Lung cancer DNA plasmid vaccine and preparation method thereof

Abstract

The invention discloses a lung cancer DNA plasmid vaccine which comprises a plasmid vector containing a fusion gene MAGE-NY-ESO of four epitopes of lung cancer antigens MAGE-3 and NY-ESO-1. The fusion gene MAGE-NY-ESO has a nucleotide sequence shown as SEQ ID NO.1; the invention also discloses a preparation method of the DNA plasmid vaccine, wherein, a DNA plasmid containing a coding sequence of a target antigen is used as a new generation of therapeutic vaccines. The vaccine induces and generates an immunoreaction with specificity aiming at the relevant coding antigen in the body and effectively restrains the development and transfer of lung cancer. Therefore, the vaccine is the DNA plasmid vaccine with simple preparation, low cost, safety and reliability as well as the effective treatment of lung cancer.

Description

technical field

[0001] The invention relates to a DNA vaccine, in particular to a lung cancer DNA plasmid vaccine and a preparation method thereof. Background technique

[0002] Lung cancer is currently one of the most common malignant tumors in the world. In recent decades, the incidence and mortality of lung cancer have increased significantly, and the upward trend ranks first among various malignant tumors. The therapeutic effect of lung cancer has not improved significantly in the past ten years. The main reason is that the biological characteristics of lung cancer are very complex, the degree of malignancy is high, and most patients are already in the advanced stage when they are diagnosed. The ideal method of cancer treatment should be able to specifically remove cancerous tissue without destroying normal tissue. At present, the three conventional treatments for cancer are surgery, chemotherapy and radiotherapy, which are not specific enough to treat cancer and often ...

Examples

Embodiment 1

[0022] Construction and purification of lung cancer DNA plasmid vaccine: According to the epitopes EVDPIGHLY and FLWGPRALY of lung cancer antigen MAGE-3 recognized by specific CTL and the epitopes SLLMWITQC and QLSLLMWIT of NY-ESO-1, a fusion gene containing the above four epitopes was designed and synthesized , and connect each epitope with flexible tetrapeptide (GPGP) (the size is about 144bp, see the results image 3 ), and subcloned it into the CMV promoter of the pNGVL4a plasmid to obtain the pNGVL-MAGE-NY-ESO plasmid, and inserted the GM-CSF gene fragment of about 432bp obtained by reverse transcription PCR into another part of the above plasmid Under a CMV promoter, constructs such as figure 1 The pNGVL-GM-CSF-MAGE-NY-ESO plasmid shown in the structure; the GM-CSF gene fragment is obtained by conventional methods: first, isolate human peripheral blood leukocytes, extract total RNA, and then design according to the known sequence (Genebank no.AAA52578) Primers, obtained...

Embodiment 2

[0026]Lung cancer DNA plasmid vaccine injection and expression analysis in mammalian cells thereof: the high-purity lung cancer DNA plasmid vaccine pNGVL-GM-CSF-MAGE-NY-ESO obtained in Example 1 and cationic liposome DOSPER (purchased from Roche company ), according to the mass ratio of DNA plasmid vaccine and cationic liposome, it is 10:1 and is dissolved in phosphate buffer solution to prepare DNA plasmid vaccine injection, and the highly purified plasmid in DNA plasmid vaccine injection is wrapped by cationic liposome .

[0027] Transfect the DNA plasmid vaccine injection into mammalian cos-7 cells, and continue culturing in DMEM medium containing 10% fetal bovine serum at 37 degrees. After harvesting the cultured cells at different periods of time, use flow cytometry (BD company) to analyze the expression of GM-CSF and the cytokines such as IL-4, IL-6 and IFN-γ stimulated by GM-CSF, and confirm that the plasmid stimulates the immune response Ability. The calculation resu...

Embodiment 3

[0029] Establishment of lung cancer model mice: According to literature reports, BALB / c mouse lung cancer was induced by diethylnitrosamine (DEN): mice were subcutaneously injected with 1% DEN aqueous solution once a week, each dose was 56mg / kg, and the total dose of DEN reached 1176mg , when the observation time is about half a year, the incidence rate of lung cancer is identified by immunohistochemistry, and the general incidence rate can reach 94%.

technical field

[0001] The invention relates to a DNA vaccine, in particular to a lung cancer DNA plasmid vaccine and a preparation method thereof. Background technique

[0002] Lung cancer is currently one of the most common malignant tumors in the world. In recent decades, the incidence and mortality of lung cancer have increased significantly, and the upward trend ranks first among various malignant tumors. The therapeutic effect of lung cancer has not improved significantly in the past ten years. The main reason is that the biological characteristics of lung cancer are very complex, the degree of malignancy is high, and most patients are already in the advanced stage when they are diagnosed. The ideal method of cancer treatment should be able to specifically remove cancerous tissue without destroying normal tissue. At present, the three conventional treatments for cancer are surgery, chemotherapy and radiotherapy, which are not specific enough to treat cancer and often ...

Examples

Embodiment 1

[0022] Construction and purification of lung cancer DNA plasmid vaccine: According to the epitopes EVDPIGHLY and FLWGPRALY of lung cancer antigen MAGE-3 recognized by specific CTL and the epitopes SLLMWITQC and QLSLLMWIT of NY-ESO-1, a fusion gene containing the above four epitopes was designed and synthesized , and connect each epitope with flexible tetrapeptide (GPGP) (the size is about 144bp, see the results image 3 ), and subcloned it into the CMV promoter of the pNGVL4a plasmid to obtain the pNGVL-MAGE-NY-ESO plasmid, and inserted the GM-CSF gene fragment of about 432bp obtained by reverse transcription PCR into another part of the above plasmid Under a CMV promoter, constructs such as figure 1 The pNGVL-GM-CSF-MAGE-NY-ESO plasmid shown in the structure; the GM-CSF gene fragment is obtained by conventional methods: first, isolate human peripheral blood leukocytes, extract total RNA, and then design according to the known sequence (Genebank no.AAA52578) Primers, obtained...

Embodiment 2

[0026]Lung cancer DNA plasmid vaccine injection and expression analysis in mammalian cells thereof: the high-purity lung cancer DNA plasmid vaccine pNGVL-GM-CSF-MAGE-NY-ESO obtained in Example 1 and cationic liposome DOSPER (purchased from Roche company ), according to the mass ratio of DNA plasmid vaccine and cationic liposome, it is 10:1 and is dissolved in phosphate buffer solution to prepare DNA plasmid vaccine injection, and the highly purified plasmid in DNA plasmid vaccine injection is wrapped by cationic liposome .

[0027] Transfect the DNA plasmid vaccine injection into mammalian cos-7 cells, and continue culturing in DMEM medium containing 10% fetal bovine serum at 37 degrees. After harvesting the cultured cells at different periods of time, use flow cytometry (BD company) to analyze the expression of GM-CSF and the cytokines such as IL-4, IL-6 and IFN-γ stimulated by GM-CSF, and confirm that the plasmid stimulates the immune response Ability. The calculation resu...

Embodiment 3

[0029] Establishment of lung cancer model mice: According to literature reports, BALB / c mouse lung cancer was induced by diethylnitrosamine (DEN): mice were subcutaneously injected with 1% DEN aqueous solution once a week, each dose was 56mg / kg, and the total dose of DEN reached 1176mg , when the observation time is about half a year, the incidence rate of lung cancer is identified by immunohistochemistry, and the general incidence rate can reach 94%.

Images