CN116688108A - Method for treating tumor by using novel influenza virus vaccine and application thereof - Google Patents
Method for treating tumor by using novel influenza virus vaccine and application thereof Download PDFInfo
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- CN116688108A CN116688108A CN202310700623.9A CN202310700623A CN116688108A CN 116688108 A CN116688108 A CN 116688108A CN 202310700623 A CN202310700623 A CN 202310700623A CN 116688108 A CN116688108 A CN 116688108A
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Classifications
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
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- C—CHEMISTRY; METALLURGY
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
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Abstract
The application relates to a method for treating tumors by using a brand-new influenza virus vaccine and application thereof, which comprises the steps of introducing influenza virus H1N1 hemagglutinin glycoprotein into a treated solid tumor, connecting an influenza virus hemagglutinin HA gene with a C3d functional sequence to construct a C3d-HA fusion protein, infecting tumor cells by recombinant oncolytic adenovirus carrying the influenza virus hemagglutinin gene, selectively replicating in the tumor, simultaneously expressing the influenza virus C3d and HA fusion protein, and anchoring HA on the surface of the tumor cells to enable the HA to become an artificial heterologous tumor antigen. When the anti-HA antibody exists in the organism, the recombinant oncolytic adenovirus carrying the influenza virus hemagglutinin gene is injected, and once the hemagglutinin protein is expressed in the tumor, the immune system of the organism can accurately identify cancer cells and completely remove tumor cells.
Description
Technical Field
The application relates to the technical field of tumor treatment, in particular to a method for treating tumors by using a brand-new influenza virus vaccine and application thereof.
Background
About 2000 tens of thousands of cancer patients are newly increased worldwide each year, and about 1000 tens of thousands die. Up to now, although new anticancer drugs or new methods are being introduced, such as monoclonal antibodies, small molecules, CART immune cell therapy techniques, oncolytic viruses, etc., satisfactory results have not been obtained for the treatment of cancer, especially for the treatment of solid tumors. Cancer remains a serious threat to human health, and the development of new anti-cancer drugs and the search for new methods of treating cancer remain challenges for scientists.
Because of the special nature of the microenvironment of solid tumors, the number of immune cells entering the solid tumor is limited. The discovery of immunodetection spots led scientists to recognize that PD-L1 on certain cancer cells binds to T cell surface PD-1, making T cells "blind". CD47, in combination with the protein sirpa on the surface of macrophages, inhibits the ability of macrophages to kill cancer cells, is widely recognized as a "do not eat me" signal. After entering solid tumors, the cells do not kill cancer cells, but become the constituent of the solid tumors. The inhibition of tumor microenvironment and the inability of immune cells to infiltrate effectively makes immune cells, including CART cells, challenging to treat solid tumors. Antigen Presenting Cells (APCs) refer to a class of immune cells that are capable of uptake, processing of a processed Antigen, and presentation of the processed Antigen to T cells. APCs mainly include dendritic cells, mononuclear phagocytes, B cells, langerhans cells, and the like, wherein the dendritic cells have the strongest antigen presenting ability. There is an increasing number of scientists now focusing on tumor specific antigens in the hope of using tumor specific antigens as tumor vaccines for the treatment of tumors. However, the lack of tumor-specific antigens and their low immunogenicity makes tumor vaccine treatment of solid tumors equally challenging. In the face of the difficult problem of treating solid tumors, the inventor is prompted to think about a strategy for treating solid tumors by introducing virus glycoprotein to artificially make specific antigens on the surfaces of cancer cells, thereby achieving the aim of treating solid tumors. The inventors also believe that antibodies may be indispensable in the treatment of solid tumors, as antibodies play a critical role in antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, altering signal transduction and immunomodulation of cancer tissues. When the antibody is combined with cancer cells, the cancer cells are more easily recognized by the human immune system, so that the immune system is guided to kill the cancer cells.
