Recombinant DNA molecules encoding human papillomavirus antigens, DNA vaccines and their applications

Abstract

The present invention provides a recombinant DNA molecule encoding human papillomavirus antigens, a DNA vaccine and applications, and relates to the field of biotechnology. The present invention optimizes the DNA sequence encoding the human papillomavirus antigen to obtain a recombinant DNA molecule with a specific nucleotide sequence. The recombinant DNA molecule can efficiently transcribe and express human papillomavirus antigens and can induce a specific cellular immune response. The DNA vaccine provided by the present invention can not only effectively transcribe and express the E6 and E7 proteins of HPV6 or HPV11 in mammalian cells; at the same time, it exhibits a high antigen-specific IFN‑γ T cell response, activates antiviral cellular immune response, and can significantly inhibit the occurrence of recurrent respiratory papilloma tumors caused by HPV6 and HPV11 viruses, and has excellent anti-tumor effects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant DNA molecule encoding human papillomavirus antigen, a DNA vaccine and applications thereof. Background Art

[0002] Recurrent respiratory papillomatosis (RRP), also known as laryngeal papilloma, is a viral disease caused by human papillomavirus (HPV) infection that can invade other parts of the respiratory tract and digestive tract. Studies have shown that RRP can lead to the growth of non-cancerous (benign) tumors, which can cause life-threatening airway obstruction, airway stenosis, anesthesia accidents, and even the development of cancer.

[0003] RRP is primarily caused by HPV6 and HPV11. Patients infected with HPV11 have more invasive lesions and are more likely to develop cancer. RRP is categorized into juvenile RRP (under 12 years of age, JoRRP) and adult RRP (AoRRP), with children being more susceptible than adults. The prevalence of RRP is 0.75–4 per 100,000 children. RRP is generally diagnosed before the age of 5 years, with an average delay of one year between symptom onset and diagnosis. Although relatively rare, it is characterized by recurrent episodes, placing a significant psychological and emotional burden on patients and their families.

[0004] Currently, RRP is primarily treated through surgical resection and CO2 laser intervention, with concurrent adjunctive use of drugs such as interferon-α and cidofovir, but these treatments are not completely curative. Currently, the main therapeutic products for this indication in China and abroad include INO-3106 (Inovio, a DNA therapeutic vaccine targeting HPV6, IIT), INO-3107 (Inovio, a DNA therapeutic vaccine targeting HPV6 and HPV11), and PRGN-2012 (Precigen, an adenovirus-based therapeutic vaccine targeting HPV6), all of which are in clinical trials. However, there are currently no research products specifically targeting RRP in China.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The first object of the present invention is to provide a recombinant DNA molecule to solve the above technical problems.

[0007] The second object of the present invention is to provide a biomaterial.

[0008] The third object of the present invention is to provide applications of the above-mentioned recombinant DNA molecules or recombinant expression vectors or biomaterials.

[0009] The fourth object of the present invention is to provide a DNA vaccine.

[0010] The fifth object of the present invention is to provide a method for preparing the above-mentioned DNA vaccine.

[0011] In order to achieve the above objectives, the following technical solutions are adopted:

[0012] In a first aspect, the present invention provides a recombinant DNA molecule comprising a first DNA molecule encoding an HPV11 type E6 protein, a second DNA molecule encoding an HPV11 type E7 protein, a third DNA molecule encoding an HPV6 type E6 protein, and a fourth DNA molecule encoding an HPV6 type E7 protein;

[0013] The nucleotide sequence of the first DNA molecule is shown as SEQ ID No.29; the nucleotide sequence of the second DNA molecule is shown as SEQ ID No.30; the nucleotide sequence of the third DNA molecule is shown as SEQ ID No.31; and the nucleotide sequence of the fourth DNA molecule is shown as SEQ ID No.32.

[0014] As a further technical solution, the recombinant DNA molecule includes a fifth DNA molecule encoding a signal peptide.

[0015] As a further technical solution, the nucleotide sequence of the fifth DNA molecule is shown as SEQ ID No.33.

[0016] As a further technical solution, the nucleotide sequence of the recombinant DNA molecule is the nucleotide sequence shown in SEQ ID No.34.

[0017] In a second aspect, the present invention provides a biomaterial comprising at least one of (a) to (b):

[0018] (a) a recombinant expression vector comprising the recombinant DNA molecule;

[0019] (b) A host cell comprising the recombinant DNA molecule or the recombinant expression vector of (a).

[0020] As a further technical solution, the recombinant expression vector is a eukaryotic expression vector, and the vector backbone of the eukaryotic expression vector is pVAX1.

[0021] In a third aspect, the present invention provides the use of the above-mentioned recombinant DNA molecules or biological materials in the following a or b:

[0022] a. Preparation of vaccines for the prevention and / or treatment of human papillomavirus infection;

[0023] b. Preparation of drugs for preventing and / or treating diseases caused by human papillomavirus;

[0024] The human papillomavirus includes HPV6 human papillomavirus and / or HPV11 human papillomavirus.

[0025] In a fourth aspect, the present invention provides a DNA vaccine, which includes the recombinant DNA molecule or recombinant expression vector.

[0026] As a further technical solution, the DNA vaccine further includes a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.

[0027] In a fifth aspect, the present invention provides a method for preparing a DNA vaccine, comprising: introducing the recombinant DNA molecule or recombinant expression vector into a host cell, culturing the host cell, extracting the recombinant DNA molecule or recombinant expression vector from the host cell, and preparing a DNA vaccine.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention optimizes the DNA sequence encoding human papillomavirus antigens to obtain a recombinant DNA molecule with a specific nucleotide sequence. The DNA molecule can efficiently transcribe and express the E6 and E7 antigens of human papillomavirus, has immunogenicity, and can induce a specific cellular immune response.

