CN117660368A - Recombinant oncolytic influenza virus expressing chemokine CCL19 and application thereof - Google Patents
Recombinant oncolytic influenza virus expressing chemokine CCL19 and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant oncolytic influenza virus for expressing a chemokine CCL19 and application thereof, wherein the recombinant oncolytic influenza virus provided by the invention is characterized in that a humanized chemokine CCL19 gene is inserted into an influenza virus genome segment, the recombinant influenza virus with replicative capacity and infectious capacity is rescued by a reverse genetics technology, and CCL19 protein can be continuously and stably expressed in tumor cells. The oncolytic influenza virus can recruit and activate immune cells such as T cells, macrophages and NK cells, promote the presentation of tumor antigens, improve the tumor microenvironment and achieve the aim of tumor immunotherapy; the influenza virus strain is influenza A virus or influenza B virus; the immunotherapeutic tumors include, but are not limited to, colorectal cancer. The recombinant influenza virus has the advantages of strong tumor killing specificity, high effectiveness and good safety, and can be used for treating tumors.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to recombinant oncolytic influenza virus for expressing a chemokine CCL19 and application thereof.
Background
The tumors seriously threaten the life health of human beings, and the traditional treatment methods comprise operations, radiotherapy, chemotherapy and the like, but the treatment methods still have the problems of incapability of radical treatment, serious toxic and side effects and the like. In recent decades, tumor immunotherapy has been greatly developed, and the purposes of controlling and eliminating tumors are achieved by restarting and maintaining tumor-immune circulation and activating normal anti-tumor immunity of organisms. However, the immunosuppressive state in the tumor microenvironment limits the effect of tumor immunotherapy, and tumor cells induce and maintain the immunosuppressive state mainly in two ways: firstly, aggregating regulatory T cells, marrow-derived suppressor cells and other immunosuppressive cells around a tumor, and inactivating cytotoxic T lymphocytes by secreting immunosuppressive factors so as to form a tumor immunosuppressive state; secondly, tumor immune escape is caused by inducing the expression of immune checkpoint molecules such as programmed death ligand 1/programmed death 1, cytotoxicity T lymphocyte related antigens and the like, and a tumor immune suppression microenvironment is formed.
Oncolytic virus therapy is an important branch of tumor immunotherapy, and has been fully confirmed to have wide application prospects in the field of tumor treatment. Oncolytic viruses are a class of natural or genetically engineered viruses that are capable of selectively infecting and killing tumor cells. The mediated antitumor activity is mainly achieved in two ways, wherein the first way is direct lysis, namely, the oncolytic virus selectively replicates in tumor cells, so that the tumor cells are subjected to lysis and necrosis, tumor-related antigens (Tumor associated antigens, TAAs) are released, tumor microenvironment immune suppression ecology is destroyed, and systemic antitumor immunity is induced; secondly, cytokines, chemokines, immune checkpoint inhibitors and the like for enhancing immune response are inserted into an oncolytic virus genome through a molecular biology technology, when the recombinant oncolytic virus replicates in host cells, exogenous genes are expressed simultaneously, the functions of recruiting and activating immune cells such as CD4+ cells, CD8+ T cells and NK cells of the host are exerted, and the activated immune cells infiltrate into tumor microenvironment, so that the tumor microenvironment can be effectively improved, and the tumor killing effect is enhanced. At present, many oncolytic viruses are adenovirus, I type herpes simplex virus, vaccinia virus, vesicular stomatitis virus and the like, but the replication efficiency of the viruses is low, and the viruses are difficult to prepare; the virus DNA is easy to integrate into a host genome, and the genetic safety is low; the virus particles are large and are easy to be cleared by a host prematurely, so that the clinical application of the virus particles is limited.
Influenza virus is an excellent oncolytic viral vector. Influenza virus is segmented RNA virus, has no reverse transcriptase activity and DNA integration activity, and has high genetic safety, and viral genes cannot be integrated into host genome. In addition, the influenza virus Hemagglutinin (HA) is combined with a receptor N-acetyl sialic acid receptor (N-acetylsialic acid receptor, SAR) and widely distributed on the surfaces of various tumor cells such as lung cancer, colorectal cancer, prostate cancer and the like, so that the influenza virus HAs natural infectivity on various tumor cells. Meanwhile, the size of the influenza virus particles is between 80 and 120nm, and the influenza virus particles are moderate in size, so that the influenza virus particles have strong immunogenicity and immunoregulation capacity, cause immunogenic death of tumor cells, are not clarified by organisms prematurely, and can play a long-term effect. In addition, the influenza virus has stronger gene plasticity, 8 gene fragments of the virus can be respectively transformed and modified by utilizing the reverse genetics technology of the influenza virus, the operation is simple and easy, more importantly, the influenza virus can be effectively replicated in chicken embryo, and the oncolytic virus is prepared by taking the influenza virus as a vector, so that the higher productivity can be obtained. Therefore, the modification of influenza virus to oncolytic virus is a new way of anti-tumor immunotherapy.
