WO2024140162A1 - Expression of human papillomavirus hpv59 l1 protein, and virus-like particle and preparation method therefor - Google Patents

Expression of human papillomavirus hpv59 l1 protein, and virus-like particle and preparation method therefor Download PDF

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WO2024140162A1
WO2024140162A1 PCT/CN2023/138114 CN2023138114W WO2024140162A1 WO 2024140162 A1 WO2024140162 A1 WO 2024140162A1 CN 2023138114 W CN2023138114 W CN 2023138114W WO 2024140162 A1 WO2024140162 A1 WO 2024140162A1
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protein
buffer
hpv59
sequence
truncated
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陈晓
高文双
薛俊莲
刘永江
张海江
张瑞霞
陈丹
伍树明
沈迩萃
张尧
银飞
王学红
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
    • 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
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    • 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
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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
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    • 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 construction and expression of human papillomavirus HPV59 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, HPV9, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, etc.): High-risk HPV is closely related to malignant tumors in 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, HPV9, HPV45, HPV51, HPV52, HPV56, HPV58, 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 co-expressed with L1 proteins and 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.
  • HPV59 type is reported in CN202110442661.X, which uses the Hansen yeast expression system to produce HPV59L1 protein.
  • the Hansen yeast expression system is a eukaryotic expression system that directly assembles into VLPs in vivo.
  • the patent does not mention whether the qualified protein can be normally expressed in the Escherichia coli prokaryotic expression system, because the Escherichia coli prokaryotic expression system does not have the post-translational modification function of the Hansen yeast expression system, so it is not suitable to express it in the prokaryotic expression system.
  • HPV59 L1 is somewhat difficult to express. Therefore, it is necessary to study and solve the problem of difficulty in expressing HPV59L1 protein in prokaryotic expression system in order to obtain more uniform VLP and lower cost in industrial application.
  • the inventors expressed the HPV59 L1 protein in a prokaryotic expression system based on the cost of the finished vaccine, and solved the problem of difficulty in expressing the HPV59 L1 protein in a prokaryotic expression system. This was achieved specifically through the following improvements: the amino acid sequence of the HPV59 L1 protein 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 amino acid sequence of HPV59L1 protein (SEQ ID NO.1) is truncated at the N/C terminal in order to obtain a better protein expression rate. 4 amino acids are truncated at the N terminal, and 31 amino acids are truncated at the C terminal. The specific amino acids after truncation are shown in SEQ ID NO.2. After N/C terminal truncation, it can be expressed on a tag-free expression vector and higher quality proteins and VLPs can be obtained.
  • sequence shown in SEQ ID NO.2 is as follows:
  • the inventors performed codon optimization 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 commonly used restriction endonuclease recognition sites.
  • the nucleotide sequence was optimized according to the above principles and screened multiple times to obtain the optimized nucleotide sequence shown in SEQ ID NO.3, and an expression cassette containing the encoding nucleic acid, an expression vector and a recombinant host cell are further provided.
  • it is E. coli.
  • sequence shown in SEQ ID NO.3 is as follows:
  • the present invention provides a tag-free expression vector of a specific SD sequence.
  • the vector pGEX expressing the fusion protein is characterized by having a 26kDa glutathione S-transferase gene (GST) on 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 HPV59 type L1 protein to form a new expression vector suitable for the HPV59 L1 protein.
  • the replaced SD sequence is AGGAGGAATTA (5'to3').
  • the present invention also provides a method for preparing HPV59 type L1 VLP, comprising the following steps: adjusting the pH and salt concentration of the buffer solution in which the HPV59 type L1 protein is obtained according to the method to enable it to self-assemble into 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; 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 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 Results of electrophoresis detection of expression in small shake flasks of XA90 pKL1-HPV59L1 (SD was not modified, i.e., the SD sequence was AGGAGATATA).
  • M marker; 1. XA90 pKL1 negative control; 2. XA90 pKL1-HPV59L1-1 whole bacteria; 3. XA90 pKL1-HPV59L1-1 supernatant; 4. XA90 pKL1-HPV59L1-1 precipitate; 5. HPV18L1; 6. XA90 pKL1-HPV59L1-2 whole bacteria; 7. XA90 pKL1-HPV59L1-2 supernatant; 8. XA90 pKL1-HPV59L1-2 precipitate.
  • XA90 is the host bacteria
  • pKL1 is the vector with the SD sequence of AGGAGATATA.
  • FIG. 1 Protein expression test results of different SD sequences.
  • M marker; 1. Uninduced XA90 pBSDm-59L1; 2. Induced whole bacteria XA90 pBSDm-59L1; 3. Induced supernatant XA90 pBSDm-59L1; 4. Induced precipitate XA90 pBSDm-59L1; 5. Uninduced XA90 pT1SDm-59L1; 6. Induced whole bacteria XA90 pT1SDm-59L1; 7. Induced supernatant XA90 pBSDm-59L1 Clear XA90 pT1SDm-59L1; 8.
