WO2019179040A1

WO2019179040A1 - Fusion protein, preparation method therefor and application thereof

Info

Publication number: WO2019179040A1
Application number: PCT/CN2018/102707
Authority: WO
Inventors: 曹广明; 张震宇
Original assignee: 首都医科大学附属北京朝阳医院
Priority date: 2018-03-19
Filing date: 2018-08-28
Publication date: 2019-09-26
Also published as: CN108484776B; CN108484776A

Abstract

A protein fused by using an HSP70 protein and an FPR1 extracellular domain protein, a plurality of amino acid sequences comprised by the FPR1 extracellular domain protein being linked by flexible linker peptides. Further provided are a nucleotide molecule that encodes the fusion protein, a vector, a recombinant bacteria and a recombinant cell comprising the nucleotide molecule, and a preparation method for and an application of the fusion protein.

Description

Fusion protein, preparation method and application thereof

Technical field

The invention belongs to the field of biotechnology, and particularly relates to a fusion protein, a preparation method and application thereof, especially in the field of tumor immunotherapy.

Background technique

Cervical cancer refers to a malignant tumor that occurs at the junction of squamous epithelial cells in the cervicovaginal or transitional zone and the columnar epithelial cells in the endocervix of the cervix. Cervical cancer is the most common malignant tumor of the female reproductive system, and the incidence of cervical cancer is second only to breast cancer. At present, the main treatment for cervical cancer is surgical treatment. At the same time, adjuvant chemotherapy is used for middle and high-risk patients. For patients with advanced cervical cancer, single or combined radiotherapy is used, but the effect does not significantly improve the prognosis of patients. Therefore, finding effective therapeutic targets and treatment methods is still a difficult point in the treatment of cervical cancer.

The present invention has been made in view of the above.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a fusion protein, a preparation method and an application thereof. The fusion protein of the invention has simple preparation, strong specificity and long duration, and can significantly enhance the effect of immunotherapy.

In order to solve the above technical problems, the basic idea of adopting the technical solution of the present invention is:

A first object of the present invention is to provide a fusion protein comprising a fusion protein of HSP70 protein and FPR1 extracellular domain protein.

Due to the discovery of HPV virus, it has provided an effective target for the prevention and treatment of cervical cancer. It has been successfully developed and started to be used for HPV virus vaccine. However, due to the limitations of the vaccination population, the effect is not clear. The most important thing is that the HPV vaccine that has been promoted can only play a preventive role, and the age of vaccination and the applicable population are limited.

This application is to treat cervical cancer by means of immunotherapy. The domestic research on immunotherapy DC vaccine is still in the initial stage. The research of this application is the first in the same industry in China. At present, it is a simple and effective method to prepare vaccines by using whole tumor cells as antigens to sensitize DCs. Animal experiments and clinical trials have confirmed that application of tumor lysates to DC cells can induce significant CTL resistance against specific HPV. Tumor response. Moreover, tumor lysates contain a large amount of heat shock proteins, which can promote the maturation of DC cells, induce the upregulation of DC cell maturation markers, and enhance their ability to activate T cells. However, the DC stimulator used is a crude extract of tumor cells, and the crude extract of tumor cells as a DC excitation substrate stimulates DC cells to induce low anti-tumor effect, poor specificity, and especially may induce autoimmune diseases due to Its safety problem cannot currently be applied to the clinic. Therefore, the key technology for preparing DC vaccines is to find and prepare specific antigen peptides for tumors. The difficulty of immunotherapy lies in the screening of specific targets for cervical cancer. In this application, the cervical cancer high expression cell membrane antigen is used in immunotherapy, which overcomes the disadvantages of poor specificity and low targeting.

In the early application group, differential protein screening was performed on cervical cancer and adjacent tissues by protein profiling technique. FPR1 is one of the differential proteins, which is highly expressed in tumor tissues.

