Fusion gene and HPV nucleic acid vaccine

Abstract

The invention provides a fusion gene and an HPV nucleic acid vaccine. A fusion gene is obtained through research and optimization, and the fusion gene is obtained by sequentially connecting an L1 gene of an HPV 16 type, an E5 gene, an E6 gene and an E7 gene of the HPV 16 type and an E5 gene, an E6 gene and an E7 gene of an HPV 52 type. The inventor finds that an HPV vaccine prepared by taking the fusion gene as an antigen component can effectively promote cellular immune response and formation of long-term protective memory, promote hair growth center response and formation of humoral immune memory, remarkably improve the proportion of NKT cells, successfully activate systemic anti-tumor cell immune response of mice, and improve the anti-tumor effect of the mice. The anti-tumor immune effect is greatly improved. The traditional Chinese medicine composition has the dual effects of prevention and treatment, has the prevention capability, can effectively inhibit tumor growth and has the tumor treatment capability. The HPV vaccine also has the advantages of high safety, good stability and the like, and has a wide application prospect.

Description

A fusion gene and HPV nucleic acid vaccine Technical Field

[0001] This invention belongs to the field of vaccine preparation technology, especially HPV vaccines, and specifically relates to a fusion gene and HPV nucleic acid vaccine. Background Technology

[0002] Cervical cancer, also known as cervical cancer, is a malignant tumor of the female reproductive tract that occurs in the cervix. The most common type is squamous cell carcinoma. The primary cause is human papillomavirus (HPV) infection. Globally, cervical cancer ranks 8th (3.3%) among all malignant tumors and 4th (6.8%) among female cancers.

[0003] Currently, there are 6 HPV preventative vaccines available globally, but no therapeutic HPV vaccines are on the market. There are 18 HPV vaccines in clinical trials, primarily targeting HPV types 6, 11, 16, and 18. With changing times, the main HPV infection types in my country are now types 16, 52, and 18, with type 52 showing a significant increase in incidence.

[0004] Therefore, there is a need in this field for a highly effective HPV vaccine that targets the main circulating strains in my country and has both preventive and therapeutic effects. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a fusion gene and an HPV nucleic acid vaccine. The HPV nucleic acid vaccine using the fusion gene as an antigen component can effectively activate humoral immunity and cellular immunity, and has both preventive and therapeutic effects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] In a first aspect, the present invention provides a fusion gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0008] A second aspect of the present invention provides a fusion protein encoded by the fusion gene as described above.

[0009] A third aspect of the invention provides a recombinant expression vector incorporating the fusion gene as described above.

[0010] In some embodiments, the backbone vector of the recombinant expression vector is selected from pcDNA3.1+C-DYK-P2A, pVAX1-C-DYK-P2A, pcDNA3.1+N-DYK-P2A, and pcDNA3.1+N-HA-C-DYK-P2A.

[0011] In some embodiments, the backbone vector of the recombinant expression vector is pcDNA3.1+C-DYK-P2A.

[0012] A fourth aspect of the present invention provides the use of the fusion gene, the fusion protein, and / or the recombinant expression vector as described above as antigens in the preparation of HPV vaccines.

[0013] In some implementations, the application includes increasing CD4 production. + TNF-α + T cells and / or CD8 + The proportion of central memory T cells.

[0014] In some implementations, the application includes increasing the proportion of germinal center B cells and / or memory B cells.

[0015] In some implementations, the application includes increasing the proportion of NKT cells.

[0016] In some embodiments, the application includes increasing the proportion of IFN-γ and / or TNF-α secreted by immune cells.

[0017] A fifth aspect of the present invention provides an HPV vaccine, wherein the antigen of the HPV vaccine comprises the fusion gene, the fusion protein and / or the recombinant expression vector as described above.

[0018] Compared with the prior art, the present invention has the following beneficial effects.

[0019] This invention, through research and optimization, has yielded a fusion gene obtained by sequentially linking the L1 gene of HPV 16, the E5, E6, and E7 genes of HPV 16, and the E5, E6, and E7 genes of HPV 52. The inventors discovered that HPV vaccines prepared using this fusion gene as an antigen component can effectively promote cellular immune responses and the formation of long-term protective memory, promote germinal center responses and humoral immune memory, significantly increase the proportion of NKT cells, successfully activate systemic anti-tumor cellular immune responses in mice, and greatly enhance anti-tumor immune efficacy. Furthermore, it possesses dual preventative and therapeutic effects, exhibiting both preventative capabilities and the ability to effectively inhibit tumor growth, thus possessing the capacity to treat tumors. The HPV vaccine also boasts advantages such as high safety and good stability, and has broad application prospects. Attached Figure Description

[0020] Figure 1 shows the map of the pcDNA3.1-C-16L1-P2A-16 / 52E5E6E7-DYK plasmid.