At present, four oncolytic virus medicines are on the market worldwide, such as oncolytic adenovirus H101 for treating nasopharyngeal carcinoma, oncolytic herpes virus I type Imlygic (T-Vec) for treating melanoma and Delytact for treating glioma, and the oncolytic virus products have a certain treatment effect clinically, but still have a great improvement space. Scientists in various countries are adopting different technical routes to construct new oncolytic viruses in order to further improve the effect of oncolytic viruses in treating solid tumors. Although oncolytic viruses may induce chemokines to turn a "cold" tumor into a "hot" tumor, thereby eliciting local and systemic anti-tumor immune responses, different oncolytic viruses, due to their individual characteristics, behave differently in oncolysis, e.g., adenovirus does not have envelope glycoproteins, and does not release the virus in a budding manner. Adenovirus E3A (gp 19K) glycoprotein binds predominantly to MHC-1 on the endoplasmic reticulum of the cell. Adenovirus is epitheliophilic, whereas most tumors in humans are derived from epithelial cells, and thus, the modification of adenovirus to oncolytic virus has the potential to treat tumors in a broad spectrum.
Disclosure of Invention
The application aims to solve the technical problems and provides a method for treating tumors by using a brand-new influenza virus vaccine and application thereof.
In order to solve the technical problems, the technical scheme provided by the application is as follows:
an application of a brand-new influenza virus vaccine in tumor treatment comprises an influenza virus hemagglutinin glycoprotein gene and influenza virus, wherein the influenza virus hemagglutinin glycoprotein gene comprises HA1, HA2 and a transmembrane region, is connected with C3d or auxiliary protein and polypeptide nucleotide, is connected with 2A or Linker in the middle, and does not generate HA fusion protein;
the influenza virus is a vaccine strain NYMCX-179A hemagglutinin glycoprotein recommended by WHO as antigen, and comprises HA or neuraminidase of all influenza virus subtypes, measles virus hemagglutinin, coronavirus surface glycoprotein and other virus glycoprotein which can be used for vaccine.
Preferably, the influenza virus hemagglutinin glycoprotein gene limits the human TERT promoter to control the HA fusion protein gene to achieve selective expression of the HA fusion protein in the tumor and anchoring at the tumor cell surface.
The human influenza virus HA gene and the CMV promoter control the HA fusion protein gene to realize the overexpression of the HA fusion protein and anchor the HA fusion protein on the surface of tumor cells.
Preferably, the restriction hamster TERT promoter controls the E1A gene to achieve selective replication of oncolytic adenoviruses in tumors and localized expression of influenza virus HA glycoproteins in tumors and anchoring to tumor cell surfaces.
A method for treating tumors by using a novel influenza virus vaccine, which comprises the following steps: the immune system can accurately identify, kill and eliminate cancer cells by injecting recombinant oncolytic virus carrying HA genes after the anti-HA antibody is generated by utilizing the existing anti-HA antibody and memory immune cells in the organism or pre-inoculating an HA protein vaccine to the organism before treatment and the standby body.
Preferably, the recombinant oncolytic virus carrying the HA gene can be injected directly into the tumor.
After the scheme is adopted, the application has the following advantages:
the foregoing summary is for the purpose of the specification only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the present application will become apparent by reference to the drawings and the following detailed description.
Drawings
In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings that are necessary for the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the application and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
Drawings
FIG. 1 is a schematic diagram showing construction of recombinant adenovirus vectors Ad-JYT-hTERT-HA, ad-JYT-CMV-HA, ad-JYT-hamsterTERT-E1A.
Fig. 2 is Marker (1). Recombinant oncolytic adenovirus Ad-JYT-hemsterT-E1A (2), ad-JYT-CMV-HA (3), ad-JYT-hTERT-HA (4) respectively infect DDT cells, cells are collected in 48 hours, and a Westernblot detection result is obtained.
FIG. 3 shows the results of Westernblot detection after CsCl ultracentrifugation purification of virus, (1) Ad-JYT-hamsterTERT-E1A, (2) Ad-JYT-CMV-HA, (3) Ad-JYT-hTERT-HA and (4) Hemagglutinin (HA) purified glycoprotein.
FIG. 4 is a flow cytometry analysis of DDT tumor cells isolated after injection of recombinant oncolytic adenoviruses Ad-JYT-CMV-HA (A, B) and Ad-JYT-hTERT-HA (C, D) into DDT solid tumors.
FIG. 5 is a graph showing the results of ELISA detection of IgG in hamster serum immunized with Hemagglutinin (HA) glycoprotein vaccine
FIG. 6 is a graph showing the survival rate of recombinant oncolytic adenoviruses Ad-JYT-hTERT-HA, ad-JYT-CMV-HA, control recombinant adenoviruses Ad-JYT-hamster T-E1A (1X 108 pfu) and PBS injected into Syrian hamster leiomyosarcoma as a result of a hamster oncolytic experiment with a Hemagglutinin (HA) protein vaccine.
FIG. 7 is a graph showing the survival rate of recombinant oncolytic adenoviruses Ad-JYT-hTERT-HA, ad-JYT-CMV-HA, control recombinant adenoviruses Ad-JYT-hamster T-E1A (1X 108 pfu) and PBS after injection of syrian hamster leiomyosarcoma, as a result of a hamster oncolysis experiment without Hemagglutinin (HA) protein vaccine.
FIG. 8 is a graph showing comparison of tumor growth size after injection of recombinant oncolytic adenoviruses Ad-JYT-hTERT-HA, ad-JYT-CMV-HA, control recombinant adenoviruses Ad-JYT-hamster T-E1A (1X 108 pfu) and PBS into Syrian hamster leiomyosarcoma in a hamster oncology experiment vaccinated with Hemagglutinin (HA) protein vaccine.
FIG. 9 is a graph showing survival of hamsters after the hamsters have been cured by recombinant oncolytic adenoviruses Ad-JYT-hTERT-HA and Ad-JYT-CMV-HA, and after the hamsters have been vaccinated with DDT cells (PBS control).
Detailed Description
Reference will now be made in detail to specific embodiments of the application. While the application will be described in conjunction with these specific embodiments, it will be understood that they are not intended to limit the application to these specific embodiments. On the contrary, these embodiments are intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the application as defined by the appended claims. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. The present application may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail in order not to unnecessarily obscure the present application.
When used in conjunction with the description herein and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. 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 to which this application belongs.
The present application will be described in further detail in connection with the following.
1-9, a brand new influenza virus vaccine is applied to tumor treatment, comprising an influenza virus hemagglutinin glycoprotein gene and influenza virus, wherein the influenza virus hemagglutinin glycoprotein gene comprises HA1, HA2 and a transmembrane region, is connected with C3d or auxiliary protein and polypeptide nucleotide, is connected with 2A or Linker in the middle, and does not generate HA fusion protein;
influenza virus is the WHO recommended vaccine strain NYMCX-179A (H1N 1)) hemagglutinin glycoprotein (HA) as antigen, including HA or neuraminidase (N) of all influenza virus subtypes, and also measles virus Hemagglutinin (HA), as well as coronavirus surface glycoprotein (Spikeglycoprotein) and other viral glycoproteins useful in vaccines.
The influenza virus hemagglutinin glycoprotein gene limits the human TERT promoter to control the HA fusion protein gene so as to realize the selective expression of the HA fusion protein in tumors and anchor the HA fusion protein on the surfaces of tumor cells.
The human influenza virus HA gene and the CMV promoter control the HA fusion protein gene to realize the overexpression of the HA fusion protein and anchor the HA fusion protein on the surface of tumor cells.
The restricted hamster TERT promoter controls the E1A gene to achieve selective replication of oncolytic adenoviruses in tumors and localized expression of influenza virus HA glycoproteins in tumors and anchoring to tumor cell surfaces.
The present application employs the human TERT promoter to control HA glycoprotein to achieve selective expression in tumors, including other promoters that are selectively expressed in tumors, such as: hamster TERT, survivin, E F, tyrosinase, prostate specific antigen, alpha fetoprotein, COX-2, and the like, and IRES downstream of the promoter control HA to achieve selective expression in tumors.
The application adopts hamster TERT to control E1A so as to realize selective replication of oncolytic adenovirus in tumor, adopts CMV to control HA fusion protein gene so as to realize selective replication of virus in tumor, and limits HA to over-expression in tumor. Other promoters are used to achieve HA-localized expression in tumors, including other technical route engineered oncolytic viruses that achieve selective replication in tumors, such as HSV oncolytic viruses that lack ICP34.5 and ICP47, such as: EF1a promoter, TK promoter, SV40 promoter, etc., especially CMV promoter.
A method for treating tumors by using a novel influenza virus vaccine, which comprises the following steps: the immune system can accurately identify, kill and eliminate cancer cells by injecting recombinant oncolytic virus carrying HA genes after the anti-HA antibody is generated by utilizing the existing anti-HA antibody and memory immune cells in the organism or pre-inoculating an HA protein vaccine to the organism before treatment and the standby body.
The recombinant oncolytic virus carrying the HA gene can be injected directly into the tumor.
The core of the present application with obvious therapeutic effect is the treatment method of expressing influenza virus surface glycoprotein in tumor and pre-inoculating HA protein vaccine to body before treatment to make body produce corresponding antibody. Thus, it is suitable for the treatment of tumors with any viral glycoprotein vaccine, including mRNA, DNA, plasmid vectors or other oncolytic viral vectors carrying viral glycoprotein genes, and for the vaccination of the body with the same antigen vaccine prior to treatment.
In order to realize the purpose of artificially manufacturing tumor specific antigens on the surface of tumor cells and further realizing the purpose of treating solid tumors, the inventor selects influenza A virus NYMCX-179A vaccine strain H1N1 Hemagglutinin (HA) glycoprotein recommended by world health organization as a tumor heterologous antigen and introduces the tumor treatment. According to the principle of antibody-dependent cell-mediated cytotoxicity, the application uses influenza virus Hemagglutinin (HA) vaccine to improve the therapeutic effect of solid tumors. The specific antibody is produced by using the obtained anti-HA antibody in the body or injecting H1N1 influenza virus Hemagglutinin (HA) vaccine into the body in advance. After the antibody exists in the body, injecting recombinant oncolytic adenovirus carrying influenza virus Hemagglutinin (HA) gene, expressing HA protein when the oncolytic adenovirus replicates in cancer cells, and anchoring on the surfaces of the cancer cells, so that the oncolytic adenovirus becomes an artificial specific antigen of tumor. Since specific antibodies are already present in the body, once hemagglutinin protein is expressed in the tumor, it causes the body's immune system to recognize the tumor and kill tumor cells. Animal tumor killing experiments show that only two needles of recombinant oncolytic adenovirus carrying HA genes are injected, so that 350mm3 tumor can be completely disappeared within one week, and 100% of cure is realized.
Influenza virus H1N1 Hemagglutinin (HA) protein is selected as an antigen vaccine, firstly, the influenza virus vaccine is easy to obtain; secondly, the safety and effectiveness of the anti-theft device are proven in long-term application; third, influenza a virus H1N1 HAs been vaccinated against influenza H1N1 vaccine many times in a worldwide fashion or in humans, where antibodies against H1N1 Hemagglutinin (HA) are prevalent. Whether or not an influenza virus H1N1 vaccine needs to be injected is determined based on the antibody level in the patient before treatment. According to animal tumor killing experiments, when no anti-HA antibody exists in vivo, aiming at 350mm3 tumor, two recombinant oncolytic adenoviruses Ad-JYT-hTERT-HA or Ad-JYT-CMV-HA are injected, the tumor killing rate is 50% to 60%, when the HA protein vaccine is inoculated in advance to animals, and after the antibody level in the antibodies in the animals reaches 4000EU, the tumor killing rate reaches 100% under the same conditions. From the first injection of oncolytic adenovirus to the complete cure of tumor, the antibody level in the cured animal is detected at the 10 th day, and the antibody level is doubled, which is 8000EU or 10000EU respectively, which indicates that memory B cells exist in the animal vaccinated with HA protein vaccine, and the recombinant oncolytic adenovirus expresses HA protein in the animal to induce, thereby playing a role of enhancing immunity, improving the anti-HAIgG level in the animal in a short time, and being beneficial to the immune system to kill tumor cells.