  • PCR primers The names and sequences of PCR primers are as follows:
  • Reverse primer 6p1-NdeImut-R sequence (5'to3'):
  • the PCR reaction system was as follows: 10 ⁇ L of 5 ⁇ phusion HF buffer, 0.5 ⁇ L of ddH 2 O3, 2 ⁇ L of 10 mM dNTP, 1 ⁇ L of 6PNE-SDm-F, 1 ⁇ L of 6PNE-SDm-R, 5 ⁇ L of pGEX-6P-2 (diluted 20 times), and 0.5 ⁇ L of Phusion HF enzyme.
  • the PCR product was digested with DpnI and transformed into E. coli DH5 ⁇ , 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, and the clones with correct sequencing results were selected. Then the clones were expanded and plasmids were extracted from them to obtain the vectors with the NdeI restriction site successfully introduced.
  • Primer information is as follows:
  • the PCR reaction system was as follows: 10 ⁇ L of 5 ⁇ phusion HF buffer, 0.5 ⁇ L of ddH 2 O3, 2 ⁇ L of 10 mM dNTP, 1 ⁇ L of 6PNE-SDm-F, 1 ⁇ L of 6PNE-SDm-R, 5 ⁇ L of the plasmid obtained in step 1 above, and 0.5 ⁇ L of Phusion HF Enzyme.
  • 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 a vector that successfully replaced the SD sequence.
  • the replaced SD sequence is AGGAGATATA (5' to 3').
  • the enzyme digestion system is as follows: Cutsmart buffer 3 ⁇ l, ddH 2 O 3 ⁇ l, vector obtained in step 2 above 20 ⁇ l, NdeI 2 ⁇ l, BamHI 2 ⁇ l.
  • the ligation product was transformed into E.coli DH5 ⁇ and monoclonal colonies were obtained after overnight culture. The monoclonal colonies were expanded and then sequenced by a professional gene sequencing company. The clones with correct sequencing results were selected and then The clone was then expanded and plasmids were extracted from it to obtain a vector that successfully replaced the SD sequence and removed the GST gene.
  • PCR primers are as follows:
  • the PCR reaction system was as follows: 10 ⁇ L of 5 ⁇ phusion HF buffer, 30.5 ⁇ L of ddH 2 O, 2 ⁇ L of 10 mM dNTP, 1 ⁇ L of 6PNE-SDm-noG-F, 1 ⁇ L of 6PNE-SDm-noG-R, 5 ⁇ L of template plasmid, and 0.5 ⁇ L of Phusion HF enzyme.
  • the PCR product was digested with DpnI template DNA, it was transformed into E. coli DH5 ⁇ and cultured overnight to obtain a monoclonal colony.
  • the monoclonal colony was expanded and cultured, and then the vector sequence was sequenced by a professional gene sequencing company, and the clone with the correct sequencing result was selected.
  • the clone was then expanded and the plasmid was extracted from it to obtain a vector that successfully replaced the SD sequence, removed the GST gene, and reintroduced the NdeI and BamHI restriction sites.
  • the vector pKL1 SD sequence 1: AGGAGATATA
  • the other three SD sequence corresponding vectors pBSDm, pT1SDm and pT2SDm vectors were constructed respectively.
  • the specific gene sequence information of the three SD sequences is as follows:
  • SD sequence 2 (pBSDm): AGGAGGAATTA;
  • SD sequence 3 (pT1SDm): AGGAATAA;
  • SD sequence 4 (pT2SDm): AGAGGTATATA.
  • Example 2 Construction of an expression vector containing a codon-optimized HPV59 L1 gene
  • HPV59L1 human papillomavirus type 59 coat protein L1
  • SEQ ID NO.1 The wild-type full-length amino acid sequence of human papillomavirus type 59 coat protein L1 (HPV59L1) is shown in SEQ ID NO.1. It was truncated, that is, 4 amino acids were truncated at the N-terminus and 31 amino acids were truncated at the C-terminus.
  • the amino acid sequence of the truncated HPV59 L1 protein is shown in SEQ ID NO.2.
  • the nucleotide sequence encoded by it was codon-optimized and artificially synthesized, as shown in SEQ ID NO.3.
  • the gene fragment of the truncated HPV59 L1 was first PCR amplified, and the L1 gene PCR fragment containing NdeI and Xho1 restriction sites and the recombinant vectors pKL1, pBSDm, pT1SDm and pT2SDm were double-digested with NdeI and Xho1, respectively. Then, the recovered gene fragments were ligated with the pKL1, pBSDm, pT1SDm and pT2SDm vectors containing the corresponding sticky ends using T4 DNA ligase, and the reaction was carried out at 16°C for 10 to 15 hours.
  • the ligation system is as follows: 6 ⁇ l of vector fragment, 2 ⁇ l of HPV59L1 gene fragment, 1 ⁇ l of T4 DNA ligase, and 1 ⁇ l of T4 DNA ligase buffer. After the ligation reaction, the ligation product is transformed into E. coli DH5 ⁇ for recombinant screening. The cloned colonies were expanded and cultured, and the plasmids were extracted and then sequenced to verify the recombinant expression vectors pKL1-HPV59L1, pBSDm-HPV59L1, pT1SDm-HPV59L1 and pT2SDm-HPV59L1.