The chemokine CCL19 is mainly expressed in peripheral lymphoid tissues and organs such as spleen and lymph node T cells, while its specific receptor CCR7 is mainly distributed on the surfaces of mature DC cells, macrophages, NK cells, B cells and T cells, and lymphocytes can directionally move towards central immune organs, peripheral lymph nodes, inflammatory sites and the like through the combination of CCL19 and CCR7, so that lymphocyte homing phenomenon occurs. In addition, CCL19 has the function of inducing specific anti-tumor immune response, the CCL19 highly expressed in tumor microenvironment can be specifically combined through a CCL19/CCR7 biological axis, chemotaxis comprises infiltration of T lymphocytes, B lymphocytes, dendritic cells, macrophages, NK and other immune cells into the tumor microenvironment, and the T cell homeostasis is activated and maintained; promoting endocytic capacity of dendritic cells; promoting natural killer cell proliferation, improving immunosuppression state in tumor microenvironment, and resisting tumor. Meanwhile, CCL19 can inhibit the expression of VEGFA by promoting tumor cells to express miR-206 and inhibiting MET/ERK/HIF-1 alpha and other channels, so that tumor angiogenesis is inhibited, and an anti-tumor effect is exerted.
Disclosure of Invention
The invention aims to solve the technical problem of improving the immune treatment effect of tumors, in particular to the treatment of colorectal cancer.
In order to solve the technical problems, the invention firstly provides a recombinant oncolytic influenza virus for expressing a chemokine CCL19, which is named rFlu-PB1-CCL19. The recombinant oncolytic influenza virus is (1) or (2):
(1) an influenza virus containing a protein known as chemokine CCL19;
(2) influenza virus expressing chemokine CCL19 protein;
the CCL19 is protein of the following 1), 2) or 3):
1) Is the amino acid sequence of SEQ ID NO:1, consisting of the protein of sequence SEQ ID NO:2 from nucleotide 2422 to 2652;
2) A protein derived from 1) having the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1;
3) Recombinant proteins obtained by ligating a tag and/or other genes to the C-terminal and/or N-terminal of 1) or 2).
The genome of the recombinant oncolytic influenza virus is single-stranded negative-strand segmented RNA, and the negative-strand RNA of the recombinant oncolytic influenza virus transcribes complete positive-strand RNA complementary to the negative-strand RNA, wherein the complete positive-strand RNA comprises PB2-RNA, PB1-CCL19-RNA, HA-RNA, PA-RNA, NP-RNA, NA-RNA, M-RNA and NS-RNA;
the PB2-RNA is positive strand RNA for encoding PB2 genes in the influenza virus strain;
the PB1-CCL19-RNA is positive strand RNA for encoding the recombinant gene PB1-CCL19 of claim 1;
the HA-RNA is positive-strand RNA for encoding an HA gene in the influenza virus strain;
the PA-RNA is positive strand RNA for encoding PA genes in the influenza virus strain;
the NP-RNA is positive strand RNA encoding an NP gene in the influenza virus strain;
the M-RNA is positive strand RNA for encoding M genes in the influenza virus strain;
the NA-RNA is positive strand RNA for encoding NA gene in the influenza virus strain;
the NS-RNA is positive strand RNA encoding an NS gene in the influenza virus strain;
in the above recombinant oncolytic influenza virus, the set of positive-strand RNAs may also consist of only the PB2-RNA, the PB1-CCL19-RNA, the HA-RNA, the PA-RNA, the NP-RNA, the M-RNA, the NA-RNA and the NS-RNA.
