Fusion protein and composition for treating animal tumors
A fusion protein and composition technology, applied in the field of anti-tumor drugs, can solve problems such as high cost, complicated preparation process, and difficult quality control, and achieve the effects of inhibiting metastasis, simple preparation process, and improving quality of life
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Embodiment 1IL12
[0037] Example 1 Construction of IL12 expressing cells
[0038] The coding region of the canine IL12 gene was synthesized, including two subunits, IL12a (Genbank ID: NM_001003293) and IL12b (Genbank ID: NM_001003292), connected with T2A sequence in the middle, and the two ends of the synthesized gene had BamHI and XhoI restriction sites respectively Spot, then digest with BamHI and XhoI, the system is as follows: 5 μg of IL12 plasmid, 4 μL of digestion buffer, 1 μL of BamHI, 1 μL of XhoI, add water to a total volume of 40 μL, and let stand at 37°C for 12 hours. Take out the EP tube, add 4.4 μL of 10× sample buffer, run electrophoresis on 1% agarose gel, recover the IL12 gene fragment after electrophoresis, and set aside.
[0039] Digest the expression vector pLentis-CMV-MCS-IRES-PURO, and the system is as follows: plasmid 2 μg, enzyme digestion buffer 3 μL, BamHI 1 μL, XhoI 1 μL, add water to a total volume of 30 μL, and stand at 37°C for 12 hours. Take out the EP tube, add...
Embodiment 2
[0043] Example 2 GMCSF expressing cell construction
[0044] Synthesize the coding region of the canine GMