Human DC-CIK immunocompetent cell and preparing method thereof

Abstract

The invention discloses a human DC-CIK immunocompetent cell. The human DC-CIK immunocompetent cell is characterized by being loaded with the vaccine oncolytic gene virus Oncopox-IL-24, wherein the vaccine oncolytic gene virus Oncopox-IL-24 is obtained through fusion of the vaccine oncolytic virus and the IL-24 gene, the IL-24 gene is inserted into a TK enzyme gene region of the vaccine oncolytic virus, and the human DC-CIK immunocompetent cell is obtained through co-culture of the vaccine oncolytic gene virus Oncopox-IL-24 and a DC-CIK immunocompetent cell. The human DC-CIK immunocompetent cell combines the advantages of the oncolytic virus, cell factors and immune cells to realize an efficient broad-spectrum tumor killing effect through cooperation, the tumor cell killing activity of the human DC-CIK immunocompetent cell is over 160 times higher than that of DC-CIK, inhibition of a neutralizing antibody on tumor cell invasion of the oncolytic virus is avoided during blood circulation, the in-vivo curative effect of the oncolytic virus is improved, the side effect of the oncolytic virus is reduced, and a new direction is provided for developing efficient and safe clinical antitumor immunity cell therapy techniques or drugs.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a human DC-CIK immunocompetent cell and a preparation method thereof. Background technique

[0002] In recent years, the morbidity and mortality of malignant tumors in my country have continued to increase. In the treatment of malignant tumors, the three conventional treatments of surgery, radiotherapy and chemotherapy have played an important role in controlling tumor progression, prolonging the life of patients, and improving the quality of life. Far from perfect.

[0003] Modern immunological studies have proved that the occurrence and development of tumors are related to the immune deficiency of the body. With the approval of the recombinant cytokine IFN-α for the treatment of hairy cell leukemia by the US FDA in the 1980s, a series of biological treatments such as cytokines, monoclonal antibody drugs, peptide protein vaccines, and cellular immunotherapy technologies ...

Examples

Embodiment 1

[0058] Example 1 , Construction of vaccinia oncolytic gene virus Oncopox-IL-24

[0059] 1.1. Construction of pCB-IL-24 plasmid

[0060] Synthesize the gene sequence of IL-24 protein, and add BglII and XbaI enzyme cutting sites at both ends of the IL-24 gene sequence, and use restriction endonucleases BglII and XbaI to double-digest the pCB plasmid (Gu Maozhi, Jiang Fumei, Cai Mingjie , Wu Xiangfu, the construction of recombinant plasmid pCB [J], "Progress in Biochemistry and Biophysics", 1980, 7(4):46-48) and the plasmid containing IL-24 gene, using the gel recovery method to recover the large fragment of pCB and The IL-24 gene fragment was purified, and then the two purified gene fragments were mixed together in proportion, then T4 ligase was added, and ligated overnight at 4°C to construct the pCB-IL-24 plasmid. Then by transforming DH5α competent cells, cultured overnight at 37°C. After picking a single clone, expand the culture and extract the corresponding plasmid. S...

Embodiment 2

[0065] Embodiment 2, Identification of vaccinia oncolytic gene virus Oncopox-IL-24

[0066] Use PCR method to detect whether the homologous recombination vaccinia virus contains the target gene, and also to identify whether the homologous recombination vaccinia virus is contaminated by wild virus, design two primers, one primer takes the upstream and downstream of the IL-24 gene Gene sequence, a primer to get the upstream and midstream gene sequence of TK enzyme gene.

[0067] 1) The primers for identifying the target gene are:

[0068] Upstream primer: 5'-CGCGCGTAATACGACTCACT-3',

[0069] Downstream primer: 5'-GAAGGCATCAGTCGGCTTGCG-3',

[0070] 2) The primers for identifying the wild-type virus are:

[0071] Upstream primer: 5'-TGTGAAGACGATAAATTAATGATC-3',

[0072] Downstream primer: 5'-GTTTGCCATACGCTCACAG-3'.

[0073] 3) The PCR reaction system is:

[0074]

[0075] 4) PCR reaction conditions are:

[0076]

[0077] If the PCR product of the viral plaque contai...

Embodiment 3

[0078] Embodiment 3, Amplification of vaccinia oncolytic gene virus Oncopox-IL-24

[0079] When the 293 cells grow to about 80% of the culture dish, add a certain amount of vaccinia virus, and then continue to put it in 37 ℃, 5% CO 2 cultured in an incubator. 2 to 3 days after the 293 cells were infected with the virus, the 293 cells were collected and repeatedly frozen and thawed between -80°C and 37°C to lyse the cells and release the virus. Finally, centrifuge using density gradient purification method, and collect the supernatant. For details, refer to the cesium chloride gradient centrifugation purification instructions for viruses (Microbix Biosystem Inc).

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a human DC-CIK immunocompetent cell and a preparation method thereof. Background technique

[0002] In recent years, the morbidity and mortality of malignant tumors in my country have continued to increase. In the treatment of malignant tumors, the three conventional treatments of surgery, radiotherapy and chemotherapy have played an important role in controlling tumor progression, prolonging the life of patients, and improving the quality of life. Far from perfect.

[0003] Modern immunological studies have proved that the occurrence and development of tumors are related to the immune deficiency of the body. With the approval of the recombinant cytokine IFN-α for the treatment of hairy cell leukemia by the US FDA in the 1980s, a series of biological treatments such as cytokines, monoclonal antibody drugs, peptide protein vaccines, and cellular immunotherapy technologies ...

Examples

Embodiment 1

[0058] Example 1 , Construction of vaccinia oncolytic gene virus Oncopox-IL-24

[0059] 1.1. Construction of pCB-IL-24 plasmid

[0060] Synthesize the gene sequence of IL-24 protein, and add BglII and XbaI enzyme cutting sites at both ends of the IL-24 gene sequence, and use restriction endonucleases BglII and XbaI to double-digest the pCB plasmid (Gu Maozhi, Jiang Fumei, Cai Mingjie , Wu Xiangfu, the construction of recombinant plasmid pCB [J], "Progress in Biochemistry and Biophysics", 1980, 7(4):46-48) and the plasmid containing IL-24 gene, using the gel recovery method to recover the large fragment of pCB and The IL-24 gene fragment was purified, and then the two purified gene fragments were mixed together in proportion, then T4 ligase was added, and ligated overnight at 4°C to construct the pCB-IL-24 plasmid. Then by transforming DH5α competent cells, cultured overnight at 37°C. After picking a single clone, expand the culture and extract the corresponding plasmid. S...

Embodiment 2

[0065] Embodiment 2, Identification of vaccinia oncolytic gene virus Oncopox-IL-24

[0066] Use PCR method to detect whether the homologous recombination vaccinia virus contains the target gene, and also to identify whether the homologous recombination vaccinia virus is contaminated by wild virus, design two primers, one primer takes the upstream and downstream of the IL-24 gene Gene sequence, a primer to get the upstream and midstream gene sequence of TK enzyme gene.

[0067] 1) The primers for identifying the target gene are:

[0068] Upstream primer: 5'-CGCGCGTAATACGACTCACT-3',

[0069] Downstream primer: 5'-GAAGGCATCAGTCGGCTTGCG-3',

[0070] 2) The primers for identifying the wild-type virus are:

[0071] Upstream primer: 5'-TGTGAAGACGATAAATTAATGATC-3',

[0072] Downstream primer: 5'-GTTTGCCATACGCTCACAG-3'.

[0073] 3) The PCR reaction system is:

[0074]

[0075] 4) PCR reaction conditions are:

[0076]

[0077] If the PCR product of the viral plaque contai...

Embodiment 3

[0078] Embodiment 3, Amplification of vaccinia oncolytic gene virus Oncopox-IL-24

[0079] When the 293 cells grow to about 80% of the culture dish, add a certain amount of vaccinia virus, and then continue to put it in 37 ℃, 5% CO 2 cultured in an incubator. 2 to 3 days after the 293 cells were infected with the virus, the 293 cells were collected and repeatedly frozen and thawed between -80°C and 37°C to lyse the cells and release the virus. Finally, centrifuge using density gradient purification method, and collect the supernatant. For details, refer to the cesium chloride gradient centrifugation purification instructions for viruses (Microbix Biosystem Inc).

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