[0021] Figure 2 is an experimental flowchart of Example 1.

[0022] Figure 3 shows CD4. + TNF-α + T cells and CD8 + Results of the detection of the proportion of TCM cells.

[0023] Figure 4 shows the results of the detection of the ratio of GC B cells and MB cells.

[0024] Figure 5 shows the results of the detection of the proportion of NKT cells.

[0025] Figure 6 is a flowchart of the experiment in Example 2.

[0026] Figure 7 shows the results of tumor appearance and size detection.

[0027] Figure 8 shows the results of L1 antibody level detection in mice.

[0028] Figure 9 shows the results of IFN-γ and TNF-α level detection in mice. Detailed Implementation

[0029] Experimental methods in the following embodiments of the present invention, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0030] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0031] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0032] The term "and / or" as used in this invention describes the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent three situations: A exists alone, A and B exist simultaneously, and B exists alone.

[0033] The following description is based on specific embodiments.

[0034] Example 1 I. Experimental Methods 1. Preparation of Antigen Components In order to improve the immune effect and make the vaccine have both preventive and therapeutic effects, this example studies and optimizes DNA as a vaccine antigen component, and compares and explains the following three fusion genes as examples.

[0035] (1) HPV 16 / 52 fusion gene, containing the full L1 gene of HPV 16, the E5, E6 and E7 genes of HPV 16, and the E5, E6 and E7 genes of HPV 52 (E is an early protein, and the transmembrane coding sequence of the E5, E6 and E7 genes has been removed), and its nucleotide sequence is shown in SEQ ID NO: 1.

[0036] (2) HPV 16 fusion gene, containing the full L1 gene of HPV 16 and the E5, E6 and E7 genes of HPV 16 (E is the early protein, and the transmembrane coding sequence of the E5, E6 and E7 genes has been removed), and its nucleotide sequence is shown in SEQ ID NO: 2.

[0037] (3) HPV 52 fusion gene, containing the full L1 gene of HPV 16 and the E5, E6 and E7 genes of HPV 52 (E is an early protein, and the transmembrane coding sequence of the E5, E6 and E7 genes has been removed), and its nucleotide sequence is shown in SEQ ID NO: 3.

[0038] The nucleotide sequences of the HPV 16 / 52 fusion gene, HPV 16 fusion gene, and HPV 52 fusion gene were synthesized by Nanjing GenScript Biotech Co., Ltd., and then ligated into the pcDNA3.1+C-DYK-P2A vector to obtain the plasmids pcDNA3.1-C-16L1-P2A-16 / 52E5E6E7-DYK, pcDNA3.1-C-16L1-P2A-16E5E6E7-DYK, and pcDNA3.1-C-16L1-P2A-52E5E6E7-DYK. The pcDNA3.1-C-16L1-P2A-16 / 52E5E6E7-DYK plasmid is shown in Figure 1 as an example.

[0039] Take 5 μL of each of the above plasmids and add them to 50 μL of DH5α competent cells. Mix well and place on ice for 30 min. Heat shock: Heat shock in a 42℃ water bath for 45 seconds, then return to ice. Take 800 μL of antibiotic-free LB liquid medium and add it to the plasmids and competent cells. Incubate at 37℃ and 220 rpm for 1 h with shaking. Centrifuge at 12000 rpm for 2 min at room temperature. Discard the supernatant in a clean bench, mix with reflux solution, and plate. Bacterial culture: Incubate the plates upright for 1 h, then invert overnight. After 12-16 h, select large, round colonies without satellite colonies, pick them, and incubate overnight for 12-16 h. Expand the bacterial culture in 100 ml Erlenmeyer flasks. Then, DNA plasmids were enriched using the Tiangen (reagent brand) plasmid endotoxin-free plasmid large-scale extraction kit. The DNA plasmids were used as antigenic components for preparing HPV vaccines, and the corresponding vaccines were named HPV16 / 52 DNA vaccine, HPV16 DNA vaccine, and HPV 52 DNA vaccine, respectively.

