CN118206665A - Fusion protein containing CCL1, preparation method and application - Google Patents
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a protein containing CCL1 fusion KRAS polypeptide antigen, a preparation method and application thereof, wherein the fusion protein sequentially comprises the following components from N end to C end: igE signal peptide, CCL1, linker, KRAS polypeptide, and PD-L1 hydrophobic peptide fragment. The invention utilizes chemotactic binding capacity of CCL1 and immune cell surface receptors such as DC cells and the like to transport and cross-present KRAS antigen proteins on the surfaces of the DC cells, improves the phagocytosis, processing and presentation efficiency of the antigen proteins by the DC cells, and achieves the effect of inhibiting the growth of related tumors. The PD-L1 hydrophobic peptide has a very strong immune enhancement effect, and can promote the presentation of DC cells to antigens so as to further excite specific cellular immune response and enhance the treatment effect of inhibiting tumor growth.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to fusion protein containing CCL1, a preparation method and application thereof.
Background
Dendritic cells (DENDRITIC CELLS, DC) are professional antigen presenting cells (ANTIGEN PRESENTING CELLS, APC) with the strongest body functions, and are involved in antigen recognition, uptake, processing and presentation with high efficiency, inducing immune responses in the body. Immature DC has strong migration capability and antigen capturing treatment capability, the DC cells are mature after being stimulated by antigen, the mature DC cells migrate to secondary lymphoid organs to be combined with T cells through various cell surface proteins, the captured antigen protein epitope peptides are crossed and presented to the T cells, and the T cells are promoted to differentiate into antigen specific cytotoxic T lymphocytes, so that antigen substances are identified and degraded in the cellular immune process.
Tumor vaccines induce effector T cell function in patients by immunopotentiating existing anti-tumor responses or activating naive T cells, and antigen-specific cd8+ Cytotoxic T Lymphocytes (CTLs) play an important role in the anti-tumor process. DC cells are the only professional antigen presenting cells capable of activating the initial CD8+ T cells, uptake and processing extracellular tumor antigens to form antigen polypeptides, and the antigen polypeptides are cross-presented after being combined by intracellular MHC molecules, so that CTL (cytotoxic T lymphocyte) is effectively activated to play a role in killing specific tumor cells. Thus, delivery of tumor antigens to DC cells by coupling to DC cell surface molecules is a tumor therapeutic strategy that is effective in inducing cd8+ T cell immune responses, playing an important role in the initiation of tumor-specific cellular immunity and throughout the immune response.
Chemokines (chemokines) are a class of cytokines secreted by cells and have the ability to induce the directional chemotaxis of nearby responding cells. APC, T cells and tumor-associated fibroblasts in the tumor microenvironment are all capable of secreting the chemokine CCL1, which, upon binding to the CCR8 receptor, plays an important role in the apoptosis, proliferation and migration of cancer cells, while being capable of recruiting T cells into the tumor microenvironment and promoting T cell activation, and also activating CCR8 receptor expression on vascular endothelial cells to promote angiogenesis. There is still a need to identify more DC cell surface molecules that can act to facilitate presentation.
The apoptosis protein 1 ligand (PD-L1) is an immune checkpoint molecule that interacts with the apoptosis protein-1 (PD-1) and negatively regulates proliferation and activation of immune cells. Under normal physiological conditions, PD-L1 mediated immunosuppression can maintain immune homeostasis, protecting normal cells from injury. In tumors, the D-L1/PD-1 signal axis mediates immune escape of the tumor. PD-1 is expressed on tumor infiltrating immune cells, and PD-L1 is expressed on the surface of tumor cells, participates in immune escape, and can be highly expressed on the surface of antigen presenting cells (DC cells, macrophages and the like) under IFN-gamma stimulation. Blocking PD-1 binding to PD-L1 can release the inhibited tumor-specific T cell killing ability. The addition of PD-L1 fragments to vaccines to induce the body to produce antibodies directed against PD-L1 may have the effect of enhancing the therapeutic effect of the vaccine.
Based on the above, the inventive fusion of chemokines with KRAS and the further addition of computationally optimized PD-L1 sequences further enhances the therapeutic effect.
