CN115611975A - Tumor antigen rich in HIF-1A protein, preparation method thereof and application of tumor antigen in preparing DC vaccine - Google Patents

Abstract

The invention provides a tumor antigen rich in HIF-1A protein, a preparation method thereof and application of preparing DC vaccine, wherein the tumor antigen is obtained by screening tumor cells transfected with lentivirus by puromycin to obtain a cell strain stably expressing the HIF-1A protein, and cracking the cells after amplifying and culturing the cell strain. The tumor antigen obtained by the invention is rich in natural molecular adjuvants HMGB-1 and CARE, which are key components for inducing tumor immunity. Compared with the prior method, the tumor antigen obtained by the method has more anti-tumor components and can induce stronger T cell proliferation.

Description

Tumor antigen rich in HIF-1A protein, preparation method thereof and application of tumor antigen in preparation of DC vaccine

Technical Field

The invention belongs to the technical field of tumor vaccines, and particularly relates to a tumor antigen rich in HIF-1A protein, a preparation method thereof and application thereof in preparing a DC vaccine.

Background

Breast cancer is a serious threat to women's life health, and triple negative breast cancer is severely limited in its treatment due to the inability to use targeted drugs. At present, conventional treatment methods such as radiotherapy, chemotherapy and operation treatment have obviously benefited patients, but the treatment effect is not ideal. Biological treatment of tumors shows great advantages and is increasingly emphasized. Among them, the tumor vaccine using dendritic cells as carrier has become a hot research point for biological treatment of tumor.

The inability of tumor-specific antigens to be efficiently presented to T cells during tumorigenesis is a significant cause of tumor immune escape. In solid tumors such as breast cancer, hypoxia is common in the middle of the tumor, and hypoxia inducible factor 1A (HIF-1A) is usually highly expressed in response to hypoxia. The cells with high expression of HIF-1A escape from the attack of organisms through various ways and then transfer, for example, the characteristics of the tumor cell stem cells with high expression of HIF-1A are enhanced, and the cells have better tolerance to chemotherapeutic drugs; the cell highly expressing HIF-1A highly expresses immunosuppressive molecules such as PD-L1 to induce T cell depletion. Tumor cells that are hypoxic in the middle of the tumor cannot efficiently take up their specific antigens by DC cells due to their long distance from the blood vessels, thus evading attack by the immune system.

Therefore, the tumor cells at the anoxic part in the middle of the tumor are obtained, and the obtained cell lysate can be used as a tumor antigen to remarkably induce the activation of DC cells and induce the proliferation of T cells. But the acquisition of tumor tissue is often difficult and there may be some contamination of non-tumor cells which will seriously affect the quality of the tumor antigen.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the defects in the prior art, the invention provides a tumor antigen rich in HIF-1A protein, a preparation method thereof and application thereof in preparing DC vaccine.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:

a tumor antigen rich in HIF-1A protein is prepared from tumor cells transfected by slow virus through screening by puromycin to obtain cell strain stably expressing HIF-1A protein, amplifying culture and cracking cells.

Preferably, the lentivirus is selected from pHBLV-HIF1A-3flag-ZsGreen-Puro; the tumor cell is selected from triple negative breast cancer cells.

The invention also provides a preparation method of the tumor antigen rich in HIF-1A protein, which comprises the following steps:

(1) Preparing a lentivirus stably expressing HIF-1A protein;

(2) Culturing tumor cells, and transfecting the lentivirus obtained in the step (1);

(3) Selecting the tumor cells transfected with the lentiviruses in the step (2), and screening by utilizing puromycin to obtain a cell strain stably expressing HIF-1A protein;

(4) And (3) performing amplification culture on the stable cell strain, and then performing cell lysis to obtain the HIF-1A protein-containing tumor antigen.

The stable expression of HIF-1A protein has GFP positive rate up to 98% or more.

Preferably, in the step (3), the screening is performed by using puromycin, wherein the optimal concentration of puromycin is that tumor cells which are not transfected with lentivirus are screened by using puromycin with different concentrations, and the lowest puromycin concentration at which the cells are just completely killed is obtained, namely the optimal concentration.

Preferably, in the step (3), the puromycin is used for screening, wherein the screening time is 14-16 days.

Preferably, in the step (4), the cell lysis is to lyse the lentivirus-transfected tumor cells by using a repeated freeze-thaw method, and finally, the obtained suspension is centrifuged, and the obtained supernatant is the tumor antigen rich in the HIF-1A protein.

The invention also provides a DC vaccine loaded with the tumor antigen rich in the HIF-1A protein.