[0040] 2. DNA vaccine preparation: The Polypuls (reagent brand) in vivo-jetPEI® kit was used to prepare the DNA vaccine (prepare fresh for each use). The specific reagent quantities are shown in Table 1 below: Table 1 3. Animal Immunization and Detection: C57 mice were used as an animal model and randomly divided into 4 groups of 5 mice each. Each group was immunized with either PBS or DNA vaccine, as shown in Table 2. Preparations A and B were prepared separately. After mixing A and B, 100 μL of the mixture was injected subcutaneously into each mouse. Immunization was performed on days 0, 7, and 14, for a total of 3 immunizations. Subcutaneous tumor bearing was performed using 1 million TC-1 cells per mouse on days 21, 22, and 23 following the first immunization. Figure 2 shows the experimental flowchart.

[0041] Table 2 Spleen and subcutaneous tumors of mice were harvested 42 days after the first immunization, and CD4+ of TNF-α produced by the spleen was detected by flow cytometry. + T cells, CD8 + Central memory T cells (CD8) + The proportions of TCM, germinal center B cells (GC B cells), and memory B cells (MB cells), and the proportion of natural killer T cells (NKT) in tumor tissue, are as follows: (1) Preparation of single-cell suspension: Take mouse spleen, grind the spleen in a cell sieve, put it into a 15ml centrifuge tube, and centrifuge at 4℃ and 1500rpm for 7min. Discard the supernatant, lyse it on ice with 1ml of Red Crack PBS for 15min, centrifuge at 4℃ and 1500rpm for 7min, discard the supernatant, and resuspend it with 1ml of PBS.

[0042] Mouse tumors were collected, ground in a cell sieve, and lymphocytes were extracted from the tumor tissue using the mouse tumor infiltrating tissue lymphocyte separation kit from Beijing Solarbio Science & Technology Co., Ltd.

[0043] (2) Cell surface molecular staining: Transfer the single cell suspension to a flow cytometer, add surface molecular staining antibody, stain for 30 min, add 1 ml PBS to stop staining, centrifuge at 4℃ and 1500 rpm for 7 min, discard the supernatant, resuspend in 200 μl PBS, and prepare for instrument detection.

[0044] II. Experimental Results 1. Cellular Immune Response and Formation of Long-Term Protective Memory As shown in Figure 3, compared with HPV16 DNA vaccine and HPV52 DNA vaccine, HPV16 / 52 DNA vaccine can significantly increase the CD8+ in the spleen of immunized mice. + TCM and CD4 + T TNF-α + The proportion of cells indicates that HPV16 / 52 DNA vaccine immunization can help the body better establish long-term, cellular immune memory against tumor antigens. HPV16 / 52 DNA vaccine can not only effectively activate CD4... + The T cell helper pathway can also successfully convert this activation signal into high-quality, long-lived CD8+. + The T-cell memory bank lays a solid cellular immune foundation for preventing tumor recurrence.

[0045] 2. Germinal Center Response and Establishment of Humoral Immune Memory: GC B cells are crucial in the germinal center process, undergoing clonal proliferation, high-frequency mutation, and affinity maturation. A significant increase in their proportion indicates that the vaccine has successfully triggered an active germinal center response, a necessary prerequisite for producing high-affinity, high-specificity antibodies. Memory B cells (MB cells) are one of the final products of the germinal center response and serve as carriers of long-term humoral immune memory; an increased proportion of MB cells indicates that the vaccine has established good humoral immune memory.

[0046] As shown in Figure 4, compared with HPV16 DNA vaccine and HPV52 DNA vaccine, HPV 16 / 52 DNA vaccine significantly increased the proportion of GC B cells and MB cells in the spleen of immunized mice. This indicates that HPV 16 / 52 DNA vaccine can more effectively drive B cells to undergo effective antigen-driven maturation and form a stable pool of memory B cells, ensuring that the body can rapidly produce high-affinity antibodies when encountering tumor antigens again.

[0047] 3. Antitumor activity of innate-like T cells: Activation of natural killer T cells (NKT) is a unique subset of T cells that possesses characteristics of both innate and adaptive immunity. They can rapidly produce large amounts of cytokines (such as IFN-γ), thereby directly killing tumor cells or regulating the tumor microenvironment.

[0048] As shown in Figure 5, compared with HPV16 DNA vaccine and HPV52 DNA vaccine, the proportion of NKT cells in the tumor local area of ​​mice immunized with HPV 16 / 52 DNA vaccine was significantly increased, indicating that HPV 16 / 52 DNA vaccine can more effectively recruit and activate anti-tumor effector cells and act directly on tumor lesions.