In the recombinant oncolytic influenza virus, the sequence of the PB1-CCL19-RNA is a hydrolysable Linker T2A nucleotide sequence sequentially inserted from 5 '. Fwdarw.3 ' before a terminal stop codon of the 5' NCR of a PB1 gene in the influenza virus strain, and the sequence SEQ ID NO:2 from nucleotide 2296 to nucleotide 2358; CCL19 signal peptide nucleotide sequence, SEQ ID NO:2 from nucleotide 2359 to 2421; CCL19 nucleotide sequence, sequence SEQ ID NO:2 and the 5' ncr terminal packaging signal sequence of the PB1 gene, the sequence of SEQ ID NO:2, and the other nucleotides are unchanged from the obtained sequence at the 2653 th to 2766 th nucleotides.
The influenza virus strain includes, but is not limited to, any subtype of influenza a virus or influenza b virus.
In order to solve the above technical problems, the present invention also provides a method for constructing and rescuing recombinant oncolytic influenza virus that expresses CCL19, comprising introducing recombinant vector pHW2000-PB2 comprising DNA molecule encoding said PB2-RNA, recombinant vector pHW2000-PA comprising DNA molecule encoding said PA-RNA, recombinant vector pHW2000-HA comprising DNA molecule encoding said HA-RNA, recombinant vector pHW2000-NP comprising DNA molecule encoding said NP-RNA, recombinant vector pHW2000-M comprising DNA molecule encoding said M-RNA, recombinant vector pHW2000-NA comprising DNA molecule encoding said NA-RNA, recombinant vector pHW2000-NS comprising DNA molecule encoding said NS-RNA, recombinant vector pHW2000-PB1-CCL19 comprising DNA molecule encoding said PB1-CCL19-RNA into packaging cells to obtain recombinant oncolytic influenza virus that expresses chemokine 19.
In order to solve the technical problems, the invention also provides any one of the following products:
(1) The genome of the recombinant oncolytic influenza virus;
(2) A kit of vectors for treating tumors, i.e., a vector containing the gene encoding CCL19;
(3) A kit of cassettes for the treatment of tumors, i.e., an cassette comprising the CCL19 encoding gene;
(4) A set of genes for treating tumors, i.e., the gene encoding CCL19;
(5) A set of proteins, the CCL19 protein, for use in the treatment of tumors;
in order to solve the technical problems, the invention also provides a biological material related to the recombinant oncolytic influenza virus, wherein the biological material is any one of the following products:
(1) A set of DNA molecules encoding said set of positive strand RNAs of said recombinant oncolytic influenza virus. The complete set of DNA molecules consists of a DNA molecule encoding the PB2-RNA, a DNA molecule encoding the PA-RNA, a DNA molecule encoding the PB1-CCL19-RNA, a DNA molecule encoding the NP-RNA, a DNA molecule encoding the NA-RNA, a DNA molecule encoding the HA-RNA, a DNA molecule encoding the M-RNA, and a DNA molecule encoding the NS-RNA.
(2) A recombinant vector set. The complete set of recombinant vectors consists of a recombinant vector containing a DNA molecule encoding the PB2-RNA, a recombinant vector containing a DNA molecule encoding the PA-RNA, a recombinant vector containing a DNA molecule encoding the HA-RNA, a recombinant vector containing a DNA molecule encoding the NP-RNA, a recombinant vector containing a DNA molecule encoding the M-RNA, a recombinant vector containing a DNA molecule encoding the NA-RNA, a recombinant vector containing a DNA molecule encoding the NS-RNA, and a recombinant vector containing a DNA molecule encoding the PB1-CCL 19-RNA.
(3) A microorganism containing the recombinant oncolytic influenza virus;
(4) An animal cell containing the recombinant oncolytic influenza virus;
(5) Animal tissue containing the recombinant oncolytic influenza virus;
(6) Animal organs containing the recombinant oncolytic influenza virus
In the above biological material, the animal cells, the animal tissue and the animal organ do not include propagation material.
In the above biological material, the microorganism, the animal cell, the animal tissue and the animal organ can be used as hosts for recombinant oncolytic viruses.
In one embodiment of the present invention, in (2), the recombinant vector containing the DNA molecule encoding the PB2-RNA is pHW2000-PB2, the recombinant vector containing the DNA molecule encoding the PA-RNA is pHW2000-PA, the recombinant vector containing the DNA molecule encoding the PB1-CCL19-RNA is pHW2000-PB1-CCL19, the recombinant vector containing the DNA molecule encoding the NP-RNA is pHW2000-NP, the recombinant vector containing the DNA molecule encoding the NA-RNA is pHW2000-NA, the recombinant vector containing the DNA molecule encoding the HA-RNA is pHW2000-HA, the recombinant vector containing the DNA molecule encoding the M-RNA is pHW2000-M, and the recombinant vector containing the DNA molecule encoding the NS-RNA is pHW2000-NS. The pHW2000-PB1-CCL19 can express the CCL19 protein shown in the sequence 1.
