT2SDm-68L1；10.未诱导XA90 pT2SDm-68L1；11.诱导全菌XA90 pT2SDm-68L1；12.诱导上清XA90 pT2SDm-68L1；13.诱导沉淀XA90 pT2SDm-68L1；14.对照XA90 pKL1。Figure 6 Protein expression test results of different SD sequences. Wherein, 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. Induced precipitate XA90 pT2SDm-68L1; 10. Uninduced XA90 pT2SDm-68L1; 11. Induced whole bacteria XA90 pT2SDm-68L1; 12. Induced supernatant XA90 pT2SDm-68L1; 13. Induced precipitate XA90 pT2SDm-68L1; 14. Control XA90 pKL1.
以上附图中箭头所指位置为目标蛋白理论位置。The position indicated by the arrow in the above figure is the theoretical position of the target protein.
具体实施方式Detailed ways
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
实施例一：含有特定SD序列的无标签HPV68L1表达载体的构建Example 1: Construction of an untagged HPV68L1 expression vector containing a specific SD sequence
pGEX-6P-1质粒载体固有的SD，不能使HPV多型别L1等外源基因有效地以非融合方式实现可溶表达，即使有表达，其表达量也很低。因此针对毒株ACX32384.1的HPV68 L1蛋白(其氨基酸序列见图3，核苷酸序列如GenBank:GQ472851.1，5515-7032bp所示)，在pGEX-6P-1质粒载体基础上，通过对SD序列的改造、构建了一簇崭新的非融合SD序列表达载体。其中改造后的SD序列为：5'-AGGAGGAATTA-3'(反向序列为3'-TAATTCCTCCT-5')。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').
1.通过突变PCR，在pGEX-6P-1质粒中引入NdeI酶切位点：1. Introduce NdeI restriction site into pGEX-6P-1 plasmid by mutation PCR:
PCR引物名称及序列如下：The names and sequences of PCR primers are as follows:
正向引物：NdeImut-F(5'to3')
Forward primer: NdeImut-F (5'to3')
反向引物：NdeImut-R序列(5'to3')
Reverse primer: NdeImut-R sequence (5'to3')
PCR反应体系如下：5×phusion HF buffer10μL，ddH2O30.5μL，10mM dNTP 2μL，6PNE-SDm-F 1μL，6PNE-SDm-R 1μL，pGEX-6P-2(稀释20倍)5μL，Phusion HF Enzyme 0.5μL。The PCR reaction system was as follows: 5× phusion HF buffer 10 μL, ddH 2 O 3 0.5 μL, 10 mM dNTP 2 μL, 6PNE-SDm-F 1 μL, 6PNE-SDm-R 1 μL, pGEX-6P-2 (diluted 20 times) 5 μL, and Phusion HF Enzyme 0.5 μL.
PCR反应程序设置：95℃ 3min；95℃ 1min，55℃ 1min，72℃ 10min；循环20次；72℃15min。PCR reaction program settings: 95℃ 3min; 95℃ 1min, 55℃ 1min, 72℃ 10min; 20 cycles; 72℃ 15min.
PCR产物用DpnI消化后转化到宿主菌DH5α中，过夜培养后获得单克隆菌落。将单克隆菌落进行扩大培养，之后由专业的基因测序公司对其中的载体序列进行测序，选出测序结果正确的克隆，然后将克隆扩繁并从中提取质粒，获得成功引入NdeI酶切位点的载体。 The PCR product was digested with DpnI and transformed into the host bacteria DH5α. 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.