FRP1 is a seven-transmembrane G-protein coupled receptor, which is the main functional receptor for inflammatory cell chemotaxis and is widely involved in human physiology and pathological processes. FPR1 plays an important role in inflammation, wound repair, and infection against pathogenic microorganisms. The bacterial formylation chemotactic polypeptide (fMLF), a classical activator of FPR1, binds to FPR1 and undergoes a series of signal transduction activities, mainly activating PI3K, PKC, MAPK, and NF-κB signaling pathways. Based on FPR1, bacterial peptides can be recognized and H ₂ O ₂ production is activated, so FPR1 plays an important role in host antimicrobial infection. Mice deficient in FPR1 are more susceptible to microbial infections. The expression level of FPR1 in various tumors is elevated and participates in the process of tumor development. In the case of malignant glioma, FPR1 is highly expressed at the tumor site, and FPR1 located on the surface of malignant glioma cells can be activated by agonists such as Anx-A1 secreted by tumor cells and normal cells. After activation, FPR1 acts on a series of regulatory molecules, p38, MAPK, ERK1/2, and transcription factors NF-κB, STAT3, HIF-1α, etc. to promote tumor cell proliferation, invasion and migration, and promoted by EGFR. The formation of blood vessels. This demonstrates that tumor cells can activate multiple important signaling pathways using FPR1 agonists in the tumor cell microenvironment, and activation of these signaling pathways ultimately promotes tumorigenesis.

The MHC class I antigen processing and presentation process is a transfer process that relies on HSPs. Therefore, heat shock proteins link tumor antigens to the body's immune system. Binding of HSP to polypeptide is one of the keys to CTL activation. Some antigenic peptides from the surface of tumor cells can only produce a limited cellular immune response, and when combined with HSP (mainly Hsp70, Hsp60, Hsp90, etc.) to form Hsp antigen peptide complex, the ability to activate CTL is a few antigenic peptides. A hundred times, significantly enhanced the effectiveness of immunotherapy.

Heat Shock Protein 70 (HSP70) can be used as a molecular chaperone to present antigenic peptides to DCs and enhance the antigenicity of antigenic peptides and ultimately activate T lymphocytes to exert specific cytotoxic effects. , producing an anti-tumor immune effect. Heat shock protein is a kind of highly conserved protein produced by biological cells stimulated by various physical and chemical factors. It has the property of molecular chaperone, that is, it can form complexes with various proteins of different functions in cells, and participate in protein folding. The composition of the subunit, the transport of the protein, and the degradation process, thereby regulating the activity and function of the target protein, but not participating in the composition of the target protein.

Heat shock protein 70 (Heat Shock Protein 70) is an important antigenic component of Mycobacterium tuberculosis, which can bind antigens to immune cells by binding to peptides similar to the MHC class I molecular peptide binding domain structure. Mycobacterial Hsp70 is highly immunogenic and has the characteristics of immunodominant antigens, which can induce specific Th1-type cellular responses, and the attack on Mycobacterium tuberculosis can produce a strong protective immune effect. Mycobacterium tuberculosis hsp70 can induce mouse macrophages to secrete various cytokines such as interleukin-1α, interleukin-1β, tumor necrosis factor and granulocyte-macrophage colony-stimulating factor, which are involved in regulating the immune response of the body. Special role. It has been experimentally confirmed that the mycobacterial hsp70 antigen gene was cloned downstream of the cytomegalovirus promoter to form a DNA vaccine, and its immune effect was similar to that of BCG, indicating that hsp70 is a protective immunogen.

Therefore, the present application selects Hsp70 protein, which is fused with cervical cancer cell membrane antigen FPR1, and expresses the purified Hsp70-FPR1 fusion protein as antigen-sensitized DC, and finds that its immunotherapy effect on cervical cancer is very significant, and The duration is greatly extended and can provide a more effective method for the treatment of cervical cancer.

In a further aspect, the FPR1 protein comprises a multi-segment extracellular segment amino acid sequence, and the amino acid sequences of the different segments of the FPR1 extracellular segment protein are linked by a flexible linker peptide.

In this application, FPR1 was selected as a target for the treatment of cervical cancer. In order to enhance the effect of immunotherapy, we expressed fusion expression of FPR1 and heat shock protein Hsp70. Since FPR1 is a seven-transmembrane protein, the transmembrane and intracellular regions of the protein are not involved in the immune recognition, that is, the antigenic effect is the extracellular domain of the FPR1 protein. If the FPR1 protein is used in immunotherapy, It will cause an increase in irrelevant epitopes and affect the specificity of immunotherapy. Therefore, this application only selects the extracellular domain of FPR1 protein for fusion with HSP70. Multiple extracellular segments of the FPR1 protein are amplified and ligated. In order not to disrupt the structure of each extracellular segment, the amino acid sequences of the different segments of the FPR1 extracellular segment protein are joined by a flexible linker.