Disclosure of Invention
Aiming at the technical problems in the related art, the invention provides a fusion protein containing CCL1, a preparation method and application thereof, which can overcome the defects in the prior art.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:
A fusion protein comprising, in order from N-terminus to C-terminus: igE signal peptide, CCL1, linker, KRAS polypeptide and PD-L1 hydrophobic peptide fragment, the amino acid sequence of the PD-L1 hydrophobic peptide fragment is shown in SEQ ID No. 2.
Preferably, the amino acid sequence of the IgE signal peptide is shown as SEQ ID NO. 4, the amino acid sequence of the CCL1 is shown as SEQ ID NO. 3, and the linker is (G5S) n, wherein n is 1-10.
The invention also provides nucleic acids encoding the fusion proteins.
The nucleotide sequence of the nucleic acid is shown as SEQ ID NO: shown at 8.
The invention also provides expression vectors containing the nucleic acids, including backbone vectors and nucleic acids encoding the fusion proteins.
The invention also provides a host transformed or transfected with the vector.
The invention also provides a preparation method of the fusion protein, which comprises the following steps: culturing a host containing a gene expression vector encoding the fusion protein to obtain a culture containing the fusion protein.
The invention also provides application of the fusion protein, the gene, the expression vector or the host in preparing products for preventing and treating diseases, wherein the prevention and treatment comprise preventing and/or treating, specifically improving the antibody level in serum, preventing the formation of tumors, inhibiting the growth of tumors and improving the immune response capability of organisms to the tumors. The disease is a tumor. The products for preventing and treating diseases comprise medicines and/or vaccines, and the administration modes comprise oral administration, injection and/or electrotransformation.
The invention has the beneficial effects that: the invention utilizes chemotactic binding capacity of CCL1 and immune cell surface receptors such as DC cells and the like to transport and cross-present KRAS antigen proteins on the surfaces of the DC cells, improves the phagocytosis, processing and presentation efficiency of the antigen proteins by the DC cells, and improves the effect of preventing and treating related diseases. The PD-L1 hydrophobic peptide has a very strong immune enhancement effect, and can promote the presentation of DC cells to antigens so as to further excite specific cellular immune response and enhance the treatment effect of inhibiting tumor growth.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of analysis of the ability of CCL1 to chemotactic various professional antigen presenting cells;
FIG. 2 is a secondary structure diagram of PD-L1 protein;
FIG. 3A is a plasmid map of pVR-CCL1-KRAS-PDL 1;
FIG. 3B is a plasmid map of pVR-CCL 1-KRAS;
FIG. 3C is a plasmid map of pVR-KRAS;
FIG. 4 shows the detection of the expression of three plasmid genes of pVR-CCL1-KRAS-PDL1, pVR-CCL1-KRAS and pVR-KRAS, and the detection of the expression of the fusion protein coding nucleotide with a Ha tag at the C-terminal by using Western blotting (Western blot);
FIG. 5 is a time axis of a prophylactic immune response of different fusion genes to mice;
FIG. 6 is a graph showing the result of detection of tumor by preventive immunity.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which are derived by a person skilled in the art based on the embodiments of the invention, fall within the scope of protection of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For definitions and terms in the art, reference is made specifically to Current Protocols in Molecular Biology (Ausubel) by the expert. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
The CCL1 is an important chemokine in a human body, belongs to one member of a CC chemokine family (CC chemokines), can express the CCL1 at a high level by APC, T cells and tumor-related fibroblasts in the human body, and can regulate apoptosis of cancer cells and activation and migration of immune cells after being combined with a unique receptor CCR 8. The CCL 1-CCR 8 signaling axis plays an important role in the migration and activation process of immune cells. Studies of the present invention demonstrate that CCL1 can be used to deliver antigen to professional antigen presenting cells, particularly DC cells, thereby enhancing the antigen presenting effect of the DC cells.
In the present invention, the CCL1 may be a human fragment, or may be a fragment derived from other animals, such as murine, rabbit, monkey, pig, etc., which may be the complete CCL1, or may be a fragment or mutant having CCL1 activity therein, which is not limited in the present invention. In the embodiment of the invention, humanized CCL1 is taken as an experimental object, and the improvement of the presentation effect of CCL1 on antigen is verified. The amino acid sequence of the humanized CCL1 is as follows:
KSMQVPFSRCCFSFAEQEIPLRAILCYRNTSSICSNEGLIFKLKRGKEAC ALDTVGWVQRHRKMLRHCPSKRK is shown as SEQ ID NO: 3).