2.设计替换SD序列的突变PCR引物，然后通过PCR方法替换原载体的SD序列2. Design a mutant PCR primer to replace the SD sequence, and then replace the SD sequence of the original vector by PCR
引物信息如下:Primer information is as follows:
SDm-F：CAATTTCACACAGGAGGAATTACATATGCCGTCTGAAGCTACSDm-F:CAATTTCACACAGGAGGAATTACATATGCCGTCTGAAGCTAC
SDm-R：GACGGCATATGTAATTCCTCCTGTGTGAAATTGTTATCCSDm-R:GACGGCATATGTAATTCCTCCTGTGTGAAATTGTTATCC
PCR反应体系如下：5×phusion HF buffer10μL，ddH2O30.5μL，10mM dNTP 2μL，SDm-F1μL，SDm-R 1μL，第1步骤获得的质粒5μL，Phusion HF Enzyme 0.5μL。The PCR reaction system was as follows: 5× phusion HF buffer 10 μL, ddH 2 O 3 0.5 μL, 10 mM dNTP 2 μL, SDm-F 1 μL, SDm-R 1 μL, plasmid obtained in step 1 5 μL, Phusion HF Enzyme 0.5 μL.
PCR反应程序设置：95℃3min；95℃1min，55℃1min，72℃10min；循环20次；72℃15min。PCR reaction program settings: 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.
PCR产物用DpnI消化模板DNA后，转化到E.coli DH5α中，过夜培养后获得单克隆菌落。将单克隆菌落进行扩大培养，之后由专业的基因测序公司对其中的载体序列进行测序，选出测序结果正确的克隆，然后将克隆扩繁并从中提取质粒，获得成功替换SD序列的载体。After the PCR product was digested with DpnI, the template DNA was transformed into E. coli DH5α 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.
3.载体进行NdeI和BamHI双酶切去除GST基因3. Double digestion of the vector with NdeI and BamHI to remove the GST gene
酶切体系如下：Cutsmart buffer3μl，ddH2O3μl，1.2获得的载体20μl，NdeI 2μl，BamHI 2μl。37℃酶切2h；0.8％琼脂糖凝胶电泳，120V，1h；切胶获得去除GST基因后的载体片段对应电泳条带，4℃保存。用琼脂糖凝胶回收试剂盒回收载体片段，所得载体片段取3μl电泳检测回收结果。然后将双酶切产物用DNA polymerase I补齐粘性末端，反应体系如下：10×T4DNA Ligase buffer2.5μl，ddH2O1.8μl，胶回收的酶切载体片段20μl，10mM dNTP0.2μl，DNA polymerase I0.5μl，25℃反应15min，加入EDTA(EDTA终浓度为10mM)并且75℃加热20min终止反应。将酶切后并末端补齐的载体进行重新连接环化，连接体系如下：T4 DNA Ligase buffer 2μl，线性平末端载体片段16μl，T4 DNA ligase2μl。16℃连接4h。连接产物消化后转化到E.coliDH5α中，过夜培养后获得单克隆菌落。将单克隆菌落进行扩大培养，之后由专业的基因测序公司对其中的载体序列进行测序，选出测序结果正确的克隆，然后将克隆扩繁并从中提取质粒，获得成功替换SD序列并去除GST基因的载体。The enzyme digestion system is as follows: Cutsmart buffer 3μl, ddH 2 O 3μl, 1.2 obtained vector 20μl, NdeI 2μl, BamHI 2μl. 37℃ 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℃. Use agarose gel recovery kit to recover the vector fragment, and take 3μl 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 2.5μl, ddH 2 O 1.8μl, gel-recovered enzyme digestion vector fragment 20μl, 10mM dNTP 0.2μl, DNA polymerase I 0.5μl, 25℃ reaction for 15min, add EDTA (EDTA final concentration is 10mM) and heat at 75℃ 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 2μl, linear blunt-end vector fragment 16μl, T4 DNA ligase 2μl. Ligation was carried out at 16℃ for 4h. The ligation product was digested and transformed into E.coliDH5α, 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.
4.PCR扩增质粒，重新引入NdeI和BamHI的酶切位点4. PCR amplification of plasmid, reintroduction of NdeI and BamHI restriction sites
PCR引物如下：PCR primers are as follows:
6PNE-SDm-noG-F(5'to3')：CAGGAGATATACATATGGGATCCCCGGAATTCCCG；6PNE-SDm-noG-F (5′to3′): CAGGAGATATACATATGGGATCCCCGGAATTCCCG;
6PNE-SDm-noG-R(5'to3')：GAATTCCGGGGATCCCATATGTATATCTCCTGTGTG。6PNE-SDm-noG-R(5′to3′):GAATTCCGGGGATCCCATATGTATATCTCCTGTGTG.
