CN1299769C - Human papilloma virus and heat shock protein recombinant protein vaccine and use thereof - Google Patents

Abstract

The invention relates to a fusion gene obtained by fusing a modified human papillomavirus type 16 (HPV-16) E6/E7 gene and a transformed mycobacterium tuberculosis heat shock protein 70 (TBhsp70) gene, and a preparation method for the fusion gene , the construction of high-efficiency expression vector containing the fusion gene and the expression vector, the recombinant protein vaccine obtained by using the fusion gene, and the application of the fusion gene and recombinant protein vaccine in the preparation of drugs for preventing and treating human papillomavirus-related tumors. Experiments have proved that the recombinant protein vaccine of the present invention has strong immune ability and anti-tumor function, and does not need to be used in combination with any other immune adjuvants.

Description

Human papillomavirus and heat shock protein recombinant protein vaccine and its application

technical field

The invention relates to a fusion gene of human papillomavirus and heat shock protein, its expression vector and recombinant protein vaccine, in particular to modified human papillomavirus type 16 (HPV-16) E6/E7 and transformed Mycobacterium tuberculosis heat Shock protein 70 (TBhsp70) fusion gene, the preparation method of the fusion gene, the construction of the high-efficiency expression vector containing the fusion gene and the expression vector, the fusion protein obtained by using the fusion gene, and the fusion gene and fusion protein in the preparation Application in the prevention and treatment of human papillomavirus-related diseases and tumor drugs.

Background technique

Malignant tumors are common and frequently-occurring diseases that seriously endanger human health. At present, the three conventional treatment methods in tumor treatment: surgery, radiotherapy and chemotherapy have been developed to a certain extent, but surgical resection is a local treatment method, while malignant tumors grow infiltrating and easily pass through blood vessels and/or lymphatic vessels Distant metastases occur, so for patients with advanced malignant tumors, surgery alone is difficult to completely remove cancer cells; radiotherapy is also a local-based treatment method, which also has defects in surgical treatment, and many malignant tumors are not affected by radiotherapy. Insensitive; Chemotherapy is a systemic treatment, but it faces the disadvantages of drug resistance and severe side effects. Therefore, the study of new treatment measures for malignant tumors and the development of new biological drugs for prevention and treatment are one of the important topics concerned by oncologists in various countries.

A large number of epidemiological and clinical studies at home and abroad have proved that: HPV, especially HPV16, is associated with cervical cancer, esophageal cancer, lung cancer, head and neck cancer, gastric cardia cancer, skin cancer, breast cancer, bladder cancer, ovarian cancer, It is closely related to the occurrence of malignant tumors such as prostate cancer, penile cancer, anal cancer and colorectal cancer. The etiological relationship between HPV and cervical cancer has been widely recognized, and HPV16 is the main viral cause of cervical cancer. A multi-center study (Int. J. Cancer, 100: 327-331, 2002) involving Shanghai, Guangzhou, Beijing, Sichuan and Hong Kong 5 provinces and regions shows that 78.7-87.7% of cervical cancer patients in my country have HPV infection, 61.7-91.8% of them were HPV16 infection. Studies on the carcinogenic mechanism of HPV have shown that after HPV infects host cells, its two oncogenes, E6 and E7, are stably integrated into the genomic DNA of the host cell and continuously expressed, which is the key to the malignant transformation of cells induced by HPV and the maintenance of malignant phenotype. Therefore, E6 and E7 have become the focus of people's research on carcinogenesis mechanism and new biological products for prevention and treatment. Studies have also shown that: using the main capsid protein L1 of HPV as a vaccine is effective for preventing HPV infection, but it has no preventive and therapeutic effect on infected persons; the preparation difficulty of this vaccine is another shortcoming, because the preparation of VLP (virus-like particles) consumes a lot of time. Time, material consumption, complicated operation, 3 times of ultracentrifugation and low yield (only 2/10,000 to 5/10,000 of the total amount of protein in the upper ultracentrifugation), thus bringing great challenges to large-scale batch production. Many difficulties. Studies in recent years have shown that using prokaryotic expressed E6 or E7 protein alone as a therapeutic vaccine has a certain preventive effect. Researchers have envisaged using unmodified E6/E7 fusion protein as a vaccine in order to improve the control effect, but due to E6 itself or The expression level of E6/E7 fusion protein is too low to be of practical value. And because it is an unmodified wild-type oncogene product, its safety has been questioned. Viruses containing E6 and E7 genes, or DNA vaccines based on plasmids, also have safety issues due to their possible integration into the cell genome. The use of synthetic peptides as vaccines is expensive and limited by MHC, and the scope of use is limited.