In the fusion protein, the PD-L1 hydrophobic peptide sequence is reconstructed from a signal peptide at the n-end and a transmembrane domain at the C-end of the PD-L1 protein, the species source belongs to a human sequence, and the sequence does not have the problem of killing other proteins of a human body after enhancing immunity. It is reported that this sequence can increase the effect of DC on antigen presentation. The PD-L1 hydrophobic peptide segment is found to be used as a polypeptide sequence after artificial transformation to have the effect of enhancing the immunity in human body. In the invention, the sequence of the PD-L1 hydrophobic peptide fragment sequence is as follows:
MRIFAVFIFMTYWHERTHLVILGAILLCLGVALTFIFRLRKGR (shown as SEQ ID NO: 2).
The number of antigens in the fusion protein of the invention is at least 1. For example, it may be 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 or more. In the study of the invention, experiments are carried out on the fusion effect of 1 antigen, and the fusion effect shows good effect.
In the invention, the KRAS polypeptide antigen is a polypeptide fragment with the strongest immunogenicity of human KRAS protein, and the amino acid sequence is as follows: IQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRT GEGF (as shown in SEQ ID NO: 1) or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the sequence. It may contain only one KRAS polypeptide sequence or may be made up of two or more KRAS polypeptide fragments in tandem.
The antigen protein is transported and cross-presented on the surface of the DC cell by utilizing chemotactic binding capacity of CCL1 and immune cell surface receptors such as the DC cell, thereby improving the phagocytosis, processing and presentation efficiency of the antigen by the DC cell and improving the effect of preventing and treating related diseases. In the embodiment of the invention, KRAS polypeptide is taken as a case, and the improvement effect of CCL1 on antigen protein presentation efficiency is proved.
In order to ensure that each functional fragment in the fusion protein is smoothly folded without being influenced by steric hindrance, a linker is added between the fragments, wherein the linker between CCL1 and KRAS polypeptide antigen is GGGGGSGGGGG. The antigens may be linked by (G 5 S) n and/or AGA.
In the present invention, in order to enhance the expression effect of the fusion protein, a signal peptide that promotes secretion of the fusion protein to the outside of the cell is added to the N-terminus of CCL 1. In some embodiments, the signal peptide is an IgE signal peptide. Its specific amino acid sequence is MDWTWILFLVAAATRVHS (shown as SEQ ID NO: 4)
In the present invention, a tag is added to the C-terminal of the fusion protein in order to facilitate purification of the fusion protein. The tag is selected from recombinant protein purification tags well known in the art. In the examples, the amino acid sequence of the tag is YPYDVPDYA, but the sequence is only a tag identifying the expression of the protein, and the immune effect of the sequence is not affected.
In a specific embodiment, the fusion protein has a structure comprising, in order from N-terminal to C-terminal: igE signal peptide, human CCL1 protein sequence, linker sequence (GGGGGSGGGGG), KRAS polypeptide sequence, PD-L1 hydrophobic polypeptide sequence, ha tag sequence. Specifically, the amino acid sequence is shown as SEQ ID NO. 14.
The nucleic acid encoding a protein according to the invention may be DNA, RNA, cDNA or PNA. In an embodiment of the invention, the nucleic acid is in the form of DNA or RNA. The DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. Nucleic acids may include nucleotide sequences having different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acids may be topologically linear or circular. The nucleic acid may be, for example, part of a vector (e.g., an expression or cloning vector), or a fragment. The nucleic acids may be obtained directly from natural sources or may be prepared by recombinant, enzymatic or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.
In the present invention, the DNA sequences expressing the fusion proteins are optimized, including but not limited to: codon usage bias, elimination of secondary structures that are detrimental to expression (e.g., hairpin structures), changes in GC content, cpG dinucleotide content, secondary structures of mRNA, cryptic splice sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA instability regions, repeat sequences (direct repeat, inverted repeat, etc.), and restriction sites that may affect cloning.
The prevention means that the drug of the invention can play a role in reducing the risk of tumorigenesis by being administered before tumorigenesis. The treatment of the invention means that the drug of the invention is administered after tumorigenesis, which can inhibit tumor growth, reduce tumor volume or delay the growth rate of tumors. In the embodiment of the invention, the mouse transplanted tumor cell CT-26 is taken as an experimental object, and the effect of the fusion protein vaccine is verified.