PCR反应体系如下：5×phusion HF buffer 10μL，ddH2O 30.5μL，10mM dNTP 2μL，6PNE-SDm-noG-F 1μL，6PNE-SDm-noG-R 1μL，模板质粒5μL，Phusion HF Enzyme 0.5μL。The PCR reaction system was as follows: 5× phusion HF buffer 10 μL, ddH 2 O 30.5 μL, 10 mM dNTP 2 μL, 6PNE-SDm-noG-F 1 μL, 6PNE-SDm-noG-R 1 μL, template plasmid 5 μL, and Phusion HF Enzyme 0.5 μL.
PCR反应程序设置：95℃3min；95℃1min，55℃1min，72℃10min；循环20次；72℃15min。PCR产物用DpnI消化模板DNA后，转化到E.coliDH5α中，过夜培养后获得单克隆 菌落。将单克隆菌落进行扩大培养，之后由专业的基因测序公司对其中的载体序列进行测序，选出测序结果正确的克隆，然后将克隆扩繁并从中提取质粒，获得成功替换SD序列、去除GST基因，并重新引入NdeI和BamHI的载体(pKL30)。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 DH5α. 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.
5.利用双酶切连接的方式***毒株ACX32384.1的HPV68L1蛋白基因序列5. Insert the HPV68L1 protein gene sequence of strain ACX32384.1 by double restriction enzyme ligation
利用NdeI和Xho1限制性内切酶分别对上一步所得载体和人工合成的毒株ACX32384.1的HPV68L1蛋白基因(核苷酸序列如GenBank:GQ472851.1，5515-7032bp所示)进行双酶切后回收，之后利用T4 DNA连接酶将回收的载体片段和基因片段进行连接反应，16℃10～15h，获得含有特定SD序列的无标签HPV68L1表达载体(pKL30-HPV68L1)。The vector obtained in the previous step and the HPV68L1 protein gene of the artificially synthesized strain ACX32384.1 (nucleotide sequence as shown in GenBank: GQ472851.1, 5515-7032bp) 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.
通过对不同SD序列的改进发现，本发明所用的SD序列(5'-AGGAGGAATTA-3')能够有效将毒株ACX32384.1的HPV68L1蛋白在大肠杆菌XA90宿主细胞表达出来，但可溶表达量偏低，见图2。而用载体原有的SD序列(5'-AGGAGATATA-3')，目的蛋白并不能表达，见图1。也就是说，通过SDS-PAGE的电泳结果显示，没有经过SD序列改进载体的目的蛋白表达明显差于经过序列改进的SD序列(5'-AGGAGGAATTA-3')载体。Through the improvement of different SD sequences, it was found that 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. However, the target protein cannot be expressed using the original SD sequence (5'-AGGAGATATA-3') of the vector, as shown in Figure 1. In other words, 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.
申请人前期根据一样的方法设计不同的SD序列，命名为：pBSDm(SD序列5'-AGGAGGAATTA-3')，pT1SDm(SD序列5'-AGGAATAA-3')，pT2SDm(SD序列5'-AGAGGTATATA-3')，从图6可以看出只有第一种SD序列有表达(泳道3)，其他2种SD序列未见表达，与未诱导的对照相比，目标位置没有多出明显的条带。The applicant previously designed different SD sequences according to the same method and named them: pBSDm (SD sequence 5'-AGGAGGAATTA-3'), pT1SDm (SD sequence 5'-AGGAATAA-3'), and pT2SDm (SD sequence 5'-AGAGGTATATA-3'). It can be seen from Figure 6 that only the first SD sequence is expressed (lane 3), and the other two SD sequences are not expressed. Compared with the uninduced control, there are no obvious additional bands at the target position.
本发明的附图中，XA90是转化的宿主菌。pKL30是实施例一去除GST基因并经过SD序列(pBSDm：5'-AGGAGGAATTA-3')改造的载体。pKL1是去除GST基因未经过SD序列改造的载体。HPV68L1指的是毒株ACX32384.1的HPV68L1蛋白序列。In the accompanying drawings of the present invention, 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.