A large number of studies have shown that immunotherapy is an effective method for tumors and other diseases that seriously endanger human health. Although a variety of highly purified new vaccines currently under development have good specificity and low toxicity, they need to be used in conjunction with highly efficient adjuvants. In-depth research on adjuvants has expanded the space of immunotherapy. However, on the one hand, different adjuvants can produce different effects even on the same antigen; , Therefore, it may cause a series of toxic and side effects (including local inflammation, long-term refractory nodules, cysts, tumorigenic effects, teratogenic effects, and cross-immune reactions, etc.). Therefore, the application of adjuvants must consider effectiveness, safety, selectivity, controllability and other theoretical, technical and manufacturing issues. For example, although the classic aluminum adjuvant has been used in combination with ordinary vaccines to improve antibody levels and safety, it has been proven in practice for a long time, but it has no effect on the ability of genetically engineered proteins or polypeptides to induce cellular immunity. Its mechanism of action is poorly understood, making it difficult to meet the needs of new vaccines; for another example, although incomplete Freund's adjuvant (IFA) has been proved to be one of the most successful adjuvants in both animals and humans, its possible People still worry about side effects, such as: nodules are often formed when applied locally, and even cysts are formed by liquefaction. It has been reported that it has tumorigenic effects in male Swiss mice (BullWHO, 1969; 41:617-621), but in BALB/c and There is no tumorigenicity in C57BL/6 mice, so safety concerns remain. Finally, although a variety of new adjuvants have been developed, the safety and mechanism of action still need to be further studied.

A large number of studies have proved that heat shock proteins have strong immunogenicity, such as: ①The hsps complex isolated from tumor or virus-infected cells can induce strong anti-tumor or anti-virus immunity; ②When mycobacteria is infected, High-frequency human CD4 ⁺ T cells targeting hsp70 and 60 appeared; ③Multiple mouse and human B cell and T cell epitopes of hsps have been identified and can be recognized by mammalian cells. This prompts researchers to use this property of hsps to develop strategies for infectious disease vaccines and tumor immunotherapy, and also provides a basis for its use as an adjuvant-free carrier. After the researchers covalently linked hsp70 to malaria small peptide (NANP) 40, HIV-1p24, ovalbumin, etc., without other adjuvant conditions, they stimulated specific humoral reactions targeting these peptides or genetically engineered proteins. and cellular immune responses. The induction of specific CD8 ⁺ CTL response plays an important role in anti-tumor immunity, and the possible mechanisms are as follows: ① The target protein covalently linked to hsp70 itself contains T cell epitopes, and the T cell epitopes of hsp70 are attached, thus immune Stronger originality; ②The target protein covalently linked to hsp70 contains a variety of known or unknown epitopes, which can fully exert its effect; ③hsp70 fusion protein can activate shared professional antigen-presenting cells (APCs), Provide specific homologous CD4 ⁺ helper T cells to activate target protein-specific CD8 ⁺ CTL; ④ hsp70 has a chaperone function, can help protein folding, and can promote the ubiquitination of covalently linked proteins to be well processed, TBhsp70 The fusion protein can be taken up by professional APCs through receptor-mediated endocytosis, and the hsp70 fusion protein can stimulate professional APCs in vivo and in vitro to up-regulate the expression of MHC I and II molecules and co-stimulatory molecules, which not only facilitates the entry of the target protein into professional APCs And presented to the corresponding cell subunits, and is conducive to the formation of MHC-peptide complexes and presented to the cell surface; ⑤Recent studies (J.Exp.Med., 191:403-408, 2000) show that: hsp70 The fusion protein induces the CD8 ⁺ CTL response of the target protein, not only does not need adjuvant, nor does it need its chaperone function, but also overcomes the limitation of MHC, and does not need the help of CD4 ⁺ T cells. In summary, TBhsp70 fusion protein immunotherapy can induce CD8 ⁺ CTL responses without the need for adjuvants, without the help of CD4 ⁺ T cells, without MHC restriction, without the need for viral vectors, and avoids the potential potential of DNA sequences in DNA vaccines. It has safety issues, and is easy to prepare in large quantities, and has good safety. Coupled with the low autoimmunity of malignant tumor patients, the down-regulation of MHC molecules and co-stimulatory molecules in cancer cells, and the significant destruction of CD4 ⁺ T cells caused by HIV infection, these all make the hsp70 fusion protein in the development of anti-tumor, anti-HIV and other infections There are obvious advantages in immunotherapy strategies for diseases.