Also provided in the present invention are transcriptional units of the fusion protein, which are DNA sequences from the start of the promoter to the end of the terminator. Promoters and terminators may also be flanked by or between them by regulatory fragments, which may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites, such as promoters' enhancers, poly (A) signals, and the like, operably linked to a nucleic acid sequence. Among the transcriptional units provided by the present invention are CMV or CMV/R promoters, CMV enhancers and nucleic acid fragments encoding fusion proteins.
The recombinant vector of the present invention, referred to as a recombinant nucleic acid vector, is a recombinant DNA molecule comprising a desired coding sequence and suitable nucleic acid sequences necessary for expression of an operably linked coding gene in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotic cells include promoters, optionally including operator sequences, ribosome binding sites and possibly other sequences. Prokaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes interchangeable, as the plasmid is the most commonly used form of vector at present. However, the present invention is intended to include such other forms of expression vectors that serve equivalent purposes, which are or become known in the art, including but not limited to: plasmids, phage particles, viral vectors and/or just potential genomic inserts. In particular embodiments, nucleic acids encoding fusion proteins provided herein can be constructed in a variety of eukaryotic expression vectors. For example, the backbone vector may be a pVAX1 series vector or a pVR series vector.
The host cell of the invention is a prokaryotic or eukaryotic host containing a nucleic acid vector and/or a target gene. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells have the ability to replicate a vector encoding the protein or express the desired protein.
In the embodiment of the invention, the preparation method of the fusion protein adopts a mode of inducing recombinant host expression, and the culture can be thalli, cell bodies, culture solution obtained by culture or substances obtained by extraction and/or purification in the culture.
The product for preventing and treating diseases provided by the invention comprises the following components: the fusion protein, the nucleic acid, the expression vector, the host, the fusion protein prepared by the preparation method and/or the culture containing the fusion protein prepared by the preparation method. In the present invention, the disease controlling products include drugs and/or vaccines. Pharmaceutically acceptable carriers, excipients and/or adjuvants are also included in the vaccine. The medicine also comprises pharmaceutically acceptable auxiliary materials.
The prevention of the invention refers to the administration of the disease prevention and treatment product before the occurrence of the disease, so that the disease prevention and treatment product can play a role in reducing the occurrence risk of the disease. The treatment of the invention means that the product for preventing and treating the diseases can improve symptoms, inhibit disease development and restore health of patients after the diseases occur. For example, the product of the invention can be used for preventing and treating tumors, can increase the antibody level in serum, inhibit the growth of tumors, reduce the tumor volume or delay the growth rate of tumors. In the embodiment of the invention, the mouse transplanted tumor cell CT-26 is taken as an experimental object, and the effect of the fusion protein vaccine is verified and a good effect is obtained.
In the examples of the present invention, the amino acid sequences of the fragments involved and the nucleic acid fragments encoded are shown in Table 1:
Amino acid sequences of the fragments of Table 1 and the nucleic acid fragments encoding the same
| SEQ ID NO:1 | Amino acid sequence of KRAS polypeptide |
| SEQ ID NO:2 | Amino acid sequence of PD-L1 hydrophobic polypeptide |
| SEQ ID NO:3 | Amino acid sequence of CCL1 |
| SEQ ID NO:4 | IgE signal peptides |
| SEQ ID NO:5 | Nucleotide sequence of plasmid pVR-CCL1-KRAS-PDL1 |
| SEQ ID NO:6 | Nucleotide sequence of plasmid pVR-CCL1-KRAS |
| SEQ ID NO:7 | Nucleotide sequence of plasmid pVR-KRAS |
| SEQ ID NO:8 | DNA sequence encoding fusion protein in plasmid pVR-CCL1-KRAS-PDL1 |
| SEQ ID NO:9 | DNA sequence encoding fusion protein in plasmid pVR-CCL1-KRAS |
| SEQ ID NO:10 | DNA sequence encoding fusion protein in plasmid pVR-KRAS |
| SEQ ID NO:11 | MRNA sequence encoding fusion protein in plasmid pVR-CCL1-KRAS-PDL1 |
| SEQ ID NO:12 | MRNA sequence encoding fusion protein in plasmid pVR-CCL1-KRAS |
| SEQ ID NO:13 | MRNA sequence encoding fusion protein in plasmid pVR-KRAS |
| SEQ ID NO:14 | Amino acid sequence of fusion protein in plasmid pVR-CCL1-KRAS-PDL1 |
| SEQ ID NO:15 | Amino acid sequence of fusion protein in plasmid pVR-CCL1-KRAS |
| SEQ ID NO:16 | Amino acid sequence of fusion protein in plasmid pVR-KRAS |
The reagent consumable adopted by the invention is a common commercial product and can be purchased in the market. The invention is further illustrated by the following examples.