Because the full-length HA protein cannot be expressed artificially due to the enrichment of hydrophobic amino acids in the influenza virus HA protein transmembrane region, the application adopts the fusion of the C3d protein and the HA protein, realizes the effective expression of the HA protein in vivo and in vitro and anchors the HA protein on the cell surface. Besides helping the expression of HA protein, the C3d protein HAs the function of enhancing the immunogenicity of the HA protein, and the effect is better than that of a chemical adjuvant. Through the detection of tumor flow type cells in animal bodies, the HA protein is successfully anchored on the cell surfaces, and shows stronger immunogenicity in vivo, so that the immune response of animal bodies is effectively stimulated, and the accurate identification of cancer cells by an immune system and the complete elimination of tumor cells are realized.
According to the principle of improving the protein expression quantity by optimizing the nucleotide sequence, the nucleotide sequence of the NYMCX-179A vaccine strain H1N1 Hemagglutinin (HA) is optimized, and the similarity of the optimized nucleotide sequence and the influenza virus wild type HA sequence is 75.8 percent.
The application adopts human TERT promoter to control HA fusion protein gene, so as to realize the selective expression of HA fusion protein in lung cancer, breast cancer, colorectal cancer, hysteromyoma and other tumors. The HA fusion protein gene is controlled by adopting a CMV promoter so as to realize the overexpression of the HA fusion protein in tumors.
The application adopts hamster TERT promoter to control the E1A gene so as to realize the selective replication of oncolytic adenovirus in tumor. Because the hamster TERT promoter is different from the human TERT promoter in nucleotide sequence, the similarity is low, the probability of recombination between the two promoters is extremely low, and the normal replication of the recombinant oncolytic adenovirus in vivo and in vitro is not affected.
According to the present application, it is presumed that most vaccine proteins can be used for tumor treatment, especially viral surface glycoproteins such as influenza virus neuraminidase (N), measles virus Hemagglutinin (HA), coronavirus spike protein (S), etc. The vast majority of humans acquire corresponding antibodies by natural infection with viruses, or by vaccination, except that the in vivo antibody levels vary from person to person, and prior to treatment, it is decided whether or not to increase the vaccinated protein based on assessing the in vivo antibody levels. When a proper amount of antibody exists in a patient, the vector carrying the corresponding gene is injected into the tumor by adopting a genetic engineering technology, or the vector reaches the tumor area by intravenous injection, for example, the target gene is transported to the tumor area by lipid, nano material wrapping mRNA, DNA, plasmid, virus vector and the like, once the target gene is expressed in the tumor, glycoprotein appears on the surface of the tumor cell, and the immune system of the organism can identify the cancer cell and kill the cancer cell. Adenovirus lacks the effect of producing viral protein antigens on the surface of the host cells, thereby reducing the tumoricidal effect of inducing the immune system of the body. By utilizing the characteristic that adenovirus can infect most tumor cells, the HA glycoprotein gene is carried by the oncolytic adenovirus, thereby overcoming the defect that the oncolytic adenovirus can not manufacture glycoprotein on the tumor surface. As the HA glycoprotein is taken as the foreign protein and is not in the structure of the oncolytic adenovirus, the rejection of the organism to the oncolytic adenovirus is not enhanced, thereby improving the oncolytic effect of the oncolytic adenovirus.