In order to solve the technical problems, the invention also provides a medicine for treating tumors, and the active ingredient of the medicine is the recombinant oncolytic influenza virus.
The active ingredients of the medicine can also be a composition obtained by combining the recombinant oncolytic influenza virus and other medicines for treating tumors.
In order to solve the technical problems, the invention also provides any one of the following applications:
(1) The application of the recombinant oncolytic influenza virus in preparing a medicine for treating tumor;
(2) The application of the product in preparing a medicine for treating tumor;
(3) The application of the biological material in preparing a medicine for treating tumor;
(4) The application of the tumor treatment medicine in preparing the tumor treatment medicine;
(5) The application of the recombinant influenza virus in treating tumors;
(6) The use of said product in the treatment of tumors;
(7) The use of said biological material in the treatment of tumors;
(8) The application of the tumor treatment medicine in treating tumors.
The tumor in the present invention includes colorectal cancer, but is not limited to colorectal cancer, and can also include lung cancer, breast cancer, liver cancer, melanoma, lymphoma, leukemia, ovarian cancer, cervical cancer, gastric cancer, renal cancer, pancreatic cancer, prostate cancer and glioma.
Experiments prove that the recombinant oncolytic influenza virus (rFlu-PB 1-CCL 19) can specifically infect tumor cells and effectively replicate in the tumor cells: after infection of cells with recombinant oncolytic influenza virus, replication efficiency is high in colorectal cancer cells, but low in normal colorectal mucosal epithelial cells. The recombinant oncolytic influenza virus has specific killing effect on tumor cells: after the recombinant oncolytic influenza virus infects cells, the recombinant oncolytic influenza virus has obvious killing effect on colorectal cancer cells, but has no obvious killing effect on normal colorectal mucosa epithelial cells. The recombinant oncolytic influenza virus can inhibit the growth and development of tumors in animals: after the recombinant oncolytic influenza virus is used for treating tumor-bearing animals, the growth of tumors is obviously inhibited, even the condition of fading occurs, the weight of mice is not obviously influenced, the survival period of the mice after treatment is obviously prolonged, and the mice have good tumor inhibiting effect and safety, and can be used for treating tumors.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly introduced below, in which the drawings 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 schematic diagram of the structure of recombinant gene PB1-CCL19 of recombinant oncolytic influenza virus rFlu-PB1-CCL19 (A: structure diagram of recombinant gene PB1-CCL19 after modification of recombinant oncolytic influenza virus PB1 gene. B: simulation diagram of mechanism of action of recombinant oncolytic influenza virus.)
FIG. 2 shows that the recombinant gene PB1-CCL19.A of the recombinant oncolytic influenza virus rFlu-PB1-CCL19 is identified, and the size of the target gene is consistent with that of the expected gene identified by RT-PCR; and B, identifying the target gene sequence to be consistent with expectations by gene sequencing.
FIG. 3 is a graph depicting the genetic stability of recombinant oncolytic influenza virus rFlu-PB1-CCL19. A: after continuous passage of rFlu-PB1-CCL19 in chick embryos for 5 passages, the hemagglutination titer of HA of the chick embryos is kept stable; b: after continuous passage of rFlu-PB1-CCL19 in chicken embryos for 5 passages, RT-PCR identified recombinant gene PB1-CCL19, the fragment size of which was consistent with that expected.
FIG. 4 shows the morphological structure and particle size of recombinant oncolytic influenza virus rFlu-PB1-CCL19 identified by electron microscopy. A: the form of rFlu-PB1-CCL19 is observed by a transmission electron microscope, so that the virus is more spherical, spike proteins such as HA, NA and the like on the surface are clearly visible, and the virus is not different from the form of the wild influenza A virus; b: the particle size distribution of rFlu-PB1-CCL19 virus is that the particle size of the rFlu-PB1-CCL19 virus is more distributed between 80 nm and 120nm, and is consistent with the size of wild type influenza A virus.