[0030] The DNA vaccine provided by the present invention can not only effectively transcribe and express the E6 and E7 proteins of HPV6 or HPV11 in mammalian cells; at the same time, it exhibits a high antigen-specific IFN-γ T cell response and activates the antiviral cellular immune response, thereby showing excellent immunogenicity against HPV6 and HPV11 viruses, and can significantly inhibit the occurrence of recurrent respiratory papilloma tumors caused by HPV6 and HPV11 viruses, and has excellent anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 The specific IFN-γ secretion levels of different DNA vaccines provided in Example 2 of the present invention on the 28th day after immunization of mice;

[0033] Figure 2 The in vitro mRNA transcription level of the DNA vaccine pAD1543 provided in Example 3 of the present invention;

[0034] Figure 3 The in vitro protein expression of the DNA vaccine pAD1543 provided in Example 4 of the present invention;

[0035] Figure 4 The specific IFN-γ secretion level of the DNA vaccine pAD1543 provided in Example 5 of the present invention on the 28th day after immunization of mice;

[0036] Figure 5 The secretion level of CD8+ T cell-specific cytokines by the DNA vaccine pAD1543 provided in Example 5 of the present invention on day 28 after immunization of mice;

[0037] Figure 6 The specific IFN-γ secretion levels of the DNA vaccine pAD1543 provided in Example 6 of the present invention at different time points after immunization of New Zealand rabbits;

[0038] Figure 7 The tumor size of tumor-bearing mice after treatment with the DNA vaccine pAD1543 provided in Example 7 of the present invention;

[0039] Figure 8 This is a visual picture of the solid tumors removed from each group of tumor-bearing mice after treatment with the DNA vaccine pAD1543 provided in Example 7 of the present invention;

[0040] Figure 9 This is a graph showing the tumor weight results of each group of tumor-bearing mice after treatment with the DNA vaccine pAD1543 provided in Example 7 of the present invention;

[0041] Figure 10 This is a graph showing the weight changes of each group of mice after tumor-bearing mice were treated with the DNA vaccine pAD1543 provided in Example 7 of the present invention. DETAILED DESCRIPTION

[0042] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0043] Generally, the nomenclature used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein and its technology are those well-known and commonly used in this area.Unless otherwise indicated, the methods and techniques of the present invention are generally according to those well-known in the art, and are carried out as described in various general and more specific references, which are cited and discussed throughout this specification.Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly achieved in this area, or as described herein.The nomenclature used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein and its laboratory procedures and technology are those well-known and commonly used in this area.

[0044] In a first aspect, the present invention provides a recombinant DNA molecule comprising a first DNA molecule encoding an HPV11 type E6 protein, a second DNA molecule encoding an HPV11 type E7 protein, a third DNA molecule encoding an HPV6 type E6 protein, and a fourth DNA molecule encoding an HPV6 type E7 protein;

[0045] The nucleotide sequence of the first DNA molecule is shown as SEQ ID No.29; the nucleotide sequence of the second DNA molecule is shown as SEQ ID No.30; the nucleotide sequence of the third DNA molecule is shown as SEQ ID No.31; and the nucleotide sequence of the fourth DNA molecule is shown as SEQ ID No.32.

[0046] The recombinant DNA molecule provided by the present invention can efficiently transcribe and express the E6 and E7 antigens of the human papillomavirus by optimizing the DNA sequence encoding the human papillomavirus antigen, and has immunogenicity and can induce a specific cellular immune response.

[0047] In some alternative embodiments, the recombinant DNA molecule includes a fifth DNA molecule encoding a signal peptide.

[0048] In some optional embodiments, the nucleotide sequence of the fifth DNA molecule is shown as SEQ ID No.33.

[0049] The present invention further improves the expression efficiency of recombinant DNA molecules by optimizing the signal peptide sequence.

[0050] In some optional embodiments, the nucleotide sequence of the recombinant DNA molecule is the nucleotide sequence shown in SEQ ID No. 34.

[0051] In a second aspect, the present invention provides a biomaterial comprising at least one of (a) to (b):

[0052] (a) A recombinant expression vector comprising the recombinant DNA molecule. The vector may be a eukaryotic expression vector, which can transform target cells by integrating into the cellular genome, producing the protein encoded by the DNA molecule through the cellular transcription and translation machinery. Optionally, the vector may contain expression signals, such as a strong promoter, a strong stop codon, adjustment of the distance between the promoter and the cloned gene, and insertion of a transcription termination sequence and a PTIS.

[0053] (b) a host cell, comprising the recombinant DNA molecule or the recombinant expression vector of (a). The cell may be a prokaryotic cell, such as a bacterial cell, typically Escherichia coli ( E. coli ), or eukaryotic cells, typically insect cells, yeast cells, avian cells or mammalian cells.

[0054] As a further technical solution, the recombinant expression vector is a eukaryotic expression vector, and the vector backbone of the eukaryotic expression vector is pVAX1.

[0055] In a third aspect, the present invention provides the use of the above-mentioned recombinant DNA molecules or biological materials in the following a or b:

[0056] a. Preparation of vaccines for the prevention and / or treatment of human papillomavirus infection;

[0057] b. Preparation of drugs for preventing and / or treating diseases caused by human papillomavirus, including but not limited to recurrent respiratory papillomatosis.