  • Example 2 The four recombinant vectors with correct sequencing results in Example 2 were transformed into Escherichia coli XA90 host cells and used as engineered bacteria to express recombinant proteins for the expression of HPV L1 protein. 0.05% of the inoculum was inoculated into LB medium (Amp+) and cultured at 37°C, 220rpm for 16h for activation. The activated bacterial solution was inoculated into 2YT medium at an inoculum of 0.5%, cultured at 30°C, 220rpm for 7h, and then IPTG was added with a final concentration of 0.2mM.
  • XA90 is the transformed host bacteria.
  • XA90 pKL1 is a negative control.
  • the XA90 pKL1-59L1 (expression vector without SD sequence modification) test sample lane corresponding to the theoretical molecular weight of the target protein (about 52.89KDa) did not show an expression band.
  • the XA90 pBSDm-59L1 i.e., the SD sequence used in the present invention, SD sequence 2: AGGAGGAATTA
  • test sample lane corresponding to the theoretical molecular weight of the target protein showed an obvious expression band (indicated by the arrow), and the target protein unit bacterial expression amount was about 0.3mg/g wet bacterial body.
  • XA90 pBSDm-59L1 bacteria Take an appropriate amount of XA90 pBSDm-59L1 bacteria and fully resuspend them in a lysis buffer (20mM PB, 20mM DTT, pH8.0) at a mass volume ratio of 1:10, and then use a high-pressure homogenizer to high-pressure rupture the bacteria.
  • the rupture conditions are: 800bar, 3 times.
  • the bacterial rupture liquid is then high-speed centrifuged (4°C, 12000rpm, 60min) to collect the supernatant.
  • the supernatant is further precipitated by ammonium sulfate with a saturation of 30%, and the precipitate is collected by centrifugation (4°C, 12000rpm, 60min).
  • the precipitate is precipitated by mass volume.
  • the mixture was fully re-dissolved with a re-dissolution buffer (20mM PB, 20mM DTT, pH8.0) at a 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 onto Superdex200 molecular sieve chromatography, molecular sieve buffer (20mM PB, 20mM DTT, pH8.0), and the fractions of the L1 target protein were collected according to the peak position of the L1 target protein.
  • the molecular sieve collected sample was loaded onto 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 fractions of the L1 target protein were collected by linear elution with 0-20% high salt buffer and 10 column volumes, which was the purified L1 protein.
  • the mass of the L1 pentamer was determined by dynamic light scattering (DLS).
  • DLS dynamic light scattering
  • the pH and salt concentration of the buffer containing the L1 protein were adjusted to allow it to self-assemble to form VLPs, and the preparation of VLPs was completed. Finally, the quality of VLPs was determined by DLS.
  • HPV59L1-VLP prepared in Example 4 was used to investigate the long-term stability data at -70°C.
  • the investigation results are as follows.

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Abstract

The expression of a human papillomavirus HPV59 L1 protein, and a virus-like particle and a preparation method therefor. The method comprises: truncating the amino acid sequence of the HPV59 L1 protein; performing codon optimization on the nucleotide sequence encoding the truncated protein to obtain an optimized encoding nucleotide sequence; and finally achieving untagged expression and purification with an untagged expression vector containing a specific SD sequence. By means of the above-mentioned improvement, a higher protein expression level can be obtained in a prokaryotic expression system such as an Escherichia coli expression system, and the VLP with more uniform quality can be obtained.

Description

人***瘤病毒HPV59 L1蛋白的表达和类病毒样颗粒及其制备方法Expression of human papillomavirus HPV59 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HPV59 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 construction and expression of human papillomavirus HPV59 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、HPV9、HPV45、HPV51、HPV52、HPV56、HPV58、HPV59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, HPV9, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, etc.): High-risk HPV is closely related to malignant tumors in 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 co-expressed with L1 proteins and 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HPV59型在CN202110442661.X中报道，采用的是汉逊酵母表达***产生HPV59L1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是否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 HPV59 L1是有一定难度的。因此，需要研究解决在原核表达***里面表达HPV59L1蛋白困难的问题，以获得更均一的VLP以及在产业应用上有更低的成本。Currently, the HPV59 type is reported in CN202110442661.X, which uses the Hansen yeast expression system to produce HPV59L1 protein. The Hansen yeast expression system is a eukaryotic expression system that directly assembles into VLPs in vivo. The patent does not mention whether the qualified protein can be normally expressed in the Escherichia coli prokaryotic expression system, because the Escherichia coli prokaryotic expression system does not have the post-translational modification function of the Hansen yeast expression system, so it is not suitable to express it in the prokaryotic expression system. HPV59 L1 is somewhat difficult to express. Therefore, it is necessary to study and solve the problem of difficulty in expressing HPV59L1 protein in prokaryotic expression system in order to obtain more uniform VLP and lower cost in industrial application.