Contents of the invention

One of the purposes of the present invention is to overcome the deficiencies of the prior art and provide a recombinant protein vaccine that can effectively treat HPV-related tumors and does not require an immune adjuvant.

One of the objectives of the present invention is achieved through the following technical approaches:

A recombinant protein vaccine of the present invention that can effectively treat HPV-related tumors and does not require an immune adjuvant, it is composed of the amino acid sequence of 161-370 positions of the ATP binding site in the hsp70 protein, the 1-120 position of the human papillomavirus E6 protein The amino acid sequence at position 1 is connected with a total of 98 amino acid sequences of E7 protein, wherein the leucine at position 50 of the E6 protein is modified to glycine, and the cysteine at position 24 and glutamic acid at position 26 of the E7 protein are modified are respectively modified to glycine; wherein the human papillomavirus E6 protein is located at the amino terminal of the recombinant protein, and the hsp70 protein is located at the carboxyl terminal of the recombinant protein.

The recombinant protein vaccine of the present invention preferably has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;

The recombinant protein vaccine of the invention has strong immune ability and anti-tumor function, and does not need immune adjuvant.

The second object of the present invention is to provide a fusion gene encoding the above-mentioned recombinant protein vaccine.

Two of the purpose of the present invention is achieved through the following technical approaches:

A fusion gene of the present invention is formed by fusion of the restructured and modified human papillomavirus E6/E7 gene and the transformed mycobacterium heat shock protein 70 gene, and the fusion gene includes hsp70 gene encoding ATP binding site The nucleotide sequence of a part (161-370 amino acids); the nucleotide sequence of the first 1-120 amino acids encoded by the E6 gene of HPV, including the complete B-epitope (1-23aa) and 2 CTLs in E6 Epitopes (29-38aa; 52-61aa); the E7 gene of HPV encodes a nucleotide sequence of a total of 98 amino acids, including 4 CTL epitopes in E7 (7-15aa; 11-20aa; 49-57aa and 79 -87aa), a Th epitope (48-54aa) and three B epitopes (1-18aa; 25-37aa and 43-60aa). In addition, the codons encoding the 50th lysine in the E6 gene, the 24th cysteine in the E7 gene, and the 26th glutamic acid in the E7 gene were mutated by site-directed mutagenesis to remove their codons. Combined with the function of the tumor suppressor gene, the safety of the expressed recombinant protein vaccine is increased.

The fusion gene of the present invention is characterized in that it can efficiently express the target fusion protein in the host and has strong immune ability and anticancer function.

The fusion gene is prepared by the following steps:

(A) obtaining TBhsp70, E6 and E7 genes;

(B) performing site-directed mutation on the codons encoding the 50th lysine of the E6 gene, the 24th cysteine of the E7 gene, and the 26th glutamic acid of the E7 gene;

(C) The nucleotide sequence of the TBhsp70 gene encoding a part of the ATP binding site (161-370 amino acids), the nucleotide sequence of the first 1-120 amino acids encoded by the E6 gene and the core of a total of 98 amino acids encoded by the E7 gene Nucleotide sequence fusion.

Another object of the present invention is to provide an expression vector containing the above fusion gene and a host cell containing the expression vector.

Another object of the present invention is to provide the above-mentioned recombinant protein vaccine and the application of the fusion gene in medicine.

A further object of the present invention is to provide the application of the above-mentioned recombinant protein vaccine in the preparation of drugs for treating and/or preventing HPV-related tumors.