FIG. 5 is a diagram showing ELISA detection of the expression of recombinant oncolytic influenza virus rFlu-PB1-CCL19 exogenous gene CCL19 in chicken embryos. A: ELISA (enzyme Linked immunosorbent assay) detection CCL19 expression standard curve; b: the results of the CCL19 expression levels of wild-type PR8 and rFlu-PB1-CCL19 show that rFlu-PB1-CCL19 is continuously passaged on chicken for 5 generations, and the CCL19 expression level is kept stable.
FIG. 6 shows HA hemagglutination potency assay after purification and concentration of recombinant oncolytic influenza virus rFlu-PB1-CCL19.
FIG. 7 is a graph depicting the replication capacity of recombinant oncolytic influenza virus rFlu-PB1-CCL19 in colorectal cancer. HA hemagglutination potency of rFlu-PB1-CCL19 and WT-PR8 after replication in HT29 cells; b: HA hemagglutination potency of rFlu-PB1-CCL19 and WT-PR8 after replication in HCT116 cells; c: HA hemagglutination potency of rFlu-PB1-CCL19 and WT-PR8 after replication in SW620 cells; d: HA hemagglutination potency of rFlu-PB1-CCL19 and WT-PR8 after replication in Lovo cells; e: HA hemagglutination potency of rbu-PB 1-CCL19 and WT-PR8 after replication in CCD841 cells; f: HA hemagglutination titers of rFlu-PB1-CCL19 and WT-PR8 after replication in CT26 cells.
FIG. 8 is a graph depicting the proliferation activity of recombinant oncolytic influenza virus rFlu-PB1-CCL19 in inhibiting colorectal cancer cells. A: inhibition effect of rFlu-PB1-CCL19 and WT-PR8 on HT29 cell viability; b: inhibition effect of rFlu-PB1-CCL19 and WT-PR8 on HCT116 cell viability; c: inhibition effect of rFlu-PB1-CCL19 and WT-PR8 on SW620 cell viability; d: inhibition effect of rFlu-PB1-CCL19 and WT-PR8 on Lovo cell viability; e: inhibition effect of rFlu-PB1-CCL19 and WT-PR8 on CCD841 cell viability; f: inhibition effect of rFlu-PB1-CCL19 and WT-PR8 on CT26 cell viability.
FIG. 9 is a graph depicting chemotactic effects of culture medium supernatant on immune cell RAW264.7 after infection of CT26 cells with recombinant oncolytic influenza virus rFlu-PB1-CCL19. A: a control group lower chamber; b: WT-PR8 group lower chamber; c: rFlu-PB1-CCL19 group lower chamber; d: statistical analysis of cell numbers in the lower chambers of each group.
FIG. 10 is a graph showing changes in body weight and tumor volume of tumor-bearing mice 21 days after treatment with recombinant oncolytic influenza virus rFlu-PB1-CCL19. A: day21 mice weight change in groups; b: day21 mice each group had tumor volume changes.
FIG. 11 is a physical image of tumor tissue after 21 days of treatment with recombinant oncolytic influenza virus rFlu-PB1-CCL19.
FIG. 12 is a diagnosis of HE cases in various tissues of mice 21 days after treatment with recombinant oncolytic influenza virus rFlu-PB1-CCL19.
FIG. 13 is a graph showing survival of tumor-bearing mice.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that illustrate the invention and are not intended to limit the scope of the invention.
Example 1 rescue and identification of recombinant oncolytic influenza Virus
The invention provides a recombinant oncolytic influenza virus named rFlu-PB1-CCL19, which expresses a protein named chemokine CCL19, wherein CCL19 is a protein with an amino acid sequence of sequence 1 and is encoded by 2422-2652 nucleotide in sequence 2.
1. Construction of recombinant bidirectional expression plasmid pHW2000-PB1-CCL19
Corresponding DNA molecules are synthesized artificially according to the structure and sequence 2 of the recombinant gene fragment PB1-CCL19 shown in the abstract diagram, and after linearizing the DNA molecules and the bicistronic vector pHW2000, the recombinant gene fragment and the vector pHW2000 fragment are connected by using an Infusion kit to construct the target recombinant plasmid pHW2000-PB1-CCL19. The target plasmid obtained by homologous recombination is transformed into escherichia coli DH-5 alpha competent cells to amplify plasmids, recombinant plasmids are identified and screened by PCR, agarose gel electrophoresis and gene sequencing technology, and the concentration and purity of the plasmids are detected by using an ultraviolet spectrophotometer.