实施例二：不同毒株HPV68L1蛋白的筛选及截短后HPV68 L1蛋白的表达Example 2: Screening of HPV68 L1 proteins from different strains and expression of truncated HPV68 L1 proteins
申请人通过很多次试验发现，并不是所有毒株的HPV68型L1蛋白能够在大肠杆菌***表达并组装成质量较好的VLP，例如毒株ACX32384.1等。因此本发明经过序列比对筛选改造，找到了与ACX32384.1毒株有29个氨基酸差异的毒株AAZ39498.1序列做表达，比对结果见图3；毒株AAZ39498.1的HPV68 L1蛋白的氨基酸如SEQ ID NO.1所示。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.
本发明将毒株AAZ39498.1的HPV68 L1蛋白(SEQ ID NO.1)的氨基酸序列做N/C端截短处理，N端截短4个氨基酸，C端截短28个氨基酸，具体的截短后的氨基酸如SEQ ID NO.2所示。具体的，毒株ACX32384.1的HPV68 L1蛋白表达结果见图2，毒株AAZ39498.1的截短的HPV68 L1蛋白(SEQ ID NO.2，标记为HPV68L1a)表达结果见图4。对比图2和图4可知，优化后毒株AAZ39498.1的截短的HPV68 L1的表达量高于毒株ACX32384.1。 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. Specifically, the expression results of the HPV68 L1 protein of the strain ACX32384.1 are shown in Figure 2, and 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.
实施例三：含密码子优化的毒株AAZ39498.1截短的HPV68 L1蛋白表达载体的构建Example 3: Construction of an expression vector containing a truncated HPV68 L1 protein of the codon-optimized strain AAZ39498.1
密码子优化后的毒株AAZ39498.1截短的HPV68L1蛋白(HPV68L1a)的基因为人工合成(序列如SEQ ID No.3所示)，利用NdeI和Xho1限制性内切酶分别对L1基因以及pKL30载体进行双酶切后回收，之后利用T4DNA连接酶将回收的L1基因片段与pKL30载体片段进行连接反应，16℃10～15h。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.
连接体系如下：pKL30载体片段6μl，L1基因片段2μl，T4 DNA ligase 1μl，T4 DNA Ligase buffer 1μl。连接反应后转化连接产物到E.coli DH5α中进行重组子的筛选。将筛选的单克隆菌落进行扩大培养并进行质粒的提取，之后进行测序验证，得到重组表达载体pKL30-HPV68L1a。The ligation system is as follows: 6μl of pKL30 vector fragment, 2μl of L1 gene fragment, 1μl of T4 DNA ligase, and 1μl of T4 DNA Ligase buffer. After the ligation reaction, the ligation product was transformed into E. coli DH5α 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.
实施例四：毒株AAZ39498.1的截短的HPV68 L1蛋白的表达Example 4: Expression of truncated HPV68 L1 protein of strain AAZ39498.1
将实施例三测序结果正确的pKL30-HPV68L1a转化大肠杆菌XA90宿主细胞，并作为表达重组蛋白质的工程菌进行截短的HPV68L1(HPV68L1a)蛋白的表达。表达过程如下：按0.05％的接种量将XA90 pKL30-HPV68L1a接种至LB培养基(Amp+)中，于37℃，220rpm培养16h进行活化。取活化后菌液按0.5％的接种量接种至2YT培养基中，于30℃，220rpm培养7h后添加终浓度为0.2mM的IPTG，于30℃，220rpm诱导表达16h后结束发酵，离心收集菌体用于表达量检测以及纯化实验。通过对SD序列(5'-AGGAGGAATTA-3')的改造结合密码子优化和氨基酸截短三种改进方式，未经改造优化的蛋白可溶表达量低、表达多在沉淀里面，目的蛋白无法纯化回收，相当于表达失败；经过改造优化后蛋白表达量明显提高，并且在上清液中，利于纯化回收，可以实现人***瘤病毒L1抗原蛋白表达从无到有。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. By modifying the SD sequence (5'-AGGAGGAATTA-3') and combining it with three improvements, namely 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. After modification and optimization, 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.