The human papillomavirus E6/E7 and mycobacterial heat shock protein 70 fusion gene of the present invention can be used to prepare a protein vaccine that is safe, effective, highly expressible, and easy to prepare in large quantities for preventing and treating HPV-related tumors such as cervical cancer. Protein vaccines induce specific CD8 ⁺ CTL responses without any adjuvant. Due to the low autoimmunity of malignant tumor patients, the expression of cancer cell MHC molecules and co-stimulatory molecules is down-regulated. Therefore, the recombinant protein vaccine of the present invention has obvious advantages in the development of immunotherapy strategies against HPV-related tumors and other infectious diseases.

Description of drawings

Figure 1 SDS-PAGE analysis of expression, purification and identification of fusion protein of mE6Δ/mE7/hsp70Δ (Western blot)

M: protein molecular weight standard; 1: purified fusion protein; 2: BL21(DE3) empty bacteria induced for 0 hours; 3, 4: BL21(DE3) bacteria transformed with PET30a(+) empty vector induced for 0 hours, 4 hours; 5 , 6: Induction of BL21(DE3) bacteria transfected with PET30a(+)-mE6Δ/mE7/TBhsp70Δ recombinant plasmid for 0 hour and 4 hours; 7, 8: E6, E7 Western blot identification

Figure 2 The recombinant mE6Δ/mE7/hsp70Δ fusion protein vaccine without adjuvant prevents the attack and re-challenge of TC-1 cells

-◆-PBS group

- -hsp70Δ group

-▲-mE6Δ/mE7 group

- - mE6Δ/mE7/hsp70Δ group

Fig. 3 Non-adjuvanted recombinant mE6Δ/mE7/hsp70Δ fusion protein vaccine prevents the attack of TC-1 tumor cells (average tumor volume 43 days after inoculation of TC-1 cells)

Figure 4 Recombinant mE6Δ/mE7/hsp70Δ fusion protein vaccine without adjuvant inhibits the growth of established solid tumors

-◆-PBS group

- - mE6Δ/mE7/hsp70Δ group

The beneficial effect of the present invention will be further described through the following examples. It should be understood that these examples are only for the purpose of illustration, and in no way limit the protection scope of the present invention.

Detailed ways

[Example]

1. Acquisition and transformation of mycobacterial heat shock protein 70 gene, human papillomavirus E6 and E7 genes and construction of mE6Δ/mE7/hsp70Δ fusion gene and recombinant prokaryotic expression plasmid vector

1. Acquisition and transformation of mycobacterial heat shock protein 70 gene

Mycobacterium tuberculosis was cultured, genomic DNA was extracted, and the nucleotide sequence (named TBhsp70Δ) of the hsp70 gene encoding a part of the ATP binding site (161-370 amino acids) was amplified by PCR, and BamH I and Xho were introduced into the two ends of the sequence respectively. The I restriction enzyme cutting site, after sequence analysis, is consistent with the sequence compared with GeneBank.

2. Acquisition of human papillomavirus E6 and E7 genes

Genomic DNA was extracted from the tumor tissue of HPV16-positive cervical cancer patients, and the E6 and E7 genes were respectively amplified by PCR. After sequence analysis, the DNA sequence of the E7 gene was consistent with foreign reports, encoding a total of 98 amino acids; the DNA sequence of the E6 gene except the first Except for T→C at position 259, the rest are consistent with foreign reports, encoding a total of 158 amino acids. This point mutation causes the coded amino acid to change from cysteine to arginine (the 87th amino acid in the E6 protein). The point mutation is located outside the known epitope of E6, so it has no effect on its immunogenicity.

3. Site-directed mutation of human papillomavirus E6 and E7 genes

Since the leucine at position 50 in the protein encoded by the E6 gene is the key site for the inactivation of the tumor suppressor gene p53, the cysteine at position 24 and glutamine at position 26 in the protein encoded by the E7 gene Acid is the key site for the inactivation of the tumor suppressor gene Rb, so these three sites were changed to glycine by site-directed mutagenesis. In order to remove its combination and inactivate the activity of tumor suppressor gene, increase the safety of the vaccine. The mutated E6 and E7 genes were named mE6 and mE7, respectively.