Specifically, the flexible linker protein was used as a link of proteins to connect them. In the experiment, GGGGGGS was selected as the flexible linker. Glycine is the smallest and chiral amino acid. As a linker, it does not destroy the spatial structure of the protein, and completely retains each protein. Epitope. And the flexible linker uses a small molecular weight and does not increase the protein epitope.

In a further aspect, the FPR1 extracellular segment protein comprises the amino acid sequence set forth in SEQ ID NO. As a preferred embodiment, the FPR1 extracellular segment protein adopts the amino acid sequence shown in SEQ ID NO. 3, which can reduce irrelevant antigenic epitopes and enhance the specificity of immunotherapy.

In a further aspect, the FPR1 extracellular segment protein and the HSP70 protein are linked by a flexible linker peptide.

In the present application, multiple extracellular segments of the FPR1 protein are amplified, ligated, and expressed in fusion with HSP70 protein. Similarly, in order not to destroy the structure of each extracellular segment and Hsp70, we utilized a flexible linker protein (GGGGGGS was selected as a flexible linker in the experiment, glycine is the smallest and no chiral amino acid, and its role as a linker does not destroy the spatial structure of the protein. The intact epitope of each protein is retained, and the flexible linker has a small molecular weight and does not increase the protein epitope. As a protein conjugate, it is expressed in tandem fusion to obtain an Hsp70-FPR1 extracellular fusion protein.

In a further aspect, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO. As a preferred embodiment, the fusion protein adopts the amino acid sequence shown in SEQ ID NO. 4, which can reduce irrelevant antigenic epitopes, enhance the specificity of immunotherapy, prolong the duration, and significantly enhance the effect of immunotherapy.

A second object of the invention is to provide a nucleotide sequence encoding a fusion protein as described above.

A third object of the present invention is to provide a vector, recombinant or recombinant cell comprising the fusion protein as described above or the nucleotide sequence.

A fourth object of the present invention is to provide a method for producing a fusion protein comprising: constructing an expression system comprising the fusion protein as described above, expressing a fusion protein of the FPR1 extracellular segment protein and the HSP70 protein; and then purifying the fusion protein.

A preferred embodiment, the nucleotide sequence expressing the amino acid sequence shown in SEQ ID NO. 4 is cloned into a proEM system, and transfected into E. coli to prepare a transfection-grade plasmid, and then the plasmid is transfected into a transfection reagent. Transient expression was carried out in mammalian cells HEK293, and the protein was purified by affinity chromatography.

More specifically, the Hsp70 and FPR1 extracellular domain genes were inserted into the expression vector proEM by double digestion, and the accuracy of the final expression vector was confirmed by restriction enzyme digestion and sequencing, and finally transferred to the DH5a clone strain through the plasmid. The transfection-grade plasmid was extracted by a kit, and then the plasmid was transfected into a mammalian cell HEK293 by a transfection reagent for transient expression, and the protein was purified by affinity chromatography to obtain a fusion protein.

A fifth object of the present invention is to provide the use of the fusion protein for the preparation of a medicament for treating and/or preventing cervical cancer.

A sixth object of the present invention is to provide a vaccine comprising a fusion protein as described above and a pharmaceutically acceptable carrier.

After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

1. The fusion protein of the present application has a single component, a clear structure, and simple preparation, which not only can reduce the cost, but also can solve the complexities of the existing tumor cell lysate components, and cannot guarantee the strength and specificity of the induced anti-tumor immunity.

2. This application only uses FPR1 extracellular segment protein, a fusion protein formed with HSP70 protein, which removes irrelevant epitopes and enhances the specificity of immunotherapy. The prepared DC vaccine lasts for a long time and can significantly enhance the effect of immunotherapy. .

The specific embodiments of the present invention are described in further detail below with reference to the accompanying drawings.