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HPV59 L1蛋白，并解决了在原核表达***里面表达HPV59L1蛋白困难的问题。具体通过以下改进实现：对HPV59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 HPV59 L1 protein in a prokaryotic expression system based on the cost of the finished vaccine, and solved the problem of difficulty in expressing the HPV59 L1 protein in a prokaryotic expression system. This was achieved specifically through the following improvements: the amino acid sequence of the HPV59 L1 protein 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，本发明将HPV59L1蛋白的氨基酸序列(SEQ ID NO.1)做N/C端截短处理，为了获得更好的蛋白表达率。N端截短4个氨基酸，且在其C端截短31个氨基酸，具体的截短后的氨基酸见SEQ ID NO.2。N/C端截短处理后，可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 amino acid sequence of HPV59L1 protein (SEQ ID NO.1) is truncated at the N/C terminal in order to obtain a better protein expression rate. 4 amino acids are truncated at the N terminal, and 31 amino acids are truncated at the C terminal. The specific amino acids after truncation are shown in SEQ ID NO.2. After N/C terminal truncation, it can be expressed on a tag-free expression vector and higher quality proteins and VLPs can be obtained.
其中，SEQ ID NO.2所示序列如下：
Wherein, the sequence shown in SEQ ID NO.2 is as follows:
其次，为了利用大肠杆菌***高效的表达HPV59L1蛋白，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，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。Secondly, in order to efficiently express HPV59L1 protein using the E. coli system, the inventors performed codon optimization 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 commonly used restriction endonuclease recognition sites. The nucleotide sequence was optimized according to the above principles and screened multiple times to obtain the optimized nucleotide sequence shown in SEQ ID NO.3, and an expression cassette containing the encoding nucleic acid, an expression vector and a recombinant host cell are further provided. Preferably, it is E. coli.
其中，SEQ ID NO.3所示序列如下：

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

最后，本发明提供特定SD序列的无标签表达载体。对于表达载体而言，表达融合蛋白的载体pGEX的特点是在载体上具有一种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HPV59型L1蛋白的SD序列，而形成新的适合HPV59 L1蛋白的表达载体。换后的SD序列为AGGAGGAATTA(5'to3')。Finally, the present invention provides a tag-free expression vector of a specific SD sequence. For the expression vector, the vector pGEX expressing the fusion protein is characterized by having a 26kDa glutathione S-transferase gene (GST) on 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 HPV59 type L1 protein to form a new expression vector suitable for the HPV59 L1 protein. The replaced SD sequence is AGGAGGAATTA (5'to3').
本发明还提供一种制备HPV59型L1VLP的方法，包括如下步骤：根据所述的方法得到的HPV59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VLP。The present invention also provides a method for preparing HPV59 type L1 VLP, comprising the following steps: adjusting the pH and salt concentration of the buffer solution in which the HPV59 type L1 protein is obtained according to the method to enable it to self-assemble into 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; 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.
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1XA90 pKL1-HPV59L1小摇瓶表达电泳检测结果(SD未改造，即SD序列为AGGAGATATA)。其中，M:marker；1.XA90pKL1阴性对照；2.XA90 pKL1-HPV59L1-1全菌；3.XA90 pKL1-HPV59L1-1上清；4.XA90 pKL1-HPV59L1-1沉淀；5.HPV18L1；6.XA90 pKL1-HPV59L1-2全菌；7.XA90 pKL1-HPV59L1-2上清；8.XA90 pKL1-HPV59L1-2沉淀。XA90是宿主菌，pKL1是SD序列为AGGAGATATA的载体。Figure 1 Results of electrophoresis detection of expression in small shake flasks of XA90 pKL1-HPV59L1 (SD was not modified, i.e., the SD sequence was AGGAGATATA). Among them, M: marker; 1. XA90 pKL1 negative control; 2. XA90 pKL1-HPV59L1-1 whole bacteria; 3. XA90 pKL1-HPV59L1-1 supernatant; 4. XA90 pKL1-HPV59L1-1 precipitate; 5. HPV18L1; 6. XA90 pKL1-HPV59L1-2 whole bacteria; 7. XA90 pKL1-HPV59L1-2 supernatant; 8. XA90 pKL1-HPV59L1-2 precipitate. XA90 is the host bacteria, and pKL1 is the vector with the SD sequence of AGGAGATATA.
图2不同SD序列的蛋白表达检测结果。其中，M:marker；1.未诱导XA90 pBSDm-59L1；2.诱导全菌XA90 pBSDm-59L1；3.诱导上清XA90 pBSDm-59L1；4.诱导沉淀XA90 pBSDm-59L1；5.未诱导XA90 pT1SDm-59L1；6.诱导全菌XA90 pT1SDm-59L1；7.诱导上清XA90 pT1SDm-59L1；8.诱导沉淀XA90 pT1SDm-59L1；9.未诱导XA90 pT2SDm-59L1；10.诱导全菌XA90 pT2SDm-59L1；11.诱导上清XA90 pT2SDm-59L1；12.诱导沉淀XA90 pT2SDm-59L1；13.对照XA90 pKL1。Figure 2 Protein expression test results of different SD sequences. Wherein, M: marker; 1. Uninduced XA90 pBSDm-59L1; 2. Induced whole bacteria XA90 pBSDm-59L1; 3. Induced supernatant XA90 pBSDm-59L1; 4. Induced precipitate XA90 pBSDm-59L1; 5. Uninduced XA90 pT1SDm-59L1; 6. Induced whole bacteria XA90 pT1SDm-59L1; 7. Induced supernatant XA90 pBSDm-59L1 Clear XA90 pT1SDm-59L1; 8. Induced precipitate XA90 pT1SDm-59L1; 9. Uninduced XA90 pT2SDm-59L1; 10. Induced whole bacteria XA90 pT2SDm-59L1; 11. Induced supernatant XA90 pT2SDm-59L1; 12. Induced precipitate XA90 pT2SDm-59L1; 13. Control XA90 pKL1.