Example 1
Monocytes, T cell subsets, eosinophils, basophils were isolated from mouse bone marrow and peripheral blood, respectively. Wherein bone marrow mononuclear cells are subjected to chemotaxis experiments after being respectively added with M-CSF and GM-CSF and IL4 to induce differentiation into macrophages and DC cells. The cells isolated as described above or induced to differentiate were placed in the upper chamber of a chemotactic cell (carbonate membrane Transwell cell: 5. Mu.m; costar, cat: 3422) and the number of cells was 1X 10 6/100. Mu.l/well based on the laboratory pre-work. A spontaneous migration control group and a CCL1 cytokine group were simultaneously set, and the number of added cells was the same. The CCL1 factor adopts E.coli to purify recombinant murine CCL1 protein, and 100ng/ml is the optimal chemotactic efficiency dose according to the existing work basis in the prior laboratory. After 4 hours, cells in the chemotactic lower chamber were collected and analyzed for chemotactic ability of CCL1 to various immune cells in a flow-through manner. As shown in FIG. 1, the results demonstrate that CCL1 is effective in recruiting various immune cells from the upper compartment to the lower compartment (P < 0.001).
Example 2
Selection of KRAS polypeptide sequence and PD-L1 polypeptide sequence
The full-length amino acid sequence of human KRAS was analyzed for immunogenicity based on an artificial neural network (ARTIFICIAL NEURAL NETWORKS, ans) algorithm using NETMHCPAN-4.1 software. Polypeptide with length of KRAS epitope set as 8mer, 9mer, 10mer and 11mer respectively, wherein the KRAS epitope predicts HLA genotype :HLa-a01:01,HLa-a02:01,HLa-a03:01,HLa-a24:02,HLa-a26:01,HLA-B07:02,HLA-B08:01,HLA-B27:05,HLA-B39:01,HLA-B40:01,HLA-B58:01,HLA-B15:01 with highest frequency in human genome to obtain polypeptide fragment with highest immunogenicity and amino acid sequence of SEQ ID NO:1, and the sequence comprises epitope ILDTAGQEEY(HLa-a*01:01),FVDEYDPTI(HLa-a*02:01),CLLDILDTA(HLa-a*02:01),QYMRTGEG(HLa-a*24:02),GETCLLDIL(HLA-B*40:01),IQNHFVDEY(HLA-B*15:01).
The domain of the PD-L1 protein was analyzed using Pymol based on the full-length amino acid sequence of human PD-L1, and the analysis found that the PD-L1 protein contained two alpha helices, PDL1 237-265 was its transmembrane hydrophobic region, PDL1 1-14 was its signal peptide region, as shown in FIG. 2. In order to improve the efficiency of antigen presentation on the cell membrane surface by DC cells, a hydrophobic signal peptide region and a C-terminal hydrophobic region of PD-L1 protein are selected and connected, the amino acid sequence of the PD-L1 hydrophobic polypeptide is subjected to codon optimization favored by mammalian cell expression, and the amino acid sequence is determined to be SEQ ID NO. 2.
Example 3
Antigen design scheme of fusion gene or protein vaccine and construction and preparation of mammal expression plasmid
Construction of pVR-CCL1-KRAS-PDL1 plasmid: fusion protein CCL1-KRAS-PDL1 is constructed according to the human KRAS polypeptide sequence, the humanized CCL1 protein and the PD-L1 hydrophobic polypeptide sequence. Connecting an IgE signal peptide with an amino acid sequence MDWTWILFLVAAATRVHS to the N end of fusion protein CCL1-KRAS-PDL 1; a Ha tag consisting of 9 amino acids of YPYYDVPDYA was ligated to the C-terminus of fusion protein CCL1-KRAS-PDL1.