The invention finally provides a preparation method of the DC vaccine, which comprises the following steps:

culturing DC cells in vitro by using culture solution containing GM-CSF and IL-4, and co-incubating the tumor antigen rich in HIF-1A protein and the cultured DC cells to obtain the DC vaccine.

Preferably, the preparation method comprises the following steps:

(1) Collecting peripheral blood of a healthy human body, and extracting mononuclear cells PBMC;

(2) Culturing PBMC, culturing adherent cells, and adding culture solution containing GM-CSF and IL-4;

(3) After culturing for a period of time, adding tumor antigen rich in HIF-1A into the cells, and incubating;

(4) Continuously culturing, and adding IL-1 beta, IL-6, TNF-a and PEG-2 into the cells to induce the DC to mature.

The invention introduces HIF-1A lentivirus into MDA-MB-231 cell strain to construct stable cell strain, thereby obtaining a large amount of effective tumor antigen with relatively stable components.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. the invention can obtain the tumor antigen rich in HIF-1A in large quantity, and the obtained antigen can be used for different patients.

2. The tumor antigen rich in HIF-1A obtained by the invention can induce more DC cells to be activated.

3. The tumor antigen rich in HIF-1A obtained by the invention can better stimulate the proliferation of T cells.

4. The tumor antigen rich in HIF-1A contains immune stimulating molecules HMGB1 and CREB.

Drawings

FIG. 1: detecting the success of the HIF-1A stable expression cell strain MDA-MB-231-HIF-1A cell strain by flow cytometry;

FIG. 2: the construction of a stable HIF-1A expression strain detected by Westernblot is successful;

FIG. 3: detecting activation of peripheral blood monocyte-derived DC cells induced by HIF-1A-rich tumor antigen by flow cytometry;

FIG. 4 is a schematic view of: detecting the proliferation of peripheral blood T cells by using HIF-1A-rich tumor antigen through flow cytometry;

FIG. 5 is a schematic view of: westernblot detects the expression of immunostimulating molecules HMGB1 and CARE in HIF-1A tumor antigen;

Detailed Description

The following is a comprehensive description of the solution of the present invention, which is the most preferred embodiment of the present invention, but the present invention is not limited to the following examples.

The Hantaan bioscience (Shanghai) limited company is entrusted to construct a HIF-1A stable and high-expression lentiviral vector pHBLV-HIF1A-3flag-ZsGreen-Puro and a control virus pHBLV-ZsGreen-Puro thereof (the difference is only that HIF-1A protein is not expressed).

Example 1: construction of HIF-1A Stable expression cell line MDA-MB-231-HIF-1A

1. Recovering MDA-MB-231 cells (purchased from Shanghai cell bank of Chinese academy of sciences), after the cells grow to logarithmic phase, digesting and counting, paving the cells in a 96-well plate, wherein 10000 cells are added into each hole, and 1, 2, 4, 8 and 10ng/ml puromycin is added to screen the cells, and after two days, the MDA-MB-231 cells with the lowest drug concentration of 2ng/ml are obtained;

2. spreading MDA-MB-231 cells in log phase in a 6-well plate, wherein each well contains 1000000 cells, transfecting the cells with lentiviruses pHBLV-HIF1A-3flag-ZsGreen-Puro and pHBLV-ZsGreen-Puro respectively after the cells are attached to the wall, naming the lentivirus transfected cells as MDA-MB-231-HIF-1A and MDA-MB-231-GFP, changing liquid on the second day, and adding 2ng/ml puromycin for screening;

3. screening the cells to the 15 th day, observing the virus transfection efficiency under a fluorescence microscope, and taking the cells for flow cytometry detection;

as shown in fig. 1: MDA-MB-231-GFP and MDA-MB-231-HIF-1A cells both express strong GFP fluorescence, and the result proves that the cell lines stably expressing HIF-1A are successfully established preliminarily.

Example 2: detection of HIF-1A expression by Westernblot

1. Recovering MDA-MB-231-GFP and MDA-MB-231-HIF-1A cells, digesting the cells until the cells grow to the logarithmic phase and counting, and paving 1000000 cells per dish in a 10cm culture dish;

2. culturing the cells in a culture dish for 2 days, removing culture solution on the third day, flushing adherent cells for 3 times by using precooled PBS, and removing PBS;

3. adding cell lysate containing protease inhibitor and phosphatase inhibitor into the washed culture dish, incubating for 15min on ice, scraping the cells from the culture dish by using a cell scraper, and carefully sucking cell debris and the cell lysate into a precooled EP tube; placing the cells obtained by the heavy suspension on ice for 15min, freezing the obtained liquid in liquid nitrogen, and then placing the liquid in a water bath at 37 ℃ for rapid thawing; repeating freeze thawing for 7 times;