图3 HPV59L1五聚体电泳检测结果M:marker；1.HPV59L1五聚体。Figure 3 HPV59L1 pentamer electrophoresis detection results M: marker; 1. HPV59L1 pentamer.
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序列的无标签表达载体的构建Example 1: Construction of a tag-free expression vector containing a specific SD sequence
1.通过突变PCR，在pGEX-6P-2质粒中引入NdeI酶切位点：1. Introduce NdeI restriction site into pGEX-6P-2 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：6p1-NdeImut-F(5'to3')：
Forward primer: 6p1-NdeImut-F (5'to3'):
反向引物：6p1-NdeImut-R序列(5'to3')：
Reverse primer: 6p1-NdeImut-R sequence (5'to3'):
PCR反应体系如下：5×phusion HF缓冲液10μL，ddH2O30.5μL，10mM dNTP 2μL，6PNE-SDm-F 1μL，6PNE-SDm-R 1μL，pGEX-6P-2(稀释20倍)5μL，Phusion HF酶0.5μL。 The PCR reaction system was as follows: 10 μL of 5× phusion HF buffer, 0.5 μL of ddH 2 O3, 2 μL of 10 mM dNTP, 1 μL of 6PNE-SDm-F, 1 μL of 6PNE-SDm-R, 5 μL of pGEX-6P-2 (diluted 20 times), and 0.5 μL of Phusion HF enzyme.
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消化后转化到大肠杆菌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 E. coli DH5α, 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, and the clones with correct sequencing results were selected. Then the clones were expanded and plasmids were extracted from them to obtain the vectors 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:
6PNE-SDm-F(5'to3')：CAATTTCACACAGGAGATATACATATGTCCCCTATACTAGG6PNE-SDm-F(5'to3'):CAATTTCACACAGGAGATATACATATGTCCCCTATACTAGG
6PNE-SDm-R(5'to3')：GTATAGGGGACATATGTATATCTCCTGTGTGAAATTGTTATCC6PNE-SDm-R(5'to3'):GTATAGGGGACATATGTATATCTCCTGTGTGAAATTGTTATCC
PCR反应体系如下：5×phusion HF缓冲液10μL，ddH2O30.5μL，10mM dNTP 2μL，6PNE-SDm-F 1μL，6PNE-SDm-R 1μL，上述步骤1获得的质粒5μL，Phusion HF Enzyme 0.5μL。The PCR reaction system was as follows: 10 μL of 5× phusion HF buffer, 0.5 μL of ddH 2 O3, 2 μL of 10 mM dNTP, 1 μL of 6PNE-SDm-F, 1 μL of 6PNE-SDm-R, 5 μL of the plasmid obtained in step 1 above, and 0.5 μL of Phusion HF Enzyme.
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.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序列的载体。替换后的SD序列为AGGAGATATA(5'to3')。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 a vector that successfully replaced the SD sequence. The replaced SD sequence is AGGAGATATA (5' to 3').
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缓冲液3μl，ddH2O3μl，上述步骤2获得的载体20μl，NdeI 2μl，BamHI 2μl。The enzyme digestion system is as follows: Cutsmart buffer 3 μl, ddH 2 O 3 μl, vector obtained in step 2 above 20 μl, NdeI 2 μl, BamHI 2 μl.
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。Enzyme digestion at 37°C for 2h; 0.8% agarose gel electrophoresis, 120V, 1h; cut the gel to obtain the electrophoresis band corresponding to the vector fragment after the GST gene is removed, and store at 4°C.
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聚合酶I补齐粘性末端，反应体系如下：10×T4 DNA连接酶缓冲液2.5μl，ddH2O1.8μl，胶回收的酶切载体片段20μl，10mM dNTP0.2μl，DNA聚合酶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。The vector fragment was recovered by agarose gel recovery kit, and 3 μl of the obtained vector fragment was taken for electrophoresis to detect the recovery result. Then the double-digested product was filled with DNA polymerase I to fill the sticky ends. The reaction system was as follows: 2.5 μl of 10×T4 DNA ligase buffer, 1.8 μl of ddH 2 O, 20 μl of gel-recovered digested vector fragment, 0.2 μl of 10 mM dNTP, 0.5 μl of DNA polymerase I, reacted at 25°C for 15 min, and EDTA (final EDTA concentration was 10 mM) was added and heated at 75°C for 20 min to terminate the reaction.
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连接酶缓冲液2μl，线性平末端载体片段16μl，T4 DNA连接酶2μl。16℃连接4h。The vector was re-ligated and circularized after restriction digestion. The ligation system was as follows: 2 μl of T4 DNA ligase buffer, 16 μl of linear blunt-end vector fragment, and 2 μl of T4 DNA ligase. Ligation was carried out at 16°C for 4 hours.