4. Construction of mE6Δ/mE7/TBhsp70Δ fusion gene and recombinant prokaryotic expression plasmid vector

Using the above mE6 as a template, amplify its nucleotide sequence encoding the first 1-120 amino acids by PCR method, and introduce an Nco I restriction enzyme site at its 5' end. Then use the PCR segmentation extension method to gradually add the nucleotide sequence of E7 gene encoding a total of 98 amino acids at the 3' end of mE6Δ, and add a BamH I restriction enzyme site at its 3' end. Sequence analysis proved that the obtained fusion gene sequence was correct and the mutation site was retained. Named mE6Δ/mE7. Then use Nco I and BamH II to double-enzyme cut PET30a (+) plasmid and mE6Δ/mE7 fusion gene respectively, after recovery, use T4 DNA ligase to connect overnight at 12°C, transform DH5α competent bacteria, and screen positive on ampicillin LB agar plate Cloning, picking a single colony to amplify and extract the recombinant plasmid vector. Named PET30a(+)-mE6Δ/mE7. Hsp70Δ and PET30a(+)-mE6Δ/mE7 were digested with BamH I and Xho I respectively, recovered, ligated, transformed, screened, single colonies picked and amplified as above, and recombinant plasmids were extracted. Named PET30a(+)-mE6Δ/mE7/TBhsp70Δ. So far, the mE6Δ/mE7/TBhsp70Δ fusion gene (see SEQ ID NO: 1 for the sequencing results) and its recombinant prokaryotic expression plasmid vector have been successfully constructed. The recombinant plasmid vector constructed at the same time containing only TBhsp70Δ was named PET30a(+)-TBhsp70Δ.

2. Construction of high-efficiency expression engineering bacteria and expression identification of fusion protein

The recombinant prokaryotic expression plasmid constructed above was transformed into Escherichia coli BL21(DE3) (Novagen Company), and 80 single colonies were randomly picked to observe the expression status. Shake the bacteria at 37°C until the OD ₆₀₀ is 0.5-0.6, induce protein expression with 1mM (final concentration) IPTG, take a sample after 4 hours for SDS-PAGE electrophoresis (12.5%), then stain with Coomassie brilliant blue, destaining solution A fusion protein band of about 52KD can be seen after decolorization (Figure 1). Then use anti-his-tag, HPV16E6 and E7 monoclonal antibodies (Santa Cruz Company) to carry out Western blot analysis and identification on the expressed protein (Figure 1), which proves that the protein is the target fusion protein, and the molecular weight is consistent with the expected. Then use the same conditions to ferment, take samples at different time points (1-5 hours) and analyze them by SDS-PAGE electrophoresis, and at the same time conduct electrophoresis with BSA of known concentration as a reference standard. After 4 hours of fermentation and cultivation, the expression of the target protein reached a saturated state, accounting for about 30% of the total amount of bacteria. Further experiments proved that the fusion protein exists in the form of inclusion bodies.

3. Purification and dialysis renaturation of protein products (take 100ml cultured bacteria as an example)

1. Induction and harvesting of cells

Cultivate high-efficiency engineering bacteria at 37°C until _OD600 is 0.5-0.6, induce protein expression with 1mM (final concentration) IPTG, harvest cells by centrifugation after 4 hours, and use lysis buffer (150mM NaCL, NP401%, deoxycholic acid 0.5 %, SDS 0.1%) to resuspend the cells and further sonicate the cells in an ice bath.

2. Preparation and dissolution of inclusion bodies

Centrifuge at 12000g at 4°C for 20 minutes, discard the supernatant, and use 10ml of denaturing extraction/washing buffer (50mM Na ₃ PO ₄ , 6M guanidine hydrochloride, 300mM NaCL) to fully dissolve the precipitate on ice; centrifuge at 12000g at 4°C for 20 minutes, Store the supernatant in a 4°C refrigerator.

3. Equilibrate resin and load fusion protein

Take 6ml of affinity chromatography medium, that is, TALON ^TM Metal affinity chromatography resin storage solution, centrifuge at 3600g at 4°C for 5 minutes; equilibrate twice with 10ml of denaturing extraction/washing buffer, mix the solution containing inclusion bodies with the resin, After washing twice with 10ml of denaturing extraction/washing buffer, carefully load it into the chromatography column.

4. Washing Miscellaneous Proteins and Eluting Fusion Proteins

Wash the chromatography column with 20ml denaturing extraction/washing buffer, flow rate: 0.5ml/min; use 20ml imidazole elution buffer (45mM Na ₃ PO ₄ , 5.4M guanidine hydrochloride, 270mM NaCL, 150ml imidazole) to elute the fusion protein, Flow rate: 0.3ml/min, collected in steps, 0.5ml per tube.