[0058] The human papillomavirus includes HPV6 human papillomavirus and / or HPV11 human papillomavirus.

[0059] Based on the beneficial effects of the above-mentioned recombinant DNA molecules encoding human papillomavirus antigens, the present invention also provides a DNA vaccine comprising the above-mentioned recombinant DNA molecules.

[0060] In a fourth aspect, the present invention provides a DNA vaccine, which includes the recombinant DNA molecule or recombinant expression vector.

[0061] The DNA vaccine provided by the present invention can not only effectively transcribe and express the E6 and E7 proteins of HPV6 or HPV11 in mammalian cells; at the same time, it exhibits a high antigen-specific IFN-γ T cell response and activates the antiviral cellular immune response, thereby showing excellent immunogenicity against HPV6 and HPV11 viruses, and can significantly inhibit the occurrence of recurrent respiratory papilloma tumors caused by HPV6 and HPV11 viruses, and has excellent anti-tumor effects.

[0062] In some optional embodiments, the DNA vaccine further comprises a pharmaceutically acceptable adjuvant, carrier, diluent or excipient to increase the ability of its active ingredient DNA molecule to produce an immune response in the subject. The pharmaceutically acceptable adjuvant can be selected from aluminum adjuvants and / or TLRs ligands and / or metal ions such as Mn 2+ 、Zn 2+ , Ca 2+ and / or cytokine and / or chemokine adjuvants, etc.

[0063] In a fifth aspect, the present invention provides a method for preparing a DNA vaccine, comprising: introducing the recombinant DNA molecule or recombinant expression vector into a host cell, culturing the host cell, extracting the recombinant DNA molecule or recombinant expression vector from the host cell, and preparing a DNA vaccine.

[0064] The DNA vaccine of the present invention can be obtained by this preparation method.

[0065] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0066] Example 1 Preparation of recombinant DNA vaccine

[0067] RRP recombinant DNA vaccine 1 (pAD1532): The nucleotide sequence encoding HPV6 E6 protein was designed as shown in SEQ ID No. 1, the nucleotide sequence encoding HPV6 E7 protein was designed as shown in SEQ ID No. 2, the nucleotide sequence encoding HPV11 E6 protein was designed as shown in SEQ ID No. 3, the nucleotide sequence encoding HPV11 E7 protein was designed as shown in SEQ ID No. 4, the nucleotide sequence encoding signal peptide 1 was designed as shown in SEQ ID No. 5, the nucleotide sequence encoding signal peptide 2 was designed as shown in SEQ ID No. 6, the restriction enzyme cleavage site 1 sequence was shown in SEQ ID No. 7, the restriction enzyme cleavage site 2 sequence was shown in SEQ ID No. 8, and the IRES sequence was shown in SEQ ID No. 9. SEQ ID No. 5, SEQ ID No. 1, SEQ ID No. 7, SEQ ID No. 2, SEQ ID No. 9, SEQ ID No. 6, SEQ ID No. 3, SEQ ID No. 8, and SEQ ID No. 4 were directly linked in order to obtain the nucleotide sequence shown in SEQ ID No. 10. SEQ ID No. 10 was inserted between the BamH I and Xba I sites of the pVAX1 vector (ThermoFisher, catalog number: V26020) to obtain a recombinant DNA vaccine, which was designated as pAD1532.

[0068] RRP recombinant DNA vaccine 2 (pAD1539): The nucleotide sequence encoding HPV11 E6 protein was designed as shown in SEQ ID No. 11, the nucleotide sequence encoding HPV11 E7 protein was designed as shown in SEQ ID No. 12, the nucleotide sequence encoding HPV6 E6 protein was designed as shown in SEQ ID No. 13, the nucleotide sequence encoding HPV6 E7 protein was designed as shown in SEQ ID No. 14, the nucleotide sequence encoding signal peptide 3 was designed as shown in SEQ ID No. 15, the sequence of restriction enzyme cleavage site 3 was shown in SEQ ID No. 16, the sequence of restriction enzyme cleavage site 4 was shown in SEQ ID No. 17, and the sequence of restriction enzyme cleavage site 5 was shown in SEQ ID No. 18. SEQ ID No. 15, SEQ ID No. 11, SEQ ID No. 16, SEQ ID No. 12, SEQ ID No. 17, SEQ ID No. 13, SEQ ID No. 18, and SEQ ID No. 14 were directly linked in order to obtain the nucleotide sequence shown in SEQ ID No. 19. SEQ ID No. 19 was inserted between the BamH I and Xho I sites of the pVAX1 vector to obtain a recombinant DNA vaccine, which was designated as pAD1539.

[0069] RRP recombinant DNA vaccine 3 (pAD1542): The nucleotide sequence encoding HPV6 E6 protein was designed as shown in SEQ ID No. 20, the nucleotide sequence encoding HPV6 E7 protein was designed as shown in SEQ ID No. 21, the nucleotide sequence encoding HPV11 E6 protein was designed as shown in SEQ ID No. 22, the nucleotide sequence encoding HPV11 E7 protein was designed as shown in SEQ ID No. 23, the nucleotide sequence encoding signal peptide 4 was designed as shown in SEQ ID No. 24, the sequence of restriction enzyme cleavage site 6 was shown in SEQ ID No. 25, the sequence of restriction enzyme cleavage site 7 was shown in SEQ ID No. 26, and the sequence of restriction enzyme cleavage site 8 was shown in SEQ ID No. 27. SEQ ID No. 24, SEQ ID No. 20, SEQ ID No. 25, SEQ ID No. 21, SEQ ID No. 26, SEQ ID No. 22, SEQ ID No. 27, and SEQ ID No. 23 were directly linked in order to obtain the nucleotide sequence shown in SEQ ID No. 28. SEQ ID No. 28 was inserted between the BamH I and Xho I sites of the pVAX1 vector to obtain a recombinant DNA vaccine, which was designated as pAD1542.