2. Rescue of recombinant oncolytic influenza virus
The recombinant vector pHW2000-PB2 containing the DNA molecule encoding the PB2-RNA, the recombinant vector pHW2000-PA containing the DNA molecule encoding the PA-RNA, the recombinant vector pHW2000-PB1-CCL19 containing the DNA molecule encoding the PB1-CCL19-RNA, the recombinant vector pHW2000-NP containing the DNA molecule encoding the NP-RNA, the recombinant vector pHW2000-NA containing the DNA molecule encoding the NA-RNA, the recombinant vector pHW2000-HA containing the DNA molecule encoding the HA-RNA, the recombinant vector pHW2000-M containing the DNA molecule encoding the M-RNA, and the recombinant vector pHW2000-NS containing the DNA molecule encoding the NS-RNA were co-transfected with 293T cells at the same ratio to obtain a culture medium supernatant containing the recombinant viruses.
3. Amplification of recombinant oncolytic influenza virus
Culture medium supernatant containing recombinant virus obtained by rescue is inoculated into chick embryo for virus amplification, allantoic fluid containing virus particles is obtained, and concentration and purification are carried out through sucrose density gradient centrifugation.
4. Biological characteristics and functional identification of recombinant oncolytic influenza virus
(1) Sequence identification is carried out on the harvested rFlu-PB1-CCL19 genome by RT-PCR, sequencing and other methods;
(2) Determining TCID50 value, plaque experiment and other methods to determine rFlu-PB1-CCL19 virus titer by a hemagglutination test;
(3) ELISA (enzyme-Linked immunosorbent assay) detection of the expression condition of exogenous gene CCL19 after rFlu-PB1-CCL19 is inoculated with chick embryo and various colorectal cancer cell lines HT29, CT26, HCT116, SW620 and Lovo, and the function of immune cells such as CCL19 chemotactic T cells, DC, NK, macrophages and the like is verified through a Transwell experiment;
(4) RT-PCR is used for respectively detecting proliferation dynamic conditions of viruses after rFlu-PB1-CCL19 is inoculated with normal human colorectal mucosa epithelial cells CCD841 and various colorectal cancer cells;
(5) After the rFlu-PB1-CCL19 is inoculated with chick embryos and a plurality of colorectal cancer cells, continuously carrying out passage for at least 5 generations, detecting genome sequences of each generation of viruses and the expression condition of the CCL19, and determining the genetic stability of the rFlu-PB1-CCL 19;
(6) Particle size and morphology of rFlu-PB1-CCL19 were observed by transmission electron microscopy.
Example 2 in vitro oncolytic Effect and mechanism Studies of recombinant oncolytic influenza Virus
1. In vitro oncolytic effect research of recombinant oncolytic influenza virus
(1) Infection of colon cancer cell line with rFlu-PB1-CCL 19: after HT29, CT26, HCT116 and SW620 and Lovo, the killing effect of rFlu-PB1-CCL19 on colorectal cancer cells was primarily determined by observing cytopathic effect (CPE);
(2) The experimental study of MTT, cellTiter-Glo and the like verifies the effect of rFlu-PB1-CCL19 on colorectal cancer cell proliferation activity;
(3) Detecting the necrosis and apoptosis proportion of tumor cells by a flow cytometry and an annexin V/PI double-staining method;
(4) After rFlu-PB1-CCL19 infection is detected by a scratch experiment, tumor cell migration conditions are detected, and the experiment is used for researching tumor targeting and oncolytic effects of rFlu-PB1-CCL19 in vitro by taking a human normal colorectal mucosal epithelial cell line CCD841 as a control.
2. In vitro oncolytic mechanism research of recombinant oncolytic influenza virus
(1) After rFlu-PB1-CCL19 infects a variety of colorectal cancer cells, ELISA detects the expression and secretion level of CCL19;
(2) The ability of CCL19 to chemotactic T cells, DCs, NK and macrophages was verified by a Transwell experiment;
(3) RT-qPCR, western Blot and the like are used for detecting the expression condition of CCR7 on the surface of tumor cells and the contents of apoptosis molecular markers Bid, bax, caspase 3 and PARP, anti-CCL 19 antibody is used for blocking CCL19/CCR7 specific binding, and then the content change of apoptosis molecular markers and the contents of the cutter are detected to evaluate the condition that CCL19/CCR7 specific binding activates exogenous apoptosis pathway.