5. Dialysis refolding and protein concentration

Put the eluted fusion protein solution into a dialysis bag, dialyze against PBS containing different concentrations of guanidine hydrochloride, and gradually reduce the concentration of guanidine hydrochloride until dialyzing PBS without guanidine hydrochloride. After dialysis, the fusion protein was concentrated to the desired concentration with PEG20000.

6. Fusion protein purity identification and quantification

Take 5-10ul of the dialyzed and concentrated protein solution for SDS-PAGE electrophoresis analysis (FIG. 1) for protein purity (>95%), and determine the protein concentration according to the Bradford method.

Repeated experiments with the above method, the recovery rate of fusion protein is 50-60%. Using 1 L of fermented broth, the above steps are scaled up to obtain 150-180 mg of the product (purity > 95%).

[Test Example 1] Tumor prevention effect test of the fusion protein vaccine of the present invention

C57BL/6 female mice (purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences, weighing 18-20 g) were randomly divided into four groups: PBS group, TBhsp70Δ group, mE6Δ/mE7 group and mE6Δ/mE7/TBhsp70Δ group, each group 10 Only. Using PBS (0.2ml), TBhsp70Δ (0.2ml PBS containing 1.5nM protein), mE6Δ/mE7 (0.2ml PBS containing 1.5nM protein), mE6/mE7/TBhsp70Δ (0.2ml PBS containing 1.5nM fusion protein) respectively Subcutaneous injection (in the groin) for immunization, 2 weeks after the first immunization, a booster immunization as above, 2 weeks after the booster immunization, attack with 6×10 ⁴ TC-1 tumor cells in the subcutaneous right rib ventral (TC-1 It is a mouse tumor cell transformed with HPV16 type E6 and E7 genes and activated Ras oncogene, donated by Dr. Wu from Johns Hopkins University in the United States), observe the time of tumor emergence in experimental animals, measure the tumor size and calculate the tumor volume: tumor volume = long diameter × short diameter ² /2. The results showed that: the mE6Δ/mE7/hsp70Δ group had a late tumor growth time, 7 had no tumor growth until the end of the experiment (43 days), and although 3 had tumors, 2 of them completely regressed, that is, only 1 tumor-bearing mice. In contrast, all experimental animals in the three control groups had tumor growth ( FIG. 2 ). On the 43rd day of the experiment, the average tumor volumes of the PBS group, hsp70Δ group, mE6Δ/mE7 group and mE6Δ/mE7/hsp70Δ group were: 3835.2±172.7mm ³ , 3685.3±158.6mm ³ , 3135.3±181.7mm ³ and 10mm ³ (Fig. 3 ). On the 51st day of the experiment, 9 tumor-free mice in the mE6Δ/mE7/hsp70Δ group were re-challenged with a larger dose of TC-1 tumor cells. The results showed that 2 mice experienced transient tumor growth, but then completely recovered. faded (Figure 2).

The above results show that after immunization with the non-adjuvanted mE6Δ/mE7/hsp70Δ fusion protein vaccine, the immunized experimental animals can produce a strong and specific anti-tumor immune response, which can repel the attack of cancer cells and prevent the formation of tumors. Has a preventive effect. It can be used to prevent the occurrence of HPV-related tumors in high-risk groups infected with HPV16.

[Test example 2] The growth effect test of fusion protein vaccine of the present invention inhibiting tumor

Since the results of prevention experiments have shown that hsp70Δ or mE6Δ/mE7 experimental animals alone cannot produce specific anti-tumor immune responses, so subsequent studies no longer set up separate groups. Experimental animals and immunization methods are the same as above. Firstly, 20 mice were subcutaneously inoculated with TC-1 tumor cells on the right flank, 1.3×105/mouse, and when the average volume of the tumor reached 5 mm ³ , they were randomly divided into 10 mice/group, and the first immunization was carried out, 2 A booster immunization was given a week later. Observe the tumor size and the survival time of the mice, and calculate the tumor volume according to the above formula. The results showed that the tumor growth in the mE6Δ/mE7/hsp70Δ group was significantly inhibited. On the 35th day of the experiment, the average tumor volumes of the PBS group and the mE6Δ/mE7/hsp70Δ group were 3508.4±156.9mm ³ and 408.5±80.3mm ³ respectively (Figure 4). The average survival time of tumor-bearing mice in PBS group and mE6Δ/mE7/hsp70Δ group was 34.2 days and 80.8 days, respectively, and 2 animals in mE6Δ/mE7/hsp70Δ group had complete tumor regression and long-term survival.