[0070] RRP recombinant DNA vaccine 4 (pAD1543): The nucleotide sequence encoding HPV11 E6 protein was designed as shown in SEQ ID No. 29, the nucleotide sequence encoding HPV11 E7 protein was designed as shown in SEQ ID No. 30, the nucleotide sequence encoding HPV6 E6 protein was designed as shown in SEQ ID No. 31, the nucleotide sequence encoding HPV6 E7 protein was designed as shown in SEQ ID No. 32, and the nucleotide sequence encoding signal peptide 5 was designed as shown in SEQ ID No. 33. Sequences SEQ ID No. 33, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, and SEQ ID No. 32 were directly linked in order to obtain the nucleotide sequence shown in SEQ ID No. 34. Sequence ID No. 34 was inserted between the BamHI and XhoI sites of the pVAX1 vector to obtain the recombinant DNA vaccine, designated pAD1543.

[0071] RRP recombinant DNA vaccine 5 (pAD1544): The nucleotide sequence encoding HPV6 E6 protein was designed as shown in SEQ ID No. 35, the nucleotide sequence encoding HPV6 E7 protein was designed as shown in SEQ ID No. 36, the nucleotide sequence encoding HPV11 E6 protein was designed as shown in SEQ ID No. 37, the nucleotide sequence encoding HPV11 E7 protein was designed as shown in SEQ ID No. 38, and the nucleotide sequence encoding signal peptide 6 was designed as shown in SEQ ID No. 39. Sequences SEQ ID No. 39, SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, and SEQ ID No. 38 were directly linked in order to obtain the nucleotide sequence shown in SEQ ID No. 40. Sequence ID No. 40 was inserted between the BamHI and XhoI sites of the pVAX1 vector to obtain the recombinant DNA vaccine, designated pAD1544.

[0072] RRP recombinant DNA vaccine 6 (pGX3024): It is the RRP recombinant DNA vaccine sequence disclosed in Inovio's patent, which is derived from sequence 3 in the patent with publication number US11744885B2. The DNA vaccine sequence corresponding to sequence 3 is the HPV6 & HPV11E6E7 full fusion protein sequence 12 expressed in the patent, which is inserted between the BamH I and Xho I sites of the pVAX1 vector to obtain a recombinant DNA vaccine.

[0073] The designs of the DNA sequences corresponding to the above-mentioned recombinant DNA vaccines are shown in Table 1.

[0074] Table 1

[0075] .

[0076] Example 2 Verification of cellular immune response to different RRP recombinant DNA therapeutic vaccines

[0077] To verify the cellular immunogenicity of each DNA vaccine prepared in Example 1, mice were immunized twice biweekly with the prepared DNA vaccines, each at a dose of 10 μg per mouse per dose. Empty pVAX1 vector served as a blank control group. Immunization was by intramuscular injection with an electric pulse, with the first day of vaccination designated as day 0. On day 28, spleen cells isolated from vaccinated mice were stimulated with the HPV6 E6 peptide pool (SEQ ID No. 41-SEQ ID No. 61), the HPV6 E7 peptide pool (SEQ ID No. 62-SEQ ID No. 74), the HPV11 E6 peptide pool (SEQ ID No. 75-SEQ ID No. 95), and the HPV11 E7 peptide pool (SEQ ID No. 96-SEQ ID No. 108), respectively, and then assayed using ELISpot assays. The mice used in this experiment were 6-8 week old female C57BL / 6 mice purchased from Shanghai Slake Co. The ELISpot assay was performed as follows:

[0078] ELISpot assay for cellular immune response: The mice were euthanized by cervical dislocation in a sterile environment. The spleen was removed and ground into a single-cell suspension. The suspension was filtered through a sterile 200-mesh copper mesh and harvested by centrifugation at 1500 rpm for 3 min. The supernatant was discarded, and the suspension was resuspended in 2 mL of red blood cell lysis buffer and lysed for 2 min. The lysis was terminated with 4 mL of 1640 medium containing FBS. The prepared single-cell suspension was counted and the ELISpot plate was plated with 5 × 10 5Cells / well were plated; 4 corresponding specific peptide pools were added for in vitro stimulation, and ELISpot was performed at 37°C, 5% CO2 culture for 18-24 hours. The next day, pour the cell supernatant in the well, add ice-cold deionized water, 200 μL / well, and place in a 4℃ refrigerator for 10 minutes; discard the supernatant, add 1× wash buffer, 260 μL / well, stay for one minute, then discard the supernatant, repeat 6 times, and pat dry on absorbent paper; dilute biotin-labeled antibody at 1:100, 100 μL / well, incubate at 37℃ for 1 hour; discard the supernatant and repeat washing; dilute enzyme-linked avidin at 1:100, 100 μL / well, incubate at 37℃ for 1 hour; discard the supernatant and repeat washing. For the last time, uncover the base, wash the bottom surface of the membrane and the base with deionized water, and pat dry on absorbent paper; prepare the color development solution, 100 μL / well, incubate at 37℃ in the dark for 30 minutes; discard the supernatant, uncover the base, wash the front and back with tap water 3-5 times to stop color development, dry and close the base; count the ELISpot plate spots.