4. 12000g of cell debris suspension obtained at the upper part is centrifuged for 15min at 4 ℃, and supernatant fluid is taken to be tumor antigen rich in HIF-1A protein and control antigen thereof;

5. adding the antigen into polyacrylamide gel at the amount of 30ug per well for SDS-PAGE, and transferring the protein onto PVDF membrane;

6. sealing the PVDF membrane with 5% skimmed milk powder for 2h, adding anti-HIF-1A monoclonal antibody or anti-GAPDH monoclonal antibody, and incubating at 4 deg.C overnight;

7. on day 2, the PVDF membrane was washed 5 times with PBST, and then HRP-labeled secondary antibody was added and incubated at room temperature for 1h;

8. washing PVDF membrane with TBST for 5 times, adding chemiluminescence reagent, and taking picture with chemiluminescence imager;

9, analyzing the gray value of the strip by ImagJ software;

as shown in FIG. 2, the expression level of HIF-1A protein in the tumor antigen obtained from MDA-MB-231-HIF-1A cell is significantly higher than that of MDA-MB-231-GFP in the control group.

Example 3: HIF-1A-rich tumor antigen-induced activation of peripheral blood-derived DCs

(1) Collecting 50ml of peripheral blood of healthy volunteers; diluting whole blood with an equal volume of PBS;

(2) 5ml of human lymphocyte separation solution is added into a 15ml centrifuge tube, and 5ml of diluted whole blood is slowly added into the upper layer of the lymphocyte separation solution;

(3) Centrifuging at 2500rpm and 25 deg.C for 25min;

(4) Carefully sucking the cells of the leucocyte layer in the middle of the centrifugal tube by using a suction tube to obtain mononuclear cells (PBMC);

(5) The PBMCs were counted and the cells plated in 6-well plates at 1000000 cells/ml with 1640 medium;

(6) Standing for 2h, washing nonadherent cells with 1640 culture solution, adding the 1640 culture solution containing GM-CSF (20 ng/ml) and IL-4 (20 ng/ml) into 6-well plate, and culturing at 37 deg.C in incubator;

(7) Culturing to day 3, removing half of the culture medium by aspiration, and replenishing GM-CSF and IL-4;

(8) Culturing to 5 days, collecting cells, counting, spreading in 48-well plate at 10000/well, adding tumor antigen rich in HIF-1A and control antigen, and protein final concentration is not 100ug/ml;

(9) Culturing to 6 days, adding IL-1 beta, IL-6, TNF-a and PEG-2 into cells to induce DC maturation;

(10) Culturing to 7 days, collecting cells, staining the cells, detecting the expression of CD40, CD80, CD83, CD86 and HLA-DR on the cell surface by a flow cytometer, and analyzing the DC activation degree.

The results are shown in FIG. 3: tumor antigens enriched for HIF-1A induce more DC cells (CD 14) ^- Cells) and is capable of inducing high expression of DC cell activation markers (CD 40, CD80, CD86, HLA-DR).

Example 4: HIF-1A-enriched tumor antigen induced T cell proliferation

(1) Collecting 50ml of peripheral blood of healthy volunteers; diluting whole blood with an equal volume of PBS;

(2) 5ml of human lymphocyte separation solution is added into a 15ml centrifuge tube, and 5ml of diluted whole blood is slowly added into the upper layer of the lymphocyte separation solution;

(3) Centrifuging at 2500rpm and 25 ℃ for 25min;

(4) Carefully sucking the cells of the leucocyte layer in the middle of the centrifugal tube by using a suction tube to obtain mononuclear cells (PBMC);

(5) The PBMCs were counted and the cells plated at 1000000/ml in 48-well plates after staining with CSFE;

(6) The stained PBMC cells are cultured by DC cells stimulated by IL-1 beta, IL-6, TNF-a and PEG-2 in the co-culture way in the embodiment 3, the DC cells are 10000 per hole, and the PBMC cells are 1000000 per hole;

(7) Co-culturing the cells for 7 days, collecting the cells on the 7 th day, respectively staining the CD4-APC and CD8-APC antibodies, and detecting the proliferation condition of the cells by a flow cytometer;

the results are shown in FIG. 4: HIF-1A-enriched tumor antigen-activated DC cells can induce more CD4 ⁺ And CD8 ⁺ T cells proliferate.