The above results show that the immunization of mE6Δ/mE7/hsp70Δ fusion protein vaccine without adjuvant can make the immunized experimental animals produce a strong and specific anti-tumor immune response, which can obviously inhibit the growth of solid tumors that have formed, so it has therapeutic potential. effect. It can be used for the treatment of HPV-related tumors.

[Test Example 3] Prevention and treatment of tumor metastasis by fusion protein vaccine of the present invention

Experimental animals and immunization methods were the same as in Test Example 2, first immunized twice, 4 weeks later, TC-1 tumor cells (2×10 ⁴ /mouse) were injected into the tail vein, and the mice were killed 5 weeks after inoculation of TC-1 cells to observe the lung metastasis (If no metastases are seen with the naked eye, the lungs are all embedded, and the metastases are observed under the microscope by serial sectioning). The results showed that all 10 mice in the PBS group developed lung metastases (7 of which had metastases with the naked eye and 3 with lung metastases under the microscope); 2 mice in the mE6Δ/mE7/hsp70Δ fusion protein group had metastases under the microscope, and 8 mice had metastases under the microscope. There was no metastases under the naked eye and microscope (Table 1).

Table 1 group naked eye transfer Microscopic transfer no transfer Transfer rate (%) mE6Δ/mE7/hsp70Δ in PBS group 70 32 08 10020

It shows that after immunization with the adjuvant-free mE6Δ/mE7/hsp70Δ fusion protein vaccine, the immunized experimental animals can produce a strong and specific anti-tumor immune response, which can prevent the metastasis of tumor cells, and thus has the effect of preventing metastasis. It can be used for the prevention of HPV-related tumor cell metastasis.

TC-1 tumor cells were injected into the tail vein (2×10 ⁴ /rat), and randomly divided into 10 rats in each group after 1 week. The treatment was divided into 2 groups. group), the other group began to immunize after 5 weeks of TC-1 cells (treatment group 2), and each group was sacrificed after 5 weeks of immunization to observe the lung metastases (if no metastases were seen with the naked eye, the lungs were completely embedded , serial section to observe the metastases under the microscope. The results showed that all 10 mice in the PBS group had lung metastases (8 of which had metastases with the naked eye, and 2 with lung metastases under the microscope); , 2 had metastases under the microscope, 7 had no metastases under the naked eye and under the microscope; in treatment group 2, 2 had metastases on the naked eye, 3 had metastases under the microscope, and 5 had no metastases on the naked eye and under the microscope (Table 2) .

Table 2 group naked eye transfer Microscopic transfer no transfer Transfer rate (%) PBS group mE6Δ/mE7/hsp70Δ (treatment group 1) mE6Δ/mE7/hsp70Δ (treatment group 2) 812 223 075 1003050

It shows that after immunization with the non-adjuvant mE6Δ/mE7/hsp70Δ fusion protein vaccine, the immunized experimental animals can produce a strong and specific anti-tumor immune response, which can inhibit the growth of metastatic tumors and thus have an effect on tumor metastasis. It can be used for the treatment of HPV-related tumor cell metastasis.

In summary, the advantage of the present invention is that human papillomavirus E6/E7 and mycobacterial heat shock protein 70 fusion gene can be used to prepare safe, effective, high-efficiency expression, easy large-scale preparation for the prevention and treatment of HPV-related tumors (such as Cervical cancer, etc.) and the fusion protein vaccine transferred thereto, the recombinant protein vaccine of the present invention induces specific CD8 ⁺ CTL responses without adjuvants. Due to the low autoimmunity of malignant tumor patients, the expression of MHC molecules and co-stimulatory molecules in cancer cells is down-regulated, and the lack of the best adjuvants and carriers for protein vaccines has seriously plagued the development of protein vaccines. Therefore, , the recombinant protein vaccine of the present invention has obvious advantages in the development of immunotherapeutic strategies against HPV-related tumors and other infectious diseases. Undoubtedly, it has important clinical application value.