[0079] Conclusion: 14 days after the second vaccination (28 days after the first vaccination), if Figure 1 As shown in the results, under the stimulation of four polypeptide pools of HPV6 E6, HPV6 E7, HPV11 E6 or HPV11 E7, it was found that the pAD1543 group and the pAD1539 group had strong specific IFN-γ secretion levels, which were significantly higher than those of the other groups. Among them, the pAD1543 group had the highest IFN-γ secretion level, showing extremely strong cellular immunogenicity, which was significantly better than the existing disclosed DNA vaccine pGX3024, indicating that the DNA vaccine designed and optimized by the present invention has better immunogenicity.

[0080] Example 3: Transcriptional Identification of RRP Recombinant DNA Therapeutic Vaccine pAD1543 in Mammalian Cells

[0081] DNA vaccine in vitro transfection: HEK293T cells were taken out of liquid nitrogen and placed in a 37°C water bath. 5 ml of DMEM containing 10% fetal bovine serum was added and centrifuged at 1500 rpm for 3 minutes to remove DMSO. The cells were then cultured for 2-3 generations at 37°C in 5% CO2. The cells were then digested with trypsin (containing 0.25% EDTA) at 37°C for 1 minute and terminated with complete medium. 4 × 10 6Cells were plated at a density of 1000 cells / well on a 60 mm culture dish, and 5 ml of growth medium (without 1% double antibody) was added and cultured in a 37°C, 5% CO2 incubator for 24 h. 4 μg of pAD1543 and sterile empty plasmid pVAX1 were added to 500 μl of OPTI-MEM serum-free reduced medium and gently mixed. At the same time, 24 μl of cationic liposomes were added to 500 μl of OPTI-MEM serum-free reduced medium, gently mixed, and incubated at room temperature for 5 min. The above plasmids were mixed with liposomes in a 1:1 ratio and incubated at room temperature for 20 min to obtain plasmid DNA / liposome complexes. The above plasmid DNA / liposome complexes were added to the 60 mm culture dish cultured for 24 h at 1 ml / dish and incubated in a 37°C, 5% CO2 incubator for up to 48 h for subsequent experiments.

[0082] RNA extraction after transfection: Digest and collect the cells transfected 48 hours after each treatment, resuspend in 1 ml of complete medium, and aspirate 100 μl of the suspension for RNA extraction. The remaining suspension was used for subsequent Western blotting sample preparation. Centrifuge the aspirated 100 μl cell suspension at 4000 rpm for 5 minutes, discard the supernatant, and add 350 μl of TRK lysis buffer (containing 20% ​​β-mercaptoethanol) to each sample for lysis. Add 350 μl of 70% ethanol (prepared with DEPC water) to each sample to stop the lysis, and mix well with a pipette; transfer the above mixture to a HiBind RNA column, centrifuge at 10,000g for 1 minute, and discard the filtrate; add 500 μl of wash buffer I to each sample column, centrifuge at 10,000g for 1 minute, and discard the filtrate; add 500 μl of wash buffer II to each sample column and wash twice, centrifuging at 10,000g for 1 minute each time, and discard the filtrate; adjust the centrifuge speed to the highest speed (17,000g) and centrifuge for 2 minutes to evaporate the ethanol in the column; transfer the column to a clean 1.5 ml centrifuge tube free of DNA and RNase, and let it stand at room temperature for 3-5 minutes. After the ethanol has completely evaporated, add 50 μl of The column was incubated with nuclease-free water at room temperature for 5 min and centrifuged at 17000 g for 1 min. The filtrate was aspirated and added to the column again, incubated at room temperature for 5 min, and centrifuged at 17000 g for 1 min to collect RNA, which was then stored at -80°C.

[0083] RNA reverse transcription and qPCR reaction: Quantify RNA concentration using a microplate reader (read using OD260 / 280). Prepare 15 μL of reaction system for each sample (3 μL of 5×g DNA digestion buffer and 100 ng of RNA, adjust the volume to 15 μL with nuclease-free water), mix gently by pipetting, and incubate at 42°C for 2 minutes. Add 5 μL of 4×Hifair to each sample. TMIII SuperMixplus, gently pipette to mix, and incubate at 25℃ for 5min, 55℃ for 15min, and 85℃ for 5min. The collected cDNA was stored at -20℃ for use. The cDNA product obtained by reverse transcription was reacted according to the qPCR kit. The reaction system is as follows: Hieff® qPCR SYBR Green Master Mix (No Rox) 10μl, 0.4μl each of the target forward and reverse primers (the four corresponding specific primers are shown in Table 2), 1μL of cDNA template, and sterile ultrapure water to make the total volume 20μl. PCR reaction conditions: 95℃, 5 min, 95℃, 10 s, 57℃, 30s, 72℃, 30s for a total of 40 cycles. The expression level of the target gene was compared with the internal reference and 2 -△△CT Method calculation.

[0084] Table 2 Primer sequence list

[0085] .

[0086] Conclusion: To further verify the in vitro mRNA expression of the RRP recombinant DNA vaccine pAD1543 designed in this invention, we transfected pAD1543 with a eukaryotic expression system and tested it with pVAX1 as a negative control. RNA was extracted from the cell products 48 hours after transfection, and specific primers designed based on the HPV6 E6, HPV6 E7, HPV11 E6, and HPV11 E7 gene sequences were used to perform gene detection of the mRNA expression of the recombinant DNA vaccine pAD1543 by qPCR. Figure 2 As shown, pAD1543 has high expression of four genes, further indicating that the DNA vaccine pAD1543 designed by the present invention can effectively transcribe the corresponding genes in eukaryotic cells.