连接产物转化到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 ligation product was transformed into E.coli DH5α and monoclonal colonies were obtained after overnight culture. The monoclonal colonies were expanded and then sequenced by a professional gene sequencing company. The clones with correct sequencing results were selected and then The clone was then expanded and plasmids were extracted from it to obtain a vector 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')：CAGGAGATATACATATGGGATCCCCGGAATTCCCG6PNE-SDm-noG-F(5'to3'):CAGGAGATATACATATGGGATCCCCGGAATTCCCG
6PNE-SDm-noG-R(5'to3')：GAATTCCGGGGATCCCATATGTATATCTCCTGTGTG6PNE-SDm-noG-R(5'to3'):GAATTCCGGGGATCCCATATGTATATCTCCTGTGTG
PCR反应体系如下：5×phusion HF缓冲液10μL，ddH2O 30.5μL，10mM dNTP 2μL，6PNE-SDm-noG-F 1μL，6PNE-SDm-noG-R 1μL，模板质粒5μL，Phusion HF酶0.5μL。The PCR reaction system was as follows: 10 μL of 5× phusion HF buffer, 30.5 μL of ddH 2 O, 2 μL of 10 mM dNTP, 1 μL of 6PNE-SDm-noG-F, 1 μL of 6PNE-SDm-noG-R, 5 μL of template plasmid, and 0.5 μL of Phusion HF enzyme.
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.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酶切位点的载体。至此，载体pKL1(SD序列1：AGGAGATATA)构建完毕。After the PCR product was digested with DpnI template DNA, it was transformed into E. coli DH5α and cultured overnight to obtain a monoclonal colony. The monoclonal colony was expanded and cultured, and then the vector sequence was sequenced by a professional gene sequencing company, and the clone with the correct sequencing result was selected. The clone was then expanded and the plasmid was extracted from it to obtain a vector that successfully replaced the SD sequence, removed the GST gene, and reintroduced the NdeI and BamHI restriction sites. At this point, the vector pKL1 (SD sequence 1: AGGAGATATA) was constructed.
按照上述方法分别构建另外3种SD序列对应载体pBSDm、pT1SDm和pT2SDm载体。3种SD序列的具体基因序列信息如下：According to the above method, the other three SD sequence corresponding vectors pBSDm, pT1SDm and pT2SDm vectors were constructed respectively. The specific gene sequence information of the three SD sequences is as follows:
SD序列2(pBSDm)：AGGAGGAATTA；SD sequence 2 (pBSDm): AGGAGGAATTA;
SD序列3(pT1SDm)：AGGAATAA；SD sequence 3 (pT1SDm): AGGAATAA;
SD序列4(pT2SDm)：AGAGGTATATA。SD sequence 4 (pT2SDm): AGAGGTATATA.
实施例二：含密码子优化的HPV59 L1基因的表达载体的构建Example 2: Construction of an expression vector containing a codon-optimized HPV59 L1 gene
人***瘤病毒59型外壳蛋白L1(HPV59L1)的野生型全长氨基酸序列如SEQ ID NO.1所示。对其进行截短处理，即N端截短4个氨基酸，且其C端截短31个氨基酸，截短的HPV59型L1蛋白的氨基酸序列如SEQ ID NO.2所示，其编码的核苷酸序列经过密码子优化，由人工合成，具体见SEQ ID NO.3，先PCR扩增截短型HPV59 L1的基因片段，将含有NdeI和Xho1酶切位点的L1基因PCR片段以及重组载体pKL1、pBSDm、pT1SDm和pT2SDm分别进行NdeI和Xho1双酶切，之后利用T4 DNA连接酶将回收的基因片段分别与含有对应粘性末端的pKL1、pBSDm、pT1SDm和pT2SDm载体进行连接反应，16℃10～15h。The wild-type full-length amino acid sequence of human papillomavirus type 59 coat protein L1 (HPV59L1) is shown in SEQ ID NO.1. It was truncated, that is, 4 amino acids were truncated at the N-terminus and 31 amino acids were truncated at the C-terminus. The amino acid sequence of the truncated HPV59 L1 protein is shown in SEQ ID NO.2. The nucleotide sequence encoded by it was codon-optimized and artificially synthesized, as shown in SEQ ID NO.3. The gene fragment of the truncated HPV59 L1 was first PCR amplified, and the L1 gene PCR fragment containing NdeI and Xho1 restriction sites and the recombinant vectors pKL1, pBSDm, pT1SDm and pT2SDm were double-digested with NdeI and Xho1, respectively. Then, the recovered gene fragments were ligated with the pKL1, pBSDm, pT1SDm and pT2SDm vectors containing the corresponding sticky ends using T4 DNA ligase, and the reaction was carried out at 16°C for 10 to 15 hours.