In example one, vector construction:
(1) The 5 'and 3' ends of the fragments of the armterEnhance terTDNA were synthesized with restriction sites Xbal and EcoRI, respectively. The shuttle vectors pDC316 and the hamster enhancement TERTDNA were digested with Xbal and EcoRI, respectively, and the synthesized hamster enhancement TERTDNA fragment was inserted into Xbal and EcoRI sites for enzyme ligation, and the constructed vector was named pDC 316-hamster.
(2) The EIA gene was synthesized with cleavage sites EcoRI and BglII at the 5 'and 3' ends, respectively. The vector pDC316-hamsterTERT and the gene E1A were digested with EcoRI and BglII, respectively, and ligated by recovery, and the vector was designated pDC316-hamsterTERT-E1A.
(3) Respectively synthesizing hTERT-C3d-2A-HA and CMV-C3d-linker-HADNA, and respectively performing PCR cloning of the hTERT-C3d-2A-HA and the CMV-C3d-linker-HA by using primers containing BgIII and SalI. And respectively recovering PCR products, carrying out double digestion on BgIII and SalI to recover DNA fragments, carrying out double digestion on the vector pDC316-hamsterTERT-E1A by using BgIII and SalI, recovering and respectively connecting the DNA fragments, and constructing vectors which are named pDC-E1A-hTERT-C3d-HA and pDC-E1A-CMV-C3d-HA respectively. Sequencing was used to identify the correctness of the DNA inserted into the recombinant vector.
(4) The recombinant vector and skeleton plasmid (pBHGloxdeltaE 1,3 Cre) are respectively transfected into 293 cells to obtain recombinant adenovirus which is named Ad-JYT-hTERT-HA, ad-JYT-CMV-HA and Ad-JYT-hemsterT-E1A. As shown in fig. 1.
Example two, plaque purification:
293 cells were cultured in 10cm cell culture dishes and recombinant adenovirus solution was added when the cells grew to 70% density. Continuously diluting recombinant adenovirus solution 10 times in a centrifuge tube, respectively taking 200 μl of 10-5 to 10-7 virus solution from 10-1 to 10-7, adding into a cell culture dish containing 5ml of fresh culture medium, gently mixing, placing into a37 ℃ and 5% CO2 incubator for 1 hour, removing virus solution, washing cells with PBS, adding 1% low melting point agarose (Lonza) and 2XDMEM medium according to 1:1, comprising 10% fbs,1% penicillin and streptomycin. Placing the recombinant adenovirus at 37 ℃ and in a 5% CO2 incubator for culturing for 10 days, picking up 10 plaques each time, and screening 1 recombinant adenovirus with the highest HA expression level by using Westernblot for the next experiment.
Example three, virus purification:
293 cells were cultured in 75cm flasks and recombinant adenovirus solution was added when the cells grew to 70% density. After 72 hours, 50ml of the culture broth and cells were collected, centrifuged at 2000rpm for 10 minutes, and the supernatant was taken for use. 5ml PBS buffer was added to the cell centrifuge tube, the cells were disrupted by sonication, and the cell debris was removed by low-speed centrifugation. Mixing the supernatant with cell supernatant, and using BeckmanOptimaXPN-100 ultracentrifuge, SW28 rotor ultracentrifuge tube, 4deg.C, 25000rpm
After centrifugation for 1 hour, the supernatant was discarded, and 1ml of PBS was added to the pellet to prepare a virus sample suspension. 3ml of 4.0mcsCl and 7ml of 2.2mcsCl gradient were applied to each centrifuge tube using a SW41 rotor, and 1ml of virus sample was added to the cesium chloride density gradient upper layer and centrifuged at 35000rpm at 4℃for 48 hours. The viral bands were aspirated by puncturing the centrifuge tube with a 1ml syringe, and the virus samples were placed in dialysis bags and dialyzed against PBS +10% glycerol. The purified recombinant adenovirus is put into a-80C refrigerator for standby.