[0087] Example 4: Expression and Identification of Antigen Protein in RRP Recombinant DNA Therapeutic Vaccine pAD1543 Mammalian Cells

[0088] In order to further verify whether the RRP recombinant DNA vaccine pAD1543 designed in the present invention can be effectively expressed in mammalian cells, the antigen protein was extracted after pAD1543 was transfected into mammalian cells and identified by Western Blot method.

[0089] Protein extraction: pVAX1 and pAD1543 plasmids were transfected into HEK293T cells, respectively. 48 hours after transfection, the culture medium was removed and the cells were rinsed with pre-chilled PBS. The PBS was discarded, and 150 μL of lysis buffer was added, mixed thoroughly, and pipetted 10 times. The cells were then centrifuged at 12,000 rpm at 4°C for 5 minutes. The supernatant was aspirated into a 1.5 mL centrifuge tube. 20 μL of the supernatant was removed from each sample, and 5 μL of 5× protein loading buffer was added. The cells were boiled at 100°C for 10 minutes and then briefly centrifuged for later use.

[0090] Sample loading and SDS-PAGE electrophoresis: Add 20 μL of the supernatant sample after boiling and centrifugation to each well of the SDS-PAGE gel. Connect the power supply to a constant voltage of 200 V and run the electrophoresis for 45 minutes. After electrophoresis, remove the sample from the SDS-PAGE gel and transfer it to the membrane. Activate the PVDF membrane by soaking it in methanol for 30 seconds. Place the PVDF membrane in 1× transfer buffer for 1 minute.

[0091] Transfer: With the positive electrode as the bottom surface, stack the membrane in the following order: eBlot L1 Transfer Mat, PVDF membrane, gel, and eBlot L1 Transfer Mat. Remove air bubbles between layers with a pipette after each stacking. Blocking: Remove the PVDF membrane and place it in a glass box filled with 1× TBST + 5% nonfat dry milk. Incubate on a shaker at 90 rpm for 1 hour at room temperature. Wash: Wash the PVDF membrane three times in 1× TBST for 10 minutes each, shaking at 90 rpm. Primary Antibody Incubation: Incubate the PVDF membrane in the primary antibody solution (HPV6 IgG antibody, mouse, 1:10,000, v / v) at room temperature at 90 rpm for 1 hour. Wash: Wash the PVDF membrane three times in 1× TBST for 10 minutes each, shaking at 90 rpm. Secondary Antibody Incubation: Place the PVDF membrane in the secondary antibody solution (goat anti-mouse HRP, abcam, Cat#ab6789, 1:5000, v / v) and incubate at room temperature for 1 hour on a shaker at 90 rpm. Washing: Wash the PVDF membrane three times in 1× TBST for 10 minutes each, shaking at 90 rpm. Color Development: Mix 3 ml of chemiluminescent solution A and 3 ml of solution B in a 1:1 ratio and apply to the PVDF membrane. Incubate for 1 minute and photograph.

[0092] Conclusion: If Figure 3 As shown, pAD1543 showed a clear target band compared with the empty vector (pVAX1) 48 hours after in vitro transfection in HEK293T cell line, indicating that pAD1543 had strong protein expression, further demonstrating that the DNA vaccine pAD1543 designed by the present invention can effectively express the antigen protein of the corresponding gene in mammalian cells.

[0093] Example 5: Immunogenicity Verification of RRP Recombinant DNA Therapeutic Vaccine pAD1543 in C57BL / 6 Mice

[0094] To further verify the cellular immunogenicity of the RRP recombinant DNA vaccine pAD1543 designed by the present invention in mice, mice were immunized twice with the prepared DNA vaccine once every two weeks. The doses of pAD1543 used were 3μg, 10μg, 30μg, 60μg, and 90μg per mouse per time. The immunization method used was intramuscular injection with electric pulses, and the first day of vaccine immunization was counted as day 0. On day 28, splenocytes isolated from mice immunized with the vaccine were stimulated with HPV6 E6, HPV6E7, HPV11 E6, and HPV11E7 specific peptide pools, respectively, and ELISpot assays were performed. The mice used in this experimental example were 6-8 week old female C57BL / 6 mice, all purchased from Shanghai Slake Company. The experimental method is as follows:

[0095] ELISpot assay for cellular immune response: The mice were euthanized by cervical dislocation in a sterile environment. The spleen was removed and ground into a single-cell suspension. The suspension was filtered through a sterile 200-mesh copper mesh and harvested by centrifugation at 1500 rpm for 3 min. The supernatant was discarded, and the suspension was resuspended in 2 mL of red blood cell lysis buffer and lysed for 2 min. The lysis was terminated with 4 mL of 1640 medium containing FBS. The prepared single-cell suspension was counted and the ELISpot plate was plated with 5 × 10 5 Cells / well were plated; 4 corresponding specific peptide pools were added for in vitro stimulation, and ELISpot was performed at 37°C, 5% CO2 culture for 18-24 hours. The next day, pour the cell supernatant in the well, add ice-cold deionized water, 200 μL / well, and place in a 4℃ refrigerator for 10 minutes; discard the supernatant, add 1× wash buffer, 260 μL / well, stay for one minute, then discard the supernatant, repeat 6 times, and pat dry on absorbent paper; dilute biotin-labeled antibody at 1:100, 100 μL / well, incubate at 37℃ for 1 hour; discard the supernatant and repeat washing; dilute enzyme-linked avidin at 1:100, 100 μL / well, incubate at 37℃ for 1 hour; discard the supernatant and repeat washing. For the last time, uncover the base, wash the bottom surface of the membrane and the base with deionized water, and pat dry on absorbent paper; prepare the color development solution, 100 μL / well, incubate at 37℃ in the dark for 30 minutes; discard the supernatant, uncover the base, wash the front and back with tap water 3-5 times to stop color development, dry and close the base; count the ELISpot plate spots.