从SDS-PAGE的结果来看，与未经SD序列改造的载体相比，经过SD序列改造的载体的目的蛋白表达量明显提高，但可溶表达量偏低，结果分别见图1及图2。经过技术方案改进之后的毒株AAZ39498.1的截短的HPV68 L1蛋白可溶表达明显提高，结果见图4。由图4可知，XA90 pKL30-HPV68L1a-1(泳道3)和XA90 pKL30-HPV68L1a-2(泳道6)可溶性目的蛋白的表达量提高。截短优化后的HPV68L1蛋白的分子量约为53kDa。From the results of SDS-PAGE, compared with the vector without SD sequence modification, the expression level of the target protein of the vector modified by SD sequence was significantly increased, but the soluble expression level was low, and the results are shown in Figures 1 and 2 respectively. After the technical solution was improved, the soluble expression of the truncated HPV68 L1 protein of strain AAZ39498.1 was significantly improved, and the results are shown in Figure 4. As shown in Figure 4, the expression level of the soluble target protein of XA90 pKL30-HPV68L1a-1 (lane 3) and XA90 pKL30-HPV68L1a-2 (lane 6) was increased. The molecular weight of the truncated and optimized HPV68L1 protein is about 53kDa.
实施例五：截短的HPV68L1蛋白(HPV68L1a)VLP组装Example 5: Truncated HPV68L1 protein (HPV68L1a) VLP assembly
取适量菌体按质量体积比1：10的比例用破菌缓冲液(20mMPB，20mMDTT，pH8.0)充分重悬，然后用高压均质机对菌体进行高压破碎，破碎条件为：800bar，3次。菌体破碎液接着进行高速离心(4℃，12000rpm，60min)收集上清。上清进一步通过饱和度为30％的硫 酸铵沉淀，离心(4℃，12000rpm，60min)收集沉淀，沉淀按质量体积比1：10的比例用复溶Buffer(20mM PB，20mM DTT，pH8.0)充分复溶后再次离心(4℃，12000rpm，60min)收集上清获得粗纯液。粗纯液先上样进行Superdex200分子筛层析，分子筛Buffer(20mM PB，20mM DTT，pH8.0)，根据L1目的蛋白的出峰位置收集其所在组分。接着将分子筛收集样品上样进行Source15Q阴离子交换层析(SQ低盐Buffer：5mM PB，10mM DTT，pH8.0，SQ高盐Buffer：5mM PB，1M NaCl，10mM DTT，pH8.0)，通过0-20％高盐Buffer，10个柱体积线性洗脱收集截短的L1目的蛋白所在组分，该组分即为纯化后截短的L1蛋白。最后调节截短的L1蛋白所在缓冲液的pH和盐浓度至其自组装形成VLP，至此截短的HPV68L1-VLP的制备完成。最后通过动态光散射(DLS)法测定VLP的质量。Take an appropriate amount of cells and resuspend them in 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 ℃, 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 ℃, 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. Then, 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).
表1截短的HPV68L1蛋白(HPV68L1a)组装前后DLS检测结果
Table 1 DLS test results of truncated HPV68L1 protein (HPV68L1a) before and after assembly
从上述表1所示纯化实验结果可知，组装缓冲液的pH在5-5.75，优选pH5，pH5.25，pH5.5，pH5.75，以及盐浓度在1.5M-3.0M之间，优选1.5M，2.0M，2.5M，3.0M时候，截短的HPV68L1(HPV68L1a)的五聚体状态良好(PdI≤0.1)，也能有效组装形成状态良好的VLP(45nm≤粒径大小≤75nm，PdI≤0.1)，纯化所得截短的HPV68L1(HPV68L1a)五聚体的电泳检测结果参见图5。 From the purification experiment results shown in Table 1 above, it can be seen that when the pH of the assembly buffer is between 5-5.75, preferably pH 5, pH 5.25, pH 5.5, pH 5.75, and the salt concentration is between 1.5M-3.0M, preferably 1.5M, 2.0M, 2.5M, 3.0M, the truncated HPV68L1 (HPV68L1a) pentamer is in good condition (PdI≤0.1) and can also be effectively assembled to form VLPs in good condition (45nm≤particle size≤75nm, PdI≤0.1). The electrophoresis detection results of the purified truncated HPV68L1 (HPV68L1a) pentamer are shown in Figure 5.