DRAWINGS

The drawings are intended to provide a further understanding of the invention, and are intended to be illustrative of the invention. It is apparent that the drawings in the following description are only a few embodiments, and other drawings can be obtained from those skilled in the art without departing from the drawings. In the drawing:

Figure 1 is a schematic diagram showing the results of sequence analysis of intracellular and extracellular structures of the FPR1 protein;

Figure 2 is a technical roadmap of the present application;

Figure 3 is a SDS-PAGE electrophoresis pattern of the purified protein of the extracellular domain of FRP1, HSP70 and HSP70-FPR1 extracellular domain;

Among them, LaneM1, M2, M3 are SDS-PAGE protein Marker; Lane1, 3, 5 is a positive control, BSA (1μg); Lane2, 4, 6 are the target proteins, and the arrow indicates the purified protein;

Figure 4 shows the morphology of dendritic cells after sensitization with different proteins;

Figure 5 is a surface marker of different protein-sensitized dendritic cells detected by flow cytometry;

Figure 6 shows the tumor position in different groups of mice during the course of taking the material;

Figure 7 is a graphical representation of each group of tumors dissected, the tumors in the protein-sensitized group were significantly smaller than the PBS control group;

Figure 8 is a graph of tumor growth curve.

It is to be understood that the drawings and the written description are not intended to limit the scope of the present invention in any way.

detailed description

The embodiments of the present invention will be clearly and completely described in the following with reference to the accompanying drawings in the embodiments of the present invention. It is not intended to limit the scope of the invention.

The main experimental materials and instruments used in the examples are as follows:

Plasmid extraction kit, DNA recovery kit, T4 ligase, Taq DNA polymerase, electrophoresis related reagents (Tris, Acr, Bis, AP, TEMED, etc.), protein concentration determination kit, ultrasonic crusher, high speed refrigerated centrifuge , mass spectrometer.

Example 1 Construction and Expression of Purified Fusion Protein

1. Hsp70, FPR1 sequence confirmation and cloning

The Hsp70 and FPR1 gene sequences were searched and analyzed in Gene Bank to obtain the nucleotide sequence and amino acid sequence of Hsp70 and FPR1 genes. Wherein the amino acid sequence of Hsp70 is shown in SEQ ID NO. The complete amino acid sequence of FPR1 is set forth in SEQ ID NO.

Since FPR1 is a seven-transmembrane protein, the present application obtains four extracellular segments of FPR1 by analysis, and the results of the analysis are shown in FIG. The specific sequence of the FPR1 protein is as follows, wherein the extra-framed portion is the extracellular portion of FPR1.

FPR1 Protein Length=350MW=38445.05PI=9.23

1

ITY LVFAVTFVLG VLGNGLVIWV AGFRMTHTVT

61 TISYLNLAVA DFCFTSTLPF FMV

VFTIVDINLF GSVFLIALIA

121 LDRCVCVLHP VWTQNHRTVS LAKKVIIGPW VMALLLTLPV II

181

FIIGF SAPMSIVAVS YGLIATKIHK QGLIKSSRPL

241 RVLSFVAAAF FLCWSPYQVV ALIATV

TSALA FFNSCLNPML

301 YVFMGQDFRE RLIHALPASL ERALTEDSTQ TSDTATNSTL PSAEVELQAK

In order to reduce irrelevant epitopes, the present application fuses the extracellular domain of the FPR1 protein with the Hsp70 protein. First, the eukaryotic expression purification system was used to obtain the four extracellular extracellular protein sequences of the extracellular domain of FPR1. In order not to change the protein structure, the protein sequence was expressed in tandem using a flexible linker, ie, a flexible linker (GGGGGS). The extracellular domain of FPR1, also known as exFPR1. Hereinafter, the collection of the FPR1 extracellular segment protein is referred to as exFPR1, and the amino acid sequence of the FPR1 extracellular segment protein (exFPR1) is shown in SEQ ID NO.

Wherein, the fusion protein of the present application, that is, the amino acid sequence of the Hsp70-exFPR1 extracellular domain fusion protein is shown in SEQ ID NO.

The nucleotide sequences of the FPR1 extracellular domain protein (exFPR1), Hsp70 protein and Hsp70-exFPR1 extracellular domain fusion protein were obtained by template amplification or artificial synthesis, respectively.

The experimental technical route of the present application is shown in FIG. 2.