Adenovirus titer assay:
adenovirus titer was determined using an immunoassay rapid detection kit manufactured by Shenzhen Leibok organism Co., ltd (Labkit), by combining an anti-adenovirus antibody with cells infected with adenovirus, combining a secondary antibody labeled with horseradish peroxidase with the primary antibody, developing with a developing solution, and measuring adenovirus titer by counting and calculating according to the following formula.
Calculate infection units per well (ifu)/ml:
(average positive cell number/field) × (field number/well)/(virus volume (ml) ×dilution })
Embodiment five, westernblot:
1, sample preparation: 293 cells were passaged at a ratio of 1:3, and the cells were infected with the viruses Ad-JYT-hemterTERT-E1A, ad-JYT-hTERT-HA, ad-JYT-CMV-HA, respectively, the cells were collected after 48 hours, the lysate RIPA was added to lyse on ice for 10 to 20 minutes, then loadingbuffer was added for 5 minutes, and 12000g was centrifuged for 5 minutes to obtain the supernatant.
2, SDS-PAGE gel electrophoresis.
3, film transfer: western blots were performed on PVDF membranes.
4, closing: incubate with 3% BSA at 37℃for 1 hour.
5, incubating primary antibody: deblocking solution, adding proper amount of primary antibody reaction solution (HA primary antibody is dissolved in 1% BSA-TBST) in an amount of 0.2-0.3ml per square centimeter; incubate on a gentle shaking table at room temperature for 1 hour.
6, incubating a secondary antibody: removing the primary antibody reaction solution, and washing the PVDF membrane with TBST for 3 times to remove unbound antibody for 10 minutes each time; a secondary antibody reaction solution (HRP-labeled secondary antibody in 1% BSA-TBST) was added at a membrane area of 0.1ml/cm2, and incubated on a flat shaker with shaking at room temperature for 1 hour.
And 7, developing: rinsing the PVDF membrane 3 times with TBST to remove unbound antibody for 10 minutes each time; immersed in freshly prepared ECL chemiluminescent reagent and developed on a chemiluminescent analyzer for photography. The results are shown in fig. 2 and 3.
Example six, flow cytometric detection:
when the tumor was grown to 600mm3, the recombinant adenovirus was injected, tumor tissue was taken 24 hours after the second injection of the recombinant adenovirus, the tumor was minced with sterile surgical scissors, 1mg/ml collagenase B (cologenaseb, roche) was contained in tumor digest PBS, tumors were treated with 0.1mg/ml Hyaluronidase (Sigma) and 0.02mg/ml dnase (dnaasei, roche), tumor-bearing hamster tumor cells were collected, and the cells were suspended in 100 μl PBS buffer. FcR ecctorBlocker (INNOVEXBiosciencesInc.California, USA) was used to block Fc, cells were washed with PBS buffer and then reacted with rabbit anti-HA antibody FACS (PBS containing 1% BSA) for 2 hours and secondary antibodies were reacted with FITC-labeled anti-rabbit antibody for 30 minutes. Tumor cells not injected with virus were used as negative controls. LX analytical flow cytometry (CytoFLEXLX) detection. The results are shown in FIG. 4.
Example seven, experimental animals were vaccinated with hemagglutinin protein vaccine:
influenza a virus NYMCX-157 (H1N 1)) strain Hemagglutinin (HA) glycoprotein (from beijing-sense baoshen) produced using baculovirus expression vectors was used to immunize 4-year-old syrian hamsters.
The prepared antigen was thoroughly mixed with an equal volume of Freund's complete adjuvant to prepare an injection for the first time, and 200. Mu.l (containing 10. Mu.g of hemagglutinin protein) of the injection was intraperitoneally injected into each hamster, and the mixture was left for several seconds after the injection to prevent outflow of the antigen. Two weeks apart, the second needle was boosted after emulsification with the same antigen plus an equal volume of Freund's incomplete adjuvant and co-injected twice. Orbital blood was collected on day 28, serum was prepared, and antibody concentration was measured. The results are shown in FIG. 5.