实施例六：截短的HPV68L1-VLP免疫原性实验结果Example 6: Experimental results of truncated HPV68L1-VLP immunogenicity
本发明的改进点在于研究截短N端或C端不多数量的氨基酸序列以提高表达量，并进一步通过密码子优化和SD序列改造，以便实现HPV68L1蛋白的高效表达。本发明对毒株AAZ39498.1的HPV68型L1蛋白(SEQ ID NO.1)的两端截短只涉及两端，并不涉及其免疫原性的核心区域，即截短后并不会影响其免疫原性；并通过以下是实验加以验证：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:
将上述实施例五获得的截短的HPV68L1-VLP蛋白原液分别与氢氧化铝佐剂吸附配制成疫苗，在4℃保存待用。取VLP蛋白疫苗，以每只0.1ml肌肉注射BAB/c小鼠，每组10只。小鼠每4周加强免疫一次，共免疫2次。二免4周后，采用假病毒细胞中和实验法(由于HPV病毒不能体外人工大量培养，假病毒法为HPV疫苗药效评价的标准方法。所用的假病毒最大限度模拟HPV病毒的结构，其为野生型HPV病毒的L1与L2蛋白共同装配成假病毒颗粒，其中L1及L2蛋白均为未修饰的全长序列)测定免疫后小鼠血清中针对68型HPV的中和抗体滴度，结果如表2。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.
表2截短的HPV68L1-VLP免疫原性研究二免4周中和抗体滴度

注：Gardasil 9为阴性对照，其为Merk公司HPV九价疫苗，不含HPV68抗原Table 2 Neutralizing antibody titers after 4 weeks of second immunization in immunogenicity study of truncated HPV68L1-VLP

Note: Gardasil 9 is a negative control, which is a nine-valent HPV vaccine from Merck and does not contain HPV68 antigen.
由表2的结果可知，本发明HPV68L1的N端或C端的截短不会影响HPV 68L1-VLP的免疫原性。It can be seen from the results in Table 2 that truncation of the N-terminus or C-terminus of HPV68L1 of the present invention will not affect the immunogenicity of HPV 68L1-VLP.
最后应说明的是：以上所述仅为本发明的优先实施例而已，并不用来限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各种实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围内。 Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned various embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

  1. 一种截短的HPV68型L1蛋白，其是在野生型HPV68型L1蛋白的基础上，在其N端截短不多于10个氨基酸，不多于5个氨基酸，优选4个氨基酸，且在其C端截短不多于30个氨基酸，优选28个氨基酸；优选地，截短的HPV68型L1蛋白的氨基酸序列如SEQ ID NO.2所示。A truncated HPV68 type L1 protein, which is based on the wild-type HPV68 type L1 protein, with no more than 10 amino acids, no more than 5 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; preferably, the amino acid sequence of the truncated HPV68 type L1 protein is as shown in SEQ ID NO.2.
  2. 编码如权利要求1所述的截短的HPV68型L1蛋白的核酸；优选地，其经过密码子优化的核酸；更优选地，其核苷酸序列如SEQ ID NO.3所示。A nucleic acid encoding the truncated HPV68 type L1 protein as described in claim 1; preferably, it is a codon-optimized nucleic acid; more preferably, its nucleotide sequence is as shown in SEQ ID NO.3.
  3. 含有SD序列和编码如权利要求1所述的截短的HPV68型L1蛋白的核苷酸序列的核酸，优选地，所述SD序列的核苷酸序列为5'-AGGAGGAATTA-3'。A nucleic acid containing an SD sequence and a nucleotide sequence encoding the truncated HPV68 type L1 protein as claimed in claim 1, preferably, the nucleotide sequence of the SD sequence is 5'-AGGAGGAATTA-3'.