The fusion protein finally obtained comprises the following components from the N end to the C end in sequence: igE signal peptide, human CCL1 protein sequence, linker sequence (GGGGGSGGGGG), KRAS polypeptide sequence, PD-L1 hydrophobic polypeptide sequence, ha tag sequence.
The amino acid sequence of the fusion protein is subjected to codon optimization favored by mammalian cell expression, the fusion gene sequence is determined to be SEQ ID NO. 8, the fusion gene sequence is subjected to gene synthesis, and then the fusion gene sequence is integrally constructed in a corresponding multi-cloning site region of a pVR plasmid vector, so that the fusion protein can be expressed in a correct codon translation sequence. The resulting plasmid was designated pVR-CCL1-KRAS-PDL1 plasmid as shown in FIG. 3A.
Construction of plasmid pVR-CCL1-KRAS: the fusion gene finally constructed comprises the following components from the N end to the C end in sequence: igE signal peptide, human CCL1 protein sequence, linker sequence (GGGGGSGGGGG), KRAS polypeptide sequence, ha tag sequence.
The amino acid sequence of the fusion protein is subjected to codon optimization favored by mammalian cell expression, the fusion gene sequence is determined to be SEQ ID NO. 9, the fusion gene sequence is subjected to gene synthesis, and then the fusion gene sequence is integrally constructed in a corresponding multi-cloning site region of a pVR plasmid vector, so that the fusion protein can be expressed in a correct codon translation sequence. The resulting plasmid was designated as pVR-CCL1-KRAS plasmid. As shown in fig. 3B.
Construction of pVR-KRAS plasmid: an IgE signal peptide of amino acid sequence MDWTWILFLVAAATRVHS was further linked before the human KRAS polypeptide sequence, followed by a Ha tag consisting of 9 amino acids of YPYDVPDYA.
So that the finally obtained fusion protein sequentially comprises the following components from the N end to the C end: igE signal peptide, KRAS polypeptide sequence, ha tag sequence, as shown in fig. 3C.
The amino acid sequence of the fusion protein is subjected to codon optimization favored by mammalian cell expression, the fusion gene sequence is determined to be SEQ ID NO.10, the fusion gene sequence is subjected to gene synthesis, and then the fusion gene sequence is integrally constructed in a corresponding multiple cloning site region of a pVR plasmid vector, so that the fusion protein can be expressed in a correct codon translation sequence. The resulting plasmid was designated as pVR-KRAS plasmid.
Specifically, the plasmid patterns constructed in the experiments set forth in this example were actually: pVR-CCL1-KRAS-PDL1, pVR-CCL1-KRAS and a control plasmid pVR-KRAS thereof.
Example 4
In vitro cell transfection experiments to construct plasmids:
24 hours prior to transfection, 2.5X10 5 HEK293T cells were inoculated in 6-well cell culture plates and the transfection assay was started when the cell density was as high as 60% -70%. The cell culture medium and the serum-free Opti-MEM medium are preheated in a water bath kettle at 37 ℃ in advance during transfection. At the time of transfection, 5. Mu.g of empty Vector (Vector), pVR-CCL1-KRAS-PDL1 expression Vector, pVR-CCL1-KRAS expression Vector, pVR-KRAS expression Vector and 20. Mu.L of PEI transfection reagent were added to 200. Mu.L of serum-free Opti-MEM in sequence, and after mixing uniformly, they were allowed to stand at room temperature for 20 minutes. The cells to be transfected were replaced with fresh medium, gently added to the transfection system described above and gently shaken. The cells were returned to the cell incubator for 6 hours and then changed. After 48 hours of transformation, the cells were harvested and the effect of expression of the KRAS polypeptide fusion gene plasmid in HEK293T cells was detected using Western Blot.
Cells were collected and 60. Mu.L of 0.5% NP40 lysis buffer containing PMSF or Cocktail protease inhibitor was added. Cells were resuspended well and lysed by spinning at 4℃for 30min. Lysates were centrifuged at 12000rpm at 4℃for 10 min, and supernatants were collected into fresh 1.5mL EP tubes, and the pellet was discarded. According to the actual volume of the sample, 5 XSDS-PAGE protein loading buffer is added, the sample is heated in an air bath at 100 ℃ for 10 minutes after being uniformly mixed, western blot is immediately carried out, and detection is carried out by using a Ha tag antibody (Sigma), and the result shows that the empty Vector (Vector) has no protein expression, the size positions of pVR-CCL1-KRAS-PDL1 and pVR-CCL1-KRAS expression proteins are obviously higher than that of pVR-KRAS, so that plasmids pVR-CCL1-KRAS-PDL1, pVR-CCL1-KRAS and plasmid pVR-KRAS of a control group can be smoothly and normally expressed in mammalian cells.
Example 5
Intervention effect of fusion gene DNA form vaccine on mouse transplanted tumor cell CT-26 allograft tumor:
In view of the fact that the fusion gene can be expressed normally in mammalian cells. We extracted pVR-CCL1-KRAS-PDL1 and pVR-KRAS plasmids alone and used the TEESA living body gene transfer instrument to perform immune plasmid electrotransformation on mice.
And observing the inhibition of fusion gene immunity to CT-26 transplanted tumor cell growth after the isotransplantation of CT-26 cells.
After determination of CT-26 cell neoplasia, we performed plasmid electrotransformation on mice according to the immunization strategy noted on the time axis of FIG. 5. BALB/c (purchased from Tonglihua) peripheral age female mice were individually injected with PBS, pVR-KRAS plasmid, pVR-CCL1-KRAS plasmid and 4 groups of pVR-CCL1-KRAS-PDL1 plasmid, five of each group, and dehairing treatment was performed on the right side of the mice near inguinal lymph node using dehairing paste. Then, the plasmid was injected into the dehairing site by using an electrotransfer apparatus, 50. Mu.g of each cell was searched for CT-26 tumor cells under the condition of tumor formation 2 weeks after the injection of the plasmid, the tumor formation time was observed, the long diameter a and the short diameter b of the tumor were measured every two days, the calculation of the tumor volume was performed according to a×b×b/2, and the tumor growth curve was drawn. As shown in FIG. 6, the groups of the immunoppVR-CCL 1-KRAS-PDL1 plasmid, the pVR-CCL1-KRAS plasmid and the pVR-KRAS plasmid can obviously inhibit the formation of the transplanted tumor in the mice compared with the control group, and the vaccine effect is obvious. This demonstrates that all four groups of vaccines have very pronounced tumor preventing effects.
In summary, by means of the technical scheme, the chemotactic binding capacity of CCL1 and immune cell surface receptors such as DC cells is utilized to transport and cross-present different antigen proteins on the surfaces of the DC cells, so that the phagocytosis, processing and presentation efficiency of the antigen proteins by the DC cells is improved, and the effect of preventing and treating related diseases is improved. The PD-L1 hydrophobic peptide has a very strong immune enhancement effect, and can promote the presentation of DC cells to antigens, so that the specific cellular immune response is further stimulated, and the treatment effect of inhibiting the growth of tumors is enhanced.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (8)
1. A fusion protein comprising, in order from the N-terminus to the C-terminus: igE signal peptide, CCL1, linker, KRAS polypeptide and PD-L1 hydrophobic peptide fragment, the amino acid sequence of the PD-L1 hydrophobic peptide fragment is shown in SEQ ID No. 2.
2. The fusion protein according to claim 1, wherein the amino acid sequence of the IgE signal peptide is shown in SEQ ID No. 4, the amino acid sequence of CCL1 is shown in SEQ ID No. 3, the linker is (G5S) n, wherein n is 1 to 10; the amino acid sequence of the KRAS polypeptide is shown as SEQ ID NO. 1.
3. A nucleic acid encoding the fusion protein of claim 1.
4. A nucleic acid according to claim 3, wherein the nucleotide sequence is as set forth in SEQ ID NO: shown at 8.
5. An expression vector comprising the nucleic acid of claim 4.
6. A host transformed or transfected with the vector of claim 5.
7. A method of preparing the fusion protein of claim 1, comprising: culturing a host comprising an expression vector encoding the fusion protein of claim 1 to obtain a culture comprising the fusion protein.
8. Use of the fusion protein of claim 1, the nucleic acid of claim 3, the expression vector of claim 5 or the host of claim 6 for the preparation of a product for the control of a disease.
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