连接体系如下：载体片段6μl，HPV59L1基因片段2μl，T4 DNA连接酶1μl，T4 DNA连接酶缓冲液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pKL1-HPV59L1、pBSDm-HPV59L1、pT1SDm-HPV59L1和pT2SDm-HPV59L1。The ligation system is as follows: 6 μl of vector fragment, 2 μl of HPV59L1 gene fragment, 1 μl of T4 DNA ligase, and 1 μl of T4 DNA ligase buffer. After the ligation reaction, the ligation product is transformed into E. coli DH5α for recombinant screening. The cloned colonies were expanded and cultured, and the plasmids were extracted and then sequenced to verify the recombinant expression vectors pKL1-HPV59L1, pBSDm-HPV59L1, pT1SDm-HPV59L1 and pT2SDm-HPV59L1.
实施例三：HPV59 L1蛋白的表达Example 3: Expression of HPV59 L1 protein
将实施例二测序结果正确的4种重组载体转化大肠杆菌XA90宿主细胞，并作为表达重组蛋白质的工程菌进行HPV L1蛋白的表达。0.05％的接种量接种至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诱导培养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。从SDS-PAGE的结果来看，未经SD序列改造的原载体pKL1-59L1(SD序列1：AGGAGATATA)不能有效表达目的蛋白(见图1)，而通过对不同SD序列的改进发现，筛选的另外3种SD序列(SD序列2(pBSDm载体)：AGGAGGAATTA，SD序列3(pT1SDm)：AGGAATAA，SD序列4(pT2SDm)：AGAGGTATATA)中仅本发明所用的SD序列，即SD序列2(pBSDm载体)：AGGAGGAATTA能够有效地将HPV59L1目的蛋白表达出来，剩余2种SD序列也不能有效表达目的蛋白(见图2)。The four recombinant vectors with correct sequencing results in Example 2 were transformed into Escherichia coli XA90 host cells and used as engineered bacteria to express recombinant proteins for the expression of HPV L1 protein. 0.05% of the inoculum was inoculated into LB medium (Amp+) and cultured at 37°C, 220rpm for 16h for activation. The activated bacterial solution was inoculated into 2YT medium at an inoculum of 0.5%, cultured at 30°C, 220rpm for 7h, and then IPTG was added with a final concentration of 0.2mM. After induction culture at 30°C, 220rpm for 16h, the fermentation was terminated and the bacteria were collected by centrifugation for expression detection and purification experiments. From the results of SDS-PAGE, it can be seen that the original vector pKL1-59L1 (SD sequence 1: AGGAGATATA) without SD sequence modification cannot effectively express the target protein (see Figure 1). However, through the improvement of different SD sequences, it was found that among the other three SD sequences screened (SD sequence 2 (pBSDm vector): AGGAGGAATTA, SD sequence 3 (pT1SDm): AGGAATAA, SD sequence 4 (pT2SDm): AGAGGTATATA), only the SD sequence used in the present invention, i.e. SD sequence 2 (pBSDm vector): AGGAGGAATTA can effectively express the HPV59L1 target protein, and the remaining two SD sequences cannot effectively express the target protein (see Figure 2).
图1和图2中XA90是转化的宿主菌。如图1中，XA90 pKL1为阴性对照。与阴性对照相比，XA90 pKL1-59L1(未经SD序列改造的表达载体)试验样品泳道对应目的蛋白理论分子量大小(约52.89KDa)的位置没有出现表达条带。如图2中，与XA90 pKL1阴性对照相比，XA90 pBSDm-59L1(即本发明所用的SD序列，SD序列2：AGGAGGAATTA)试验样品泳道对应目的蛋白理论分子量大小的位置出现了明显的表达条带(箭头所指)，目的蛋白单位菌体表达量约为0.3mg/g湿菌体。而XA90 pT1SDm-59L1(SD序列3：AGGAATAA)和XA90 pT2SDm-59L1(SD序:4：AGAGGTATATA)试验样品泳道对应目的蛋白理论分子量大小的位置也没有出现表达条带。In Figures 1 and 2, XA90 is the transformed host bacteria. As shown in Figure 1, XA90 pKL1 is a negative control. Compared with the negative control, the XA90 pKL1-59L1 (expression vector without SD sequence modification) test sample lane corresponding to the theoretical molecular weight of the target protein (about 52.89KDa) did not show an expression band. As shown in Figure 2, compared with the XA90 pKL1 negative control, the XA90 pBSDm-59L1 (i.e., the SD sequence used in the present invention, SD sequence 2: AGGAGGAATTA) test sample lane corresponding to the theoretical molecular weight of the target protein showed an obvious expression band (indicated by the arrow), and the target protein unit bacterial expression amount was about 0.3mg/g wet bacterial body. However, no expression bands appeared at the positions corresponding to the theoretical molecular weights of the target proteins in the experimental sample lanes of XA90 pT1SDm-59L1 (SD sequence 3: AGGAATAA) and XA90 pT2SDm-59L1 (SD sequence: 4: AGAGGTATATA).
从SDS-PAGE的结果可以看出，经过技术方案的改进实现了HPV59L1蛋白的可溶高效表达(图2，泳道2及3)。From the results of SDS-PAGE, it can be seen that the soluble and efficient expression of HPV59L1 protein was achieved through the improvement of the technical solution (Figure 2, lanes 2 and 3).