Example 5: detection of immune stimulating molecules HMGB-1 and CARE in HIF-1A tumor antigen

1. Recovering MDA-MB-231-GFP and MDA-MB-231-HIF-1A cells, digesting the cells until the cells grow to the logarithmic phase and counting, and paving 1000000 cells per dish in a 10cm culture dish;

2. culturing the cells in a culture dish for 2 days, removing culture solution on the third day, washing adherent cells for 3 times by using precooled PBS, and removing PBS;

3. adding cell lysate containing protease inhibitor and phosphatase inhibitor into the washed culture dish, incubating for 15min on ice, scraping cells from the culture dish by using a cell scraper, and carefully sucking cell debris and cell lysate into a precooled EP tube; placing the cells obtained by the heavy suspension on ice for 15min, freezing the obtained liquid in liquid nitrogen, then placing the liquid in a water bath at 37 ℃ for rapid thawing, and repeating the freezing and thawing for 7 times;

4. centrifuging the cell debris suspension obtained from the upper part at 12000g and 4 ℃ for 15min, and sucking the supernatant to obtain the tumor antigen rich in HIF-1A protein and a control antigen thereof;

5. adding the antigen into polyacrylamide gel at the amount of 30ug per well for SDS-PAGE, and transferring the protein onto PVDF membrane;

6. sealing the PVDF membrane by using 5% skimmed milk powder for 2h, adding an anti-HMGB 1 or CARE monoclonal antibody or an anti-GAPDH monoclonal antibody, and incubating overnight at 4 ℃;

7. on day 2, the PVDF membrane was washed 5 times with PBST, then HRP-labeled secondary antibody was added and incubated at room temperature for 1h;

8. washing PVDF membrane with TBST for 5 times, adding chemiluminescence reagent, and taking picture with chemiluminescence imager;

9, analyzing the gray value of the strip by ImagJ software;

as shown in FIG. 5, the expression levels of the immunostimulating molecules HMGB1 and CARE in the tumor antigen obtained from MDA-MB-231-HIF-1A cell are significantly higher than those of the control group MDA-MB-231-GFP.

Claims (9)

1. A tumor antigen rich in HIF-1A protein is characterized in that the tumor antigen is obtained by screening tumor cells transfected with lentivirus with puromycin to obtain a cell strain stably expressing the HIF-1A protein, amplifying and culturing the cell strain and then cracking the cell.

2. The HIF-1A protein-rich tumor antigen of claim 1, wherein the lentivirus is selected from the group consisting of pHBLV-HIF1A-3flag-ZsGreen-Puro; the tumor cells are selected from triple negative breast cancer cells.

3. The method of producing a tumor antigen enriched in HIF-1A protein according to claim 1 or 2, comprising the steps of:

(1) Preparing a lentivirus stably expressing HIF-1A protein;

(2) Culturing tumor cells, and transfecting the lentivirus obtained in the step (1);

(3) Selecting the tumor cells transfected with the lentiviruses in the step (2), and screening by utilizing puromycin to obtain a cell strain stably expressing HIF-1A protein;

(4) And (3) performing amplification culture on the stable cell strain, and then performing cell lysis to obtain the HIF-1A protein-containing tumor antigen.

4. The method according to claim 3, wherein the optimal concentration of puromycin in the step (3) is selected by screening the tumor cells without transfection of lentivirus with puromycin at different concentrations to obtain the lowest puromycin concentration at which the cells are completely killed.

5. The method for producing a HIF-1A protein-enriched tumor antigen according to claim 3, wherein the selection in step (3) is carried out using puromycin, wherein the selection is carried out for 14 to 16 days.

6. The method according to claim 3, wherein the step (4) of lysing the cells comprises lysing the lentivirus-transfected tumor cells by repeated freeze-thawing, and centrifuging the suspension to obtain the HIF-1A protein-enriched tumor antigen.

7. A DC vaccine comprising tumor antigens enriched in HIF-1A protein according to claim 1 or 2.

8. The method of preparing the DC vaccine of claim 7, comprising the steps of:

culturing DC cells in vitro by using culture solution containing GM-CSF and IL-4, and co-incubating the tumor antigen rich in HIF-1A protein and the cultured DC cells to obtain the DC vaccine.

9. The method for preparing the DC vaccine according to claim 8, comprising the steps of:

(1) Collecting peripheral blood of a healthy human body, and extracting mononuclear cell PBMC;

(2) Culturing PBMC, culturing adherent cells, and adding culture solution containing GM-CSF and IL-4;

(3) Culturing for a period of time, adding HIF-1A-rich tumor antigen to the cells, and incubating;

(4) Continuously culturing, and adding IL-1 beta, IL-6, TNF-a and PEG-2 into the cells to induce the DC to mature.

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