WO2024140160A1 - Expression de protéine l1 du papillomavirus humain hpv39, particules pseudo-virales et procédé de préparation associé - Google Patents

Expression de protéine l1 du papillomavirus humain hpv39, particules pseudo-virales et procédé de préparation associé Download PDF

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WO2024140160A1
WO2024140160A1 PCT/CN2023/138104 CN2023138104W WO2024140160A1 WO 2024140160 A1 WO2024140160 A1 WO 2024140160A1 CN 2023138104 W CN2023138104 W CN 2023138104W WO 2024140160 A1 WO2024140160 A1 WO 2024140160A1
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WIPO (PCT)
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protein
buffer
hpv39
truncated
expression
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PCT/CN2023/138104
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English (en)
Chinese (zh)
Inventor
伍树明
刘永江
薛俊莲
王学红
高文双
张海江
陈晓
张尧
银飞
沈迩萃
陈丹
王丽英
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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Publication of WO2024140160A1 publication Critical patent/WO2024140160A1/fr

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  • 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, HPV39, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, 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, HPV39, HPV39, HPV45, HPV51, HPV52, HPV
  • the inventors expressed the HPV39 L1 protein in a prokaryotic expression system based on the cost of the finished vaccine, and solved the difficulty in expressing the HPV39 L1 protein in a prokaryotic expression system. This was achieved specifically through the following improvements: the amino acid sequence of the HPV39 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.
  • sequence shown in SEQ ID NO.2 is as follows:
  • sequence shown in SEQ ID NO.3 is as follows:
  • the present invention removes the GST tag of the vector and replaces the SD sequence that can efficiently express the HPV39 type L1 protein to form a new expression vector suitable for the HPV39 L1 protein.
  • the replaced SD sequence is AGGAGATATA (5' to 3').
  • the present invention also provides a method for preparing HPV39 L1 VLP, comprising the following steps: adjusting the pH and salt concentration of the buffer solution in which the HPV39 L1 protein is located to enable it to self-assemble into VLP according to the step of obtaining the HPV39 L1 protein according to the method.
  • 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 is between 4.75 and 5.25, and the salt concentration is between 2.0 and 4.0 M, preferably pH 4.75, pH 5.0, and pH 5.25;
  • the salt concentration in the mixture is between 2.0-4.0 M, preferably 2.0 M, 2.5 M, 3.0 M, 3.5 M, 4.0 M;
  • Figure 3 XA90 pKL1-HPV39-N9L1 expression electrophoresis detection results in small shake flasks.
  • M marker; 1. XA90 pKL1 negative control; 2. XA90 pKL1-HPV39L1-N9-1 whole cells; 3. XA90 pKL1-HPV39L1-N9-1 supernatant; 4. XA90 pKL1-HPV39L1-N9-1 precipitate; 5. XA90 pKL1-HPV39L1-N9-2 whole cells; 6. XA90 pKL1-HPV39L1-N9-2 supernatant; 7. XA90 pKL1-HPV39L1-N9-2 precipitate; 8. XA90 pKL1 negative control.
  • FIG. 4 HPV39-N9 L1 pentamer electrophoresis detection results.
  • M marker; 1.
  • Example 1 Construction of a tag-free expression vector containing a specific SD sequence
  • 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.
  • 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 vector fragment was recovered by agarose gel recovery kit, and 3 ⁇ l 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: 10 ⁇ T4 DNA ligase buffer 2.5 ⁇ l, ddH 2 O1.8 ⁇ l, gel-recovered digested vector fragment 20 ⁇ l, 10 mM dNTP0.2 ⁇ l, DNA polymerase I0.5 ⁇ l, 25°C for 15 min, EDTA (final EDTA concentration was 10 mM) was added and heated at 75°C for 20 min to terminate the reaction.
  • 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-noG-F, 1 ⁇ L of 6PNE-SDm-noG-R, 5 ⁇ L of template plasmid, and 0.5 ⁇ L of Phusion HF enzyme.
  • the template DNA 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 clones were expanded and plasmids were extracted from them to obtain a vector that successfully replaced the SD sequence, removed the GST gene, and reintroduced NdeI and BamHI. At this point, the vector pKL1 was constructed.
  • Example 2 Construction of an expression vector containing a codon-optimized HPV39L1 gene
  • the ligation product is transformed into E.coli DH5 ⁇ for recombinant screening.
  • the screened monoclonal colonies are expanded and cultured, and the plasmids are extracted, and then sequenced and verified to obtain the corresponding recombinant expression vector pKL1-HPV39L1 (the corresponding three truncated forms of the vectors include truncated forms: pKL1-HPV39L1 is truncated by 4 amino acids at the N-terminus and 29 at the C-terminus).
  • 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 break the bacteria, and the breaking conditions are: 800bar, 3 times.
  • the bacterial cell break 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).
  • HPV39-N9 L1-VLP prepared in Example 4 was investigated for long-term stability data at -70°C. The results are as follows.

Abstract

L'invention concerne une expression de la protéine L1 du papillomavirus humain HPV39, des particules pseudo-virales et un procédé de préparation associé. Le procédé de préparation comprend : la troncature de la séquence d'acides aminés de la protéine L1 de HPV39, la réalisation d'une optimisation de codon sur une séquence nucléotidique codante de la protéine tronquée pour obtenir une séquence nucléotidique codante optimisée, et enfin la mise en correspondance avec un vecteur d'expression sans étiquette ayant une séquence SD spécifique pour obtenir une expression et une purification sans étiquette. L'amélioration décrite permet d'obtenir une expression de protéine supérieure dans des systèmes d'expression procaryotes tels qu'un système d'expression d'Escherichia coli, et des VLP ayant une qualité plus constante.
PCT/CN2023/138104 2022-12-28 2023-12-12 Expression de protéine l1 du papillomavirus humain hpv39, particules pseudo-virales et procédé de préparation associé WO2024140160A1 (fr)

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CN202211702935.5 2022-12-28

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WO2024140160A1 true WO2024140160A1 (fr) 2024-07-04

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