Example 3 in vivo oncolytic Effect and mechanism Studies of recombinant oncolytic influenza Virus
1. In vivo oncolytic effect research of recombinant oncolytic influenza virus
(1) Establishing a Balb/C mouse colorectal cancer cell line CT26 subcutaneous transplantation tumor model, injecting RFLU-PB1-CCL19 into the tumor, measuring and drawing a tumor growth curve, calculating a tumor inhibition rate, performing histopathological and immunohistochemical examination on main organs and the like, and evaluating the effectiveness of the RFLU-PB1-CCL19 in inhibiting colorectal cancer growth and metastasis;
(2) The targeting and safety of RFLU-PB1-CCL19 were evaluated by measuring the body weight of mice, drawing survival curves, RT-qPCR detection of the biological distribution of viruses in the mice and pathological detection of the damaging effects of important tissues and organs.
2. In vivo oncolytic mechanism research of recombinant oncolytic influenza virus
(1) Detecting the expression level of CCL19 in tumor tissues by WesternBlot;
(2) Histopathology, immunohistochemistry, multicolor immunofluorescence and the like are used for detecting infiltration of T cells, DC, NK and macrophages in a tumor microenvironment; detecting the expression and secretion levels of immune cell effector molecule granzyme B, perforin, IFN-gamma, IL-2 and the like in the tumor microenvironment by using RT-qPCR, western Blot and other methods, and evaluating the activation condition of immune cells in the tumor microenvironment;
(3) Flow cytometry is used for detecting immune cell phenotypes such as T cells, DC, NK, macrophages and the like in the spleen of the mouse, and evaluating the ability of RFLU-PB1-CCL19 to regulate tumor-specific immune response of the organism;
(4) Through single cell genome sequencing and bioinformatic analysis, the signal path of RFLU-PB1-CCL19 effect is enriched, the cellular interaction of tumor cells and cell components in the tumor microenvironment is analyzed, and the functions of inhibiting tumor growth, invasion and metastasis are biologically verified.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (9)
1. The recombinant oncolytic influenza virus is characterized in that: the recombinant oncolytic influenza virus is an influenza virus expressing chemokine CCL19 protein, and the PB1 gene of the recombinant oncolytic influenza virus is modified into recombinant genes PB1-CCL19;
the CCL19 protein has an amino acid sequence of SEQ ID NO: 1;
the nucleotide sequence of the recombinant gene PB1-CCL19 is SEQ ID NO:2, and a recombinant gene thereof.
2. The recombinant oncolytic influenza virus of claim 1, wherein the genome of the recombinant oncolytic influenza virus is comprised of 8 single-stranded negative-strand, segmented RNAs, the negative-strand RNAs of the recombinant oncolytic influenza virus transcribed into a set of positive-strand RNAs complementary to the negative-strand RNAs, the set of positive-strand RNAs comprising PB2-RNA, PA-RNA, PB1-CCL19-RNA, NP-RNA, NA-RNA, HA-RNA, M-RNA, NS-RNA;
the PB2-RNA is positive strand RNA for encoding PB2 genes in the influenza virus strain;
the PA-RNA is positive strand RNA for encoding PA genes in the influenza virus strain;
the PB1-CCL19-RNA is positive strand RNA for encoding the recombinant gene PB1-CCL19 of claim 1;
the NP-RNA is positive strand RNA encoding an NP gene in the influenza virus strain;
the NA-RNA is positive strand RNA for encoding NA gene in the influenza virus strain;
the HA-RNA is positive strand RNA of NP gene in the encoding influenza virus strain;
the M-RNA is positive strand RNA for encoding M genes in the influenza virus strain;
the NS-RNA is a positive strand RNA encoding the NP gene in the influenza virus strain.
3. The recombinant oncolytic influenza virus of any one of claims 1-2, characterized in that: the sequence of the recombinant gene PB1-CCL19-RNA is that a hydrolyzable Linker T2A nucleotide sequence is inserted from 5 '. Fwdarw.3 ' in front of a terminal stop codon of the 5' NCR of the PB1 gene in the influenza virus strain, and the sequence SEQ ID NO:2 from nucleotide 2296 to nucleotide 2358; CCL19 signal peptide nucleotide sequence, SEQ ID NO:2 from nucleotide 2359 to 2421; CCL19 nucleotide sequence, sequence SEQ ID NO:2 and the 5' ncr terminal packaging signal sequence of the PB1 gene, the sequence of SEQ ID NO:2, the 2653 th-2766 th nucleotides in the sequence, and other nucleotides are unchanged;
the recombinant gene may be obtained by embedding a CCL19 gene in the influenza virus gene PB1, or may be obtained by embedding a CCL19 gene in other genes on the influenza virus genome.