2. Protein expression and purification

The exFPR1, HSP70 and Hsp70-exFPR1 genes were inserted into the expression vector proEM by double enzyme digestion, and the accuracy of the final expression vector was confirmed by enzyme digestion and sequencing, and finally transferred to the DH5a clone strain, and the plasmid was extracted by the plasmid. The transfection-grade plasmid was extracted, and then the plasmid was transfected into mammalian cells HEK293 by transfection reagent for transient expression, and the protein was purified by affinity chromatography.

The purified protein was identified by SDS-PAGE, and the results are shown in Fig. 3. In Fig. 3, LaneM1, M2, M3 are SDS-PAGE protein Marker; Lane1, 3, 5 are positive controls, BSA (1 μg);

Lane

2, 4, 6 are proteins of interest, and the arrow indicates the purified protein. From this, it was found that the obtained exFPR1, HSP70 and Hsp70-exFPR1 proteins were all correct.

3. Acquisition of DC cells and activation of T cells

1) Umbilical cord blood mononuclear cell separation

Take heparin anticoagulated venous blood and mix well with the equivalent amount of Hank's solution or RPMI1640, and slowly add it to the equal amount of lymphocyte separation surface along the tube wall with a dropper, paying attention to maintain a clear interface. Centrifuge horizontally at 2000 rpm for 20 minutes. After centrifugation, the tube is divided into three layers, the upper layer is plasma and Hank's solution, and the lower layer is mainly red blood cells and granulocytes. The middle layer is a lymphocyte separation solution, and there is a white cloud layer narrow band mainly composed of single nuclear cells at the interface between the upper and middle layers, and the mononuclear cells include lymphocytes and monocytes. In addition, it also contains platelets. Capillaries are inserted into the cloud layer to absorb individual nuclear cells. Insert into another short tube, add more than 5 times the volume of Hank's solution or RPMI1640, centrifuge at 1500 rpm for 10 minutes, and wash the cells twice. That is, the mononuclear cells are relatively pure.

Mononuclear cells were then cultured in medium containing 10% fetal bovine serum IMDM, and after 4 hours, adherent cells were used to induce differentiation into DC cells, and the suspension cells were mainly T cells.

2) DC cell culture and identification

Adherent cells (mainly CD14+ monocytes), serum-free medium containing recombinant human GM-CSF 500-1,000 U/ml and recombinant human IL-4 500 U/ml, 37 ° C, 5% CO 2 incubator Cultured, induced monocytes to differentiate into DC cells; changed fluid once every 3d and supplemented with cytokines; added tumor antigen 50ng/ml on the 5th day of culture, and carried on antigen load on DC.

The experiment was divided into 5 groups according to different antigens: PBS group, exFPR1 protein group, Hsp70 protein group, exFPR1 protein + Hsp70 protein group and Hsp70-exFPR1 fusion protein group. Among them, the exFPR1 protein + Hsp70 protein group is a mixture of two proteins, and Hsp70-exFPR1 is a fusion protein.

On the 6th day of culture, recombinant human TNF-α (500 U/ml) was added to induce DC cell maturation; on the 7th or 8th day of culture, DC cells were harvested in an amount of 1×10 ⁶ or more. The DC cells are then subjected to a quality test.

Quality inspection method:

(1) Cell viability was measured by trypan blue staining, and cell morphology was observed. The test results are shown in Figure 4. Among them, the number of living cells is above 80%. Figure 4 shows the morphology of dendritic cells sensitized with different proteins. Compared with the control group of PBS, the dendritic cells of exFPR1 protein group, Hsp70 protein group, Hsp70+exFPR1 protein group and Hsp70-exFPR1 fusion protein group were obviously mature, scattered and distributed, and the cell volume increased significantly. Dendrites.

(2) Flow cytometry was used to detect the expression of HLA-DR, CD83 and CD86 on the surface of DC cells to determine whether the DC was mature. The detection results are shown in Fig. 5. Among them, H-F is Hsp70-exFPR1 fusion protein, and H+F is Hsp70 and exFPR1 mixed protein.

The dendritic cells of the exFPR1 protein group, the Hsp70 protein group, the Hsp70+exFPR1 protein group and the Hsp70-exFPR1 fusion protein group were significantly matured compared with the PBS group by HLA-DR, CD83, CD1a and CD11C staining. In addition, it can be seen from the results that the Hsp70-exFPR1 fusion protein group has higher DC maturation rate than the other groups, indicating that the Hsp70-exFPR1 fusion protein group can effectively promote DC maturation.