Example eight, animal oncology experiment:
hamsters (8 weeks old) vaccinated with H1N1 Hemagglutinin (HA) protein vaccine and positive for anti-HA antibodies were tested by ELISA, and animal tumor killing experiments were performed on day 28 post-vaccine. Control oncologic experiments were also performed with 8 week old non-vaccinated hamsters. 100 μl of 5X106DDT cells (hamster leiomyosarcoma cells) were inoculated on the dorsal right side of syrian hamsters, the vaccinated hamsters were divided into four groups, and the unvaccinated hamsters were divided into four groups of 10 hamsters each. Until the tumor grows to 350mm 3 Mu.l of recombinant adenovirus containing 1X108pfu was injected into each tumor, 100. Mu.l of PBS buffer was injected into the tumor-bearing hamster of the blank group, and the injections were twice on days 0 and 2. The recombinant adenovirus samples are Ad-JYT-hTERT-HA, ad-JYT-CMV-HA and Ad-JYT-hamsterTERT-E1A. The health status of the hamsters was observed daily, and the tumor growth size and the survival number were recorded, and the results are shown in fig. 6 to 8.
Experiment of tumor cells re-inoculated in animals after cure:
the animals after cure were re-vaccinated with tumor cells, and the hamsters after cure with recombinant oncolytic adenovirus Ad-JYT-hTERT-HA or Ad-JYT-CMV-HA were continuously fed, 8 animals were each selected, and 100. Mu.l of 1X107 DDT-containing cells were vaccinated on the right-back side of the hamsters after 70 days. Control groups selected 8 healthy syrian hamsters 15 weeks old, and were inoculated on the back-right side with 100 μl of 1X107DDT cells. The growth and survival number of each group of tumors are observed. The results are shown in FIG. 9.
The application and its embodiments have been described above with no limitation, and the actual construction is not limited to the embodiments of the application as shown throughout. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present application.
Claims (6)
1. An application of a brand-new influenza virus vaccine in tumor treatment is characterized in that: the influenza virus hemagglutinin glycoprotein gene comprises HA1, HA2 and a transmembrane region, is connected with C3d or auxiliary protein and polypeptide nucleotide, is connected with 2A or Linker in the middle, and does not generate HA fusion protein;
the influenza virus is a vaccine strain NYMCX-179A hemagglutinin glycoprotein recommended by WHO as antigen, and comprises HA or neuraminidase of all influenza virus subtypes, measles virus hemagglutinin, coronavirus surface glycoprotein and other virus glycoprotein which can be used for vaccine.
2. Use of a completely new influenza virus vaccine according to claim 1 in tumor treatment, characterized in that: the influenza virus hemagglutinin glycoprotein gene limits the human TERT promoter to control the HA fusion protein gene so as to realize the selective expression of the HA fusion protein in tumors and anchor the HA fusion protein on the surfaces of tumor cells.
3. A human influenza virus HA gene, characterized in that: the CMV promoter controls the HA fusion protein gene to achieve overexpression and anchoring of the HA fusion protein on the tumor cell surface.
4. A human influenza virus HA gene according to claim 3, wherein: the restricted hamster TERT promoter controls the E1A gene to achieve selective replication of oncolytic adenoviruses in tumors and localized expression of influenza virus HA glycoproteins in tumors and anchoring to tumor cell surfaces.
5. A novel method for treating tumors by using influenza virus vaccine, which is characterized by comprising the following steps: the method comprises the following steps: the immune system can accurately identify, kill and eliminate cancer cells by injecting recombinant oncolytic virus carrying HA genes after the anti-HA antibody is generated by utilizing the existing anti-HA antibody and memory immune cells in the organism or pre-inoculating an HA protein vaccine to the organism before treatment and the standby body.
6. The method for treating tumors with a completely new influenza virus vaccine according to claim 5, wherein: the recombinant oncolytic virus carrying the HA gene can be injected directly into the tumor.
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