[0049] In conclusion, HPV 16 / 52 DNA vaccines using the HPV 16 / 52 fusion gene as an antigen component can significantly enhance immunization efficacy, and further research on HPV 16 / 52 DNA vaccines is warranted.

[0050] Example 2 I. Experimental Methods 1. Subcutaneous Tumors in Mice C57 mice were used as an animal model and randomly divided into two groups of five mice each. Each group was immunized with PBS and HPV 16 / 52 DNA vaccine, respectively. Subcutaneous tumor-bearing mice were immunized with 1 million TC-1 cells per mouse on days 1, 2, and 3, followed by immunization on days 4, 11, and 18, for a total of three immunizations. The immunization method was the same as in Example 1. The experimental protocol is shown in Figure 6.

[0051] 2. Appearance and size of tumor tissue: Subcutaneous tumors from 39-day-old C57 mice were immersed in PBS at 4°C to maintain tumor cell viability. The tumor tissues, after removing excess PBS, were placed on clean white paper from largest to smallest and photographed under natural light.

[0052] 3. ELISA detection of serum antibody, IFN-γ and TNF-α levels (1) Take orbital blood from C57 mice 39 days after immunization, let stand for 4 hours, centrifuge at 4℃ and 2500 rpm for 30 min, and take the supernatant; (2) Take out the required strips (Boyan Bio mouse papillomavirus type 16 L1 antibody ELISA kit, mouse IFN-γ ELISA kit, mouse TNF-α ELISA kit) from the aluminum foil bag, and seal the remaining strips in a self-sealing bag and put them back in the refrigerator.

[0053] (3) Set up standard wells and sample wells. Add 50 μL of standard of different concentration to each standard well; (4) Add 50 μL of the sample to be tested (sample diluted 10 times) to the sample well; do not add to the blank well.

[0054] (5) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 60 min.

[0055] (6) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350μL), let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times.

[0056] (7) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0057] (8) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0058] II. Experimental Results 1. The appearance and size of the tumor tissue are shown in Figure 7. Compared with the control group, the HPV 16 / 52 DNA vaccine can significantly inhibit tumor growth and has a good therapeutic effect.

[0059] 2. The ELISA results are shown in Figure 8. The HPV 16 / 52 DNA vaccine effectively activated L1 antibody levels in mice 39 days after the initial immunization, and the difference remained significant at day 39 (p < 0.01). This indicates that the vaccine has good antibody protection and can effectively prevent HPV 16.

[0060] As shown in Figure 9, 39 days after the initial immunization, the levels of IFN-γ and TNF-α in mice were significantly increased. The results indicate that the HPV 16 / 52 DNA vaccine can successfully activate a systemic anti-tumor cellular immune response in mice, achieving a better anti-tumor immune effect.

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[0064] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0065] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A fusion gene, characterized in that, The nucleotide sequence of the fusion gene is shown in SEQ ID NO:

1.

2. The fusion protein encoded by the fusion gene as described in claim 1.

3. Insert a recombinant expression vector containing the fusion gene as described in claim 1.

4. The recombinant expression vector as described in claim 3, characterized in that, The backbone vector of the recombinant expression vector is selected from pcDNA3.1+C-DYK-P2A, pVAX1-C-DYK-P2A, pcDNA3.1+N-DYK-P2A, and pcDNA3.1+N-HA-C-DYK-P2A; preferably, the backbone vector of the recombinant expression vector is pcDNA3.1+C-DYK-P2A.

5. The use of the fusion gene as described in claim 1, the fusion protein as described in claim 2, and / or the recombinant expression vector as described in any one of claims 3 to 4 as antigens in the preparation of HPV vaccines.

6. The application as described in claim 5, characterized in that, The application includes improving CD4 + TNF-α + T cells and / or CD8 + The proportion of central memory T cells.

7. The application as described in claim 5, characterized in that, The application includes increasing the proportion of germinal center B cells and / or memory B cells.

8. The application as described in claim 5, characterized in that, The application includes increasing the proportion of NKT cells.

9. The application as described in claim 5, characterized in that, The applications include increasing the proportion of IFN-γ and / or TNF-α secreted by immune cells.

10. An HPV vaccine, characterized in that, The antigen of the HPV vaccine comprises the fusion gene as described in claim 1, the fusion protein as described in claim 2, and / or the recombinant expression vector as described in any one of claims 3 to 4.

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