4. A method for constructing and rescuing the recombinant oncolytic influenza virus of any one of claims 1-3, characterized in that: introducing a recombinant vector containing a DNA molecule encoding the PB2-RNA, a recombinant vector containing a DNA molecule encoding the PA-RNA, a recombinant vector containing a DNA molecule encoding the PB1-CCL19-RNA, a recombinant vector containing a DNA molecule encoding the NP-RNA, a recombinant vector containing a DNA molecule encoding the NA-RNA, a recombinant vector containing a DNA molecule encoding the HA-RNA, a recombinant vector containing a DNA molecule encoding the M-RNA, and a recombinant vector containing a DNA molecule encoding the NS-RNA into a packaging cell to obtain a recombinant oncolytic influenza virus.
The vector is a pHW2000 plasmid, but is not limited to pHW2000 plasmid.
5. A medicament for treating colorectal cancer, the active ingredient of which is the recombinant oncolytic influenza virus of any one of claims 1-3.
6. A recombinant oncolytic influenza virus according to any one of claims 1-3 as any one of the following products, characterized in that: the product is as follows:
(1) The genome of the recombinant oncolytic influenza virus;
(2) A kit of vectors for treating tumors, i.e., a vector containing the gene encoding CCL19;
(3) A kit of cassettes for the treatment of tumors, i.e., an cassette comprising the CCL19 encoding gene;
(4) A set of genes for treating tumors, i.e., the gene encoding CCL19;
(5) A complete set of proteins, the CCL19 protein, for use in the treatment of tumors.
7. A biological material associated with the recombinant oncolytic influenza virus of claim 1, characterized in that: the biological material is any one of the following products:
(1) A set of DNA molecules encoding the set of positive strand RNAs of the recombinant oncolytic influenza virus; the complete set of DNA molecules consists of a DNA molecule encoding the PB2-RNA, a DNA molecule encoding the PA-RNA, a DNA molecule encoding the PB1-CCL19-RNA, a DNA molecule encoding the NP-RNA, a DNA molecule encoding the NA-RNA, a DNA molecule encoding the HA-RNA, a DNA molecule encoding the M-RNA, and a DNA molecule encoding the NS-RNA;
(2) A set of recombinant vectors; the complete set of recombinant vectors consists of a recombinant vector containing a DNA molecule encoding the PB2-RNA, a recombinant vector containing a DNA molecule encoding the PA-RNA, a recombinant vector containing a DNA molecule encoding the HA-RNA, a recombinant vector containing a DNA molecule encoding the NP-RNA, a recombinant vector containing a DNA molecule encoding the M-RNA, a recombinant vector containing a DNA molecule encoding the NA-RNA, a recombinant vector containing a DNA molecule encoding the NS-RNA, a recombinant vector containing a DNA molecule encoding the PB1-CCL 19-RNA;
(3) A microorganism containing the recombinant oncolytic influenza virus;
(4) An animal cell containing the recombinant oncolytic influenza virus;
(5) Animal tissue containing the recombinant oncolytic influenza virus;
(6) Animal organs containing the recombinant oncolytic influenza virus
In the above biological material, the animal cells, the animal tissue and the animal organ do not include propagation material;
in the above biological material, the microorganism, the animal cell, the animal tissue and the animal organ can be used as hosts for recombinant oncolytic viruses.
8. Use of a recombinant oncolytic influenza virus according to any one of claims 1-3 as any one of the following products, characterized in that: the application is as follows:
the application of the recombinant oncolytic influenza virus in preparing a medicine for treating tumor;
such tumors include, but are not limited to, colorectal cancer, lung cancer, breast cancer, liver cancer, melanoma, lymphoma, leukemia, ovarian cancer, cervical cancer, gastric cancer, renal cancer, pancreatic cancer, prostate cancer, glioma.
9. The recombinant oncolytic influenza virus of any one of claims 1-2, characterized in that: influenza virus strains include, but are not limited to, influenza a virus or influenza b virus.
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