Sequence Listing

SEQUENCE LISTING

<110> Institute of Cancer, Cancer Hospital, Chinese Academy of Medical Sciences

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<213>Homo sapiens

<400>2

Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro

1 5 10 15

Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp

20 25 30

Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu

35 40 45

Val Gly Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly

50 55 60

Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile

65 70 75 80

Ser Glu Tyr Arg His Tyr Arg Tyr Ser Leu Tyr Gly Thr Thr Leu Glu

85 90 95

Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn

100 105 110

Cys Gln Lys Pro Leu Cys Pro Glu Met His Gly Asp Thr Pro Thr Leu

115 120 125

His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Gly

130 135 140

Tyr GlyGln Leu Asn Asp Ser Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly

145 150 155 160

Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr

165 170 175

Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr

180 185 190

His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly

195 200 205

Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Lys Leu Glu Lys Glu Gln

210 215 220

Arg Ile Leu Val Phe Asp Leu Gly Gly Gly Thr Phe Asp Val Ser Leu

225 230 235 240

Leu Glu Ile Gly Glu Gly Val Val Glu Val Arg Ala Thr Ser Gly Asp

245 250 255

Asn His Leu Gly Gly Asp Asp Trp Asp Gln Arg Val Val Asp Trp Leu

260 265 270

Val Asp Lys Phe Lys Gly Thr Ser Gly Ile Asp Leu Thr Lys Asp Lys

275 280 285

Met Ala Met Gln Arg Leu Arg Glu Ala Ala Glu Lys Ala Lys Ile Glu

290 295 300

Leu Ser Ser Ser Gln Ser Thr Ser Ile Asn Leu Pro Tyr Ile Thr Val

305 310 315 320

Asp Ala Asp Lys Asn Pro Leu Phe Leu Asp Glu Gln Leu Thr Arg Ala

325 330 335

Glu Phe Gln Arg Ile Thr Gln Asp Leu Leu Asp Arg Thr Arg Lys Pro

340 345 350

Phe Gln Ser Val Ile Ala Asp Thr Gly Ile Ser Val Ser Glu Ile Asp

355 360 365

His Val Val Leu Val Gly Gly Ser Thr Arg Met Pro Ala Val Thr Asp

370 375 380

Leu Val Lys Glu Leu Thr Gly Gly Lys Glu Pro Asn Lys Gly Val Asn

385 390 395 400

Pro Asp Glu Val Val Ala Val Gly Ala Ala Leu Gln Ala Gly Val Leu

405 410 415

Lys Gly Glu Val Lys Asp Val Leu Leu Leu Asp Val Thr Pro

420 425 430

Claims (9)

1. A recombinant protein vaccine comprising a total of 98 amino acids consisting of the 161-370 amino acid sequence of the ATP binding site in the hsp70 protein, the 1-120 amino acid sequence of the human papillomavirus type 16 E6 protein and the E7 protein The sequence is connected, wherein the leucine at the 50th position of the E6 protein is modified to glycine, and the cysteine at the 24th position and the glutamic acid at the 26th position of the E7 protein are respectively modified to glycine. 2. The recombinant protein vaccine according to claim 1, characterized in that it is the amino acid sequence shown in SEQ ID NO:2. 3. The recombinant protein vaccine according to claim 1, wherein the human papillomavirus type 16 E6 protein is located at the amino terminal of the recombinant protein, and the hsp70 protein is located at the carboxyl terminal of the recombinant protein. 4. The nucleotide sequence encoding any one of the recombinant protein vaccines of claims 1-3. 5. The nucleotide sequence according to claim 4, characterized in that it is the nucleotide sequence shown in SEQ ID NO:1. 6. An expression vector comprising the nucleotide sequence according to any one of claims 4-5. 7. A host cell containing the expression vector of claim 6. 8. The use of the recombinant protein vaccine according to any one of claims 1-3 in the preparation of medicines for treating and/or preventing human papillomavirus-related diseases and tumors. 9. Use of the nucleotide sequence according to any one of claims 4-5 in the preparation of drugs for treating and/or preventing human papillomavirus-related diseases and tumors.

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