  4. 含有如权利要求2或3所述的编码核酸的表达盒或表达载体。An expression cassette or expression vector containing the encoding nucleic acid as claimed in claim 2 or 3.
  5. 如权利要求4所述的表达盒或表达载体，其特征在于，其是原核表达载体，更优选地是在载体pGEX基础上去除了GST标签序列，并且整合所述SD序列的截短的HPV68型L1蛋白的核酸分子而得到。The expression cassette or expression vector according to claim 4 is characterized in that it is a prokaryotic expression vector, more preferably obtained by removing the GST tag sequence from the vector pGEX and integrating the nucleic acid molecule of the truncated HPV68 type L1 protein of the SD sequence.
  6. 含有如权利要求2或3所述的编码核酸的表达盒或表达载体的重组宿主细胞。A recombinant host cell containing an expression cassette or expression vector encoding the nucleic acid as claimed in claim 2 or 3.
  7. 如权利要求6所述的重组宿主细胞，其特征在于，其是大肠杆菌。The recombinant host cell according to claim 6, characterized in that it is Escherichia coli.
  8. 一种表达如权利要求1截短的HPV68型L1蛋白的方法，其特征在于，培养如权利要求6或7所述的重组宿主细胞以产生截短的HPV68型L1蛋白，任选地，包括纯化步骤，优选地所述纯化步骤为：取所述的重组宿主细胞的菌体用破菌缓冲液充分重悬，然后用高压均质机对菌体进行高压破碎，离心收集上清；上清进一步通过硫酸铵沉淀，硫酸铵的最终饱和度为30％，沉淀复溶后再次离心收集上清获得粗纯液；A method for expressing the truncated HPV68 type L1 protein according to claim 1, characterized in that the recombinant host cell according to claim 6 or 7 is cultured to produce the truncated HPV68 type L1 protein, and optionally, a purification step is included, 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, the final saturation of ammonium sulfate is 30%, and the precipitate is re-dissolved and the supernatant is collected again by centrifugation to obtain a crude pure liquid;
    粗纯液先上样进行Superdex200分子筛层析，根据L1目的蛋白的出峰位置收集其所在组分；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;
    接着将分子筛收集样品上样进行Source15Q阴离子交换层析，通过NaCl线性洗脱收集L1目的蛋白所在组分，得到截短的HPV68型L1蛋白。Then, the sample collected by the molecular sieve was loaded onto Source15Q anion exchange chromatography, and the fraction containing the L1 target protein was collected by NaCl linear elution to obtain a truncated HPV68 type L1 protein.
  9. 一种制备截短的HPV68型L1蛋白VLP的方法，其特征在于，包括如下步骤：根据如权利要求8所述的方法得到的截短的HPV68型L1蛋白的步骤，调节其所在缓冲液的pH和盐浓度，使其自组装形成截短的HPV68型L1蛋白VLP。A method for preparing a truncated HPV68 type L1 protein VLP, characterized in that it comprises the following steps: the step of obtaining the truncated HPV68 type L1 protein according to the method of claim 8, adjusting the pH and salt concentration of the buffer in which the truncated HPV68 type L1 protein is located, so that it self-assembles to form a truncated HPV68 type L1 protein VLP.
  10. 如权利要求9所述的方法，其特征在于，所述缓冲液包括但不限于Tris缓冲液，磷酸盐缓冲液，醋酸缓冲液，HEPES缓冲液，MOPS缓冲液，枸橼酸缓冲液、组氨酸缓冲液， 硼酸缓冲液，优选磷酸盐缓冲液；The method according to claim 9, characterized in that 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;
    缓冲液的pH在5-5.75，优选pH5，pH5.25，pH5.5，pH5.75；盐浓度在1.5M-3.0M之间，优选1.5M，2.0M，2.5M，3.0M；The pH of the buffer is between 5 and 5.75, preferably pH 5, pH 5.25, pH 5.5, and pH 5.75; 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;
    任选地，还包括纯化所得截短的HPV68型L1蛋白VLP的步骤。 Optionally, the method further comprises the step of purifying the obtained truncated HPV68 type L1 protein VLP.
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