[0096] FACs flow cytometry assay for cellular immune response: mice were euthanized by cervical dislocation in a sterile environment, and spleens were removed. The cells were ground into a single-cell suspension, filtered through a sterile 200-mesh copper mesh, and harvested by centrifugation at 1500 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in 2 mL of red blood cell lysis buffer and lysed for 2 min. The lysis was terminated with 4 mL of 1640 medium containing FBS. The prepared single-cell suspension was counted, and the flow cytometry plate was used with 1×10 6 150 μL / well of PBS (1:1000) and CD16 / 32 blocking agent (1:500) were added and incubated at 4°C in the dark for 30 min; 150 μL / well of PBS (containing 2% FBS) was added and centrifuged at 1500 rpm for 3 min at 4°C and the supernatant was discarded; 50 μL / well of extracellular dye (anti-mouse CD8 antibody) was added and incubated at 4°C in the dark for 30 min; 150 μL / well of PBS (containing 2% FBS) was added and centrifuged at 1500 rpm for 3 min at 4°C and the supernatant was discarded; 200 μL / well of fixative (fixative: diluent = 1:3 (v:v)) was added and incubated at 4°C in the dark for 1 minute. h; 1500 rpm, 4 ° C, centrifuge for 3 min, and discard the supernatant; add 200 μL / well permeabilization buffer (permeabilization buffer: ultrapure water 1:9 (v:v)), wash once; add 50 μL / well intracellular cytokine dye (anti-mouse IFN-γ antibody), incubate at 100 rpm at room temperature in the dark for 1 h; add 150 μL / well PBS (containing 2% FBS), centrifuge at 1500 rpm, 4 ° C for 3 min, and discard the supernatant; add 200 μL / well PBS to resuspend and detect on flow cytometry.

[0097] Conclusion: 14 days after the second vaccination (28 days), if Figure 4 As shown in the figure, under the stimulation of four peptide pools, HPV6 E6, HPV6E7, HPV11 E6, and HPV11E7, pAD1543 had a strong level of specific IFN-γ secretion at different doses, showing extremely strong cellular immunogenicity. As the dosage of pAD1543 continued to increase, the immunogenicity showed a normal distribution. Among them, when the dose was 30µg / dose, its cellular immune response was the strongest. Figure 5 As shown, under the stimulation of four peptide pools, pAD1543 + T cells (cytotoxic T cells) showed a high secretion level of specific IFN-γ, indicating that they have strong cell killing ability. The above results show that the pAD1543 designed by the present invention can stimulate a strong specific cellular immune response after immunization.

[0098] Example 6: Immunogenicity Verification of RRP Recombinant DNA Therapeutic Vaccine pAD1543 in New Zealand Rabbits

[0099] To further validate the cellular immunogenicity of the RRP recombinant DNA vaccine pAD1543 designed in this invention in New Zealand rabbits, the animals were immunized with the prepared DNA vaccine every three weeks for a total of six doses. The pAD1543 doses used were 0.3 mg, 1 mg, 3 mg, 6 mg, and 9 mg per rabbit per dose. Immunization was by intramuscular injection with an electric pulse, with the first day of vaccination designated as day 0. On day 14 after each immunization, peripheral blood mononuclear cells (PBMCs) isolated from the vaccinated New Zealand rabbits were stimulated with pools of HPV6 E6, HPV6 E7, HPV11 E6, and HPV11 E7-specific peptides, respectively, and then analyzed by ELISpot assay. The New Zealand rabbits used in this experiment weighed 2-3 kg and were purchased from the Shanghai Institute of Product Research.

[0100] ELISpot assay for cellular immune response: Aseptically collect blood from New Zealand rabbits, separate PBMCs from the rabbit whole blood using Ficoll (Ficoll-Paque PLUS), and wash twice with PBS; count the prepared cell suspension and use 5×10 5 Plate 100 cells / well and add the corresponding specific peptide pool for in vitro stimulation. Incubate at 37°C, 5% CO2 for 18-24 hours before detection by ELISpot. The next day, pour the cell supernatant from the wells, add PBS (200 μL / well), discard the supernatant, repeat five times, and pat dry on absorbent paper. Incubate the wells with detection antibody (MT318-biotin) at a 1:5000 dilution, 100 μL / well, at room temperature for 1 hour, discard the supernatant, and repeat the wash. Incubate the wells with Streptavidin-ALP at a 1:1000 dilution, 100 μL / well, at room temperature for 1 hour, discard the supernatant, and repeat the wash. Filter the colorimetric solution through a 0.45 µm filter, add 100 μL / well, and incubate at room temperature in the dark for 5 minutes. Discard the supernatant, remove the base, wash both sides with tap water 3-5 times to stop color development, air dry, and close the base. Count the spots on the ELISpot plate.