3) Preparation of DC activated T cells

The DC cells and T cells obtained in step 1) and step 2) were collected and co-cultured at a ratio of 1:10 (number ratio), and recombinant human IL-2 (300 U/ml) was added to the serum-free medium; every 3 days Half the solution was changed once and supplemented with recombinant human IL-2 (300 U/ml). The cells were collected on the 7th day.

4. DC-T cells were subjected to adoptive input into pre-neoplastic NOG immunodeficient mice to evaluate the effect of immunotherapy.

On day 0, 200 μL of tumor cells (1 × 10 ⁷ ) were subcutaneously injected into the right side of a 4-week-old female NOG mouse. On day 14, on days 15 and 21, 3 x ¹⁰⁶ human lymphocytes stimulated with different proteins were injected by tail vein and immunized twice. Tumor diameters were measured in a two-dimensional lengthwise manner using a sliding caliper every 3 days. Mice were sacrificed in CO ₂ chamber on day 36 and tumors were dissected to assess their volume and weight. Tumor volume was then calculated by the following formula: tumor volume = tumor length x tumor width x tumor width x 0.5.

Results: The tumors were obtained as shown in Fig. 6 and Fig. 7. The growth curve is shown in Fig. 8.

Fig. 6 is a photograph of different groups of mice in the samples, and the tumor position can be clearly observed. Figure 7 shows that the tumors of each group were photographed, and the tumors in the protein-sensitized group were significantly smaller than the PBS control group. Figure 8 is a graph showing the tumor growth curve.

It can be seen from the tumor growth curve that after two injections of tumor vaccine in the NOG mouse tumor formation model, each group of DC vaccines produced better immune effects. The tumor growth rate of Hsp70-exFPR1 fusion protein group and Hsp70+exFPR1 group was significantly inhibited, and the therapeutic effect was better than Hsp70 and FPR1 protein group, especially after the second immunotherapy on the 21st day. The Hsp70-exFPR1 fusion protein group has the best persistence, indicating that the fusion protein has a great advantage in immunotherapy and is better than the mixed effect of different proteins.

The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the present invention, and any technology that is familiar with the present patent. Those skilled in the art can make some modifications or modifications to equivalent embodiments by using the technical content of the above-mentioned hints without departing from the technical scope of the present invention, but the technology according to the present invention does not deviate from the technical solution of the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments are still within the scope of the present invention.

Claims

A fusion protein, characterized in that the fusion protein comprises a fusion protein of an HSP70 protein and an FPR1 extracellular domain protein.
The fusion protein according to claim 1, wherein the FPR1 extracellular segment protein comprises a plurality of amino acid sequences, and the amino acid sequences of the different segments of the FPR1 extracellular segment protein are linked by a flexible linker peptide.
A fusion protein according to claim 1 or 2, wherein the FPR1 extracellular segment protein comprises the amino acid sequence set forth in SEQ ID NO.
A fusion protein according to any one of claims 1 to 3, wherein the FPR1 extracellular segment protein and the HSP70 protein are linked by a flexible linker peptide.
A fusion protein according to any one of claims 1 to 4, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO.
A nucleotide sequence encoding the fusion protein of any of claims 1-5.
A vector, recombinant or recombinant cell comprising the fusion protein of any one of claims 1 to 5 or the nucleotide sequence of claim 6.
A method for producing a fusion protein, comprising: constructing an expression system comprising the fusion protein of any one of claims 1 to 5, expressing a fusion protein of an extracellular domain of FPR1 and an HSP70 protein; and then the fusion protein Purification was carried out.

Preferably, the nucleotide sequence expressing the amino acid sequence shown in SEQ ID NO. 4 is cloned into a proEM system, and transfected into E. coli to prepare a transfection-grade plasmid, and then the plasmid is transfected into a lactation by a transfection reagent. The animal cells were transiently expressed in HEK293, and the protein was purified by affinity chromatography.
Use of the fusion protein of any of claims 1-5 for the manufacture of a medicament for the treatment and/or prevention of cervical cancer.
A vaccine comprising a fusion protein according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier;

Preferably, the vaccine comprises a DC vaccine for treating and/or preventing cervical cancer.