实施例四：HPV59L1蛋白纯化及VLP组装Example 4: HPV59L1 protein purification and VLP assembly
取适量XA90 pBSDm-59L1菌体按质量体积比1：10的比例用破菌缓冲液(20mM PB，20mM DTT，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的比例用复溶缓冲液(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，分子筛缓冲液(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低盐缓冲液：5mM PB，10mM DTT，pH8.0，SQ高盐缓冲液：5mM PB，1M NaCl，10mM DTT，pH8.0)，通过0-20％高盐缓冲液，10个柱体积线性洗脱收集L1目的蛋白所在组分，该组分即为纯化后L1蛋白。通过动态光散射(DLS)法测定L1五聚体的质量。最后调节L1蛋白所在缓冲液的pH和盐浓度至其自组装形成VLP，至此VLP的制备完成。最后通过DLS测定VLP的质量。Take an appropriate amount of XA90 pBSDm-59L1 bacteria and fully resuspend them in a lysis buffer (20mM PB, 20mM DTT, pH8.0) at a mass volume ratio of 1:10, and then use a high-pressure homogenizer to high-pressure rupture the bacteria. The rupture conditions are: 800bar, 3 times. The bacterial rupture liquid is then high-speed centrifuged (4°C, 12000rpm, 60min) to collect the supernatant. The supernatant is further precipitated by ammonium sulfate with a saturation of 30%, and the precipitate is collected by centrifugation (4°C, 12000rpm, 60min). The precipitate is precipitated by mass volume. The mixture was fully re-dissolved with a re-dissolution buffer (20mM PB, 20mM DTT, pH8.0) at a 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 onto Superdex200 molecular sieve chromatography, molecular sieve buffer (20mM PB, 20mM DTT, pH8.0), and the fractions of the L1 target protein were collected according to the peak position of the L1 target protein. Then, the molecular sieve collected sample was loaded onto 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 fractions of the L1 target protein were collected by linear elution with 0-20% high salt buffer and 10 column volumes, which was the purified L1 protein. The mass of the L1 pentamer was determined by dynamic light scattering (DLS). Finally, the pH and salt concentration of the buffer containing the L1 protein were adjusted to allow it to self-assemble to form VLPs, and the preparation of VLPs was completed. Finally, the quality of VLPs was determined by DLS.
表1 HPV59L1蛋白组装前后DLS检测结果
Table 1 DLS test results of HPV59L1 protein 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，HPV59L1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HPV59L1五聚体的电泳检测结果参见图3。From the purification experiment results shown in Table 1 above, it can be seen that when the pH of the assembly buffer is 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 pentamer of HPV59L1 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 HPV59L1 pentamer are shown in Figure 3.
实施例五：蛋白长期稳定性实验Example 5: Protein long-term stability experiment
取实施例四制备的HPV59L1-VLP，在-70℃条件下，进行长期稳定性数据的考察，考察结果如下。
The HPV59L1-VLP prepared in Example 4 was used to investigate the long-term stability data at -70°C. The investigation results are as follows.
“-”表示无此项规定或者该项未进行试验。“-” means that there is no provision for this item or the item has not been tested.
可以看出，经过9个月的长期考察，HPV59L1-VLP抗原蛋白的外观性状，pH值，VLP平均粒径及分散系数，纯度以及体外效价等，都没有明显变化，抗原蛋白相当稳定。It can be seen that after 9 months of long-term observation, the appearance properties, pH value, VLP average particle size and dispersion coefficient, purity and in vitro potency of the HPV59L1-VLP antigen protein have not changed significantly, and the antigen protein is quite stable.
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HPV59型L1蛋白，其是在野生型HPV59型L1蛋白的基础上，在其N端截短4个氨基酸，且在其C端截短31个氨基酸；优选地，截短的HPV59型L1蛋白的氨基酸序列如SEQ ID NO.2所示。A truncated HPV59 type L1 protein, which is based on the wild-type HPV59 type L1 protein, with 4 amino acids truncated at its N-terminus and 31 amino acids truncated at its C-terminus; preferably, the amino acid sequence of the truncated HPV59 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HPV59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 HPV59 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2所述的截短的HPV59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 HPV59 type L1 protein as described in claim 2, 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HPV59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 HPV59 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，其是原核细胞，优选为大肠杆菌。The recombinant host cell according to claim 6, characterized in that it is a prokaryotic cell, preferably Escherichia coli.
  8. 一种表达如权利要求1截短的HPV59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HPV59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 HPV59 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 HPV59 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HPV59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 the HPV59 type L1 protein.
  9. 一种表达如权利要求1截短的HPV59型L1蛋白的方法，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HPV59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VLP。A method for expressing the truncated HPV59 type L1 protein as claimed in claim 1, characterized in that it comprises the following steps: the step of obtaining the HPV59 type L1 protein according to the method as claimed in claim 8, adjusting the pH and salt concentration of the buffer in which it is located to make it self-assemble to form VLPs.
  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，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 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; The salt concentration is between 1.5M and 3.0M, preferably 1.5M, 2.0M, 2.5M, 3.0M;
    任选地，还包括纯化所得HPV59L1五聚体的步骤。 Optionally, the method further comprises a step of purifying the obtained HPV59L1 pentamer.
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