[0101] Conclusion: If Figure 6As shown, under the stimulation of the four peptide pools of HPV6 E6, HPV6 E7, HPV11 E6, and HPV11 E7, pAD1543 induced strong specific IFN-γ secretion levels at different doses in New Zealand rabbits, demonstrating extremely strong cellular immunogenicity. Among them, in the early stage, when the pAD1543 dose was 3 mg / dose, its cellular immune response was the strongest. In the later stage, as the number of doses continued to increase, the cellular immunity levels of almost all groups reached a peak, and there was no significant difference between the groups. The above results further demonstrate that the pAD1543 designed by the present invention can stimulate a strong specific cellular immune response after immunization.

[0102] Example 7 Pharmacodynamic Verification of RRP Recombinant DNA Therapeutic Vaccine pAD1543 in the Mouse MOC-1-HPV6 / 11 Tumor Model

[0103] In order to verify the efficacy of the RRP recombinant DNA vaccine pAD1543 designed in the present invention, the mouse oral squamous cell carcinoma MOC-1-HPV6 / 11 stable cell tumor model (the tumor cell concentration of each mouse was 3x10 6 Mice were immunized with the prepared DNA vaccine once weekly for a total of six times (the first day of tumor cell inoculation was counted as day 0, and the first immunization began on day 7). Doses of pAD1543 were 10μg, 30μg, 60μg, and 90μg per mouse per dose. Immunization was by intramuscular injection with electric pulses. Tumor size and body weight were monitored three times weekly. The mice used in this experiment were 6-8 week old C57BL / 6 mice purchased from Shanghai Slake Co., Ltd.

[0104] Conclusion: If Figure 7 As shown, the tumor volume in the control group (pVAX1) increased significantly over time. At different doses, the RRP recombinant DNA vaccine pAD1543 exhibited significant tumor suppression, with complete tumor regression occurring with increasing dosing frequency. The dose trend of pAD1543 at different doses exhibited a normal distribution. When pAD1543 was 60µg / dose, seven of the eight tumor-bearing mice had their tumors disappear after six doses, demonstrating the best tumor suppression effect. Images of the exfoliated solid tumors from each group are shown below. Figure 8 Tumor weights were as shown. Figure 9 The results show that the pAD1543 designed by the present invention can significantly treat recurrent respiratory papillomavirus caused by HPV6 and HPV11 viruses and has excellent anti-tumor effects. At the same time, there was no significant change in the body weight of each group of mice (such as Figure 10 The overall body weight data remained stable and slightly increased, and there was no statistical difference in the body weight of mice in each group, indicating that the pAD1543 designed by the present invention has good safety.

[0105] In summary, the present invention provides a DNA vaccine that can express the full fusion protein of HPV6 and HPV11 E6 and E7 in vivo after sequence optimization, which can stimulate a more efficient immune response. The present invention uses a DNA optimization algorithm to design a DNA sequence with high expression potential encoding the E6 and E7 fragments of HPV6 and HPV11, and constructs the RRP DNA therapeutic vaccine pAD1543 to enhance its expression level and immunogenicity; the optimized DNA sequence is cloned into the expression vector pVAX1, and after transformation by Escherichia coli DH10B, the vaccine candidate DNA molecule pAD1543 is obtained, and the candidate DNA molecule is digested and sequenced to confirm that it is correctly constructed and can correctly express the target antigen. The cellular immune response specific to HPV6 and HPV11 E6 or E7 is used as an evaluation indicator to conduct immunogenicity studies of the DNA therapeutic vaccine in a mammalian model. As can be seen, the RRP DNA therapeutic vaccine pAD1543 designed in the present invention has superior cellular immunogenicity. Due to its excellent sequence design, it exhibits excellent immunogenicity against HPV6 and HPV11 viruses, and can significantly inhibit the occurrence of recurrent respiratory papilloma tumors caused by HPV6 and HPV11 viruses, showing excellent anti-tumor effects. It is reasonable to expect that the DNA therapeutic vaccine pAD1543 designed in the present invention can be further developed for clinical trials and, based on the results of clinical trials, can be marketed.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant DNA molecule, characterized in that The nucleotide sequence of the recombinant DNA molecule is the nucleotide sequence shown in SEQ ID No.

34.

2. Biomaterial, characterized in that The biomaterial includes at least one of (a) to (b): (a) a recombinant expression vector comprising the recombinant DNA molecule of claim 1; (b) A host cell comprising the recombinant DNA molecule of claim 1 or the recombinant expression vector of (a).

3. The biomaterial according to claim 2, characterized in that The recombinant expression vector is a eukaryotic expression vector, and the vector skeleton of the eukaryotic expression vector is pVAX1.

4. Use of the recombinant DNA molecule according to claim 1 or the biomaterial according to any one of claims 2 to 3 in the following a or b: a. Preparation of vaccines for the prevention and / or treatment of human papillomavirus infection; b. Preparation of drugs for preventing and / or treating diseases caused by human papillomavirus; The human papillomavirus includes HPV6 human papillomavirus and / or HPV11 human papillomavirus.

5. A DNA vaccine, characterized in that The DNA vaccine comprises the recombinant DNA molecule according to claim 1 or the recombinant expression vector according to any one of claims 2 to 3.

6. The DNA vaccine according to claim 5, characterized in that The DNA vaccine further includes a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.

7. The method for preparing the DNA vaccine according to any one of claims 5 to 6, characterized in that: include: The recombinant DNA molecule according to claim 1 or the recombinant expression vector according to any one of claims 2 to 3 is introduced into a host cell, the host cell is cultured, and the recombinant DNA molecule or recombinant expression vector in the host cell is extracted to prepare a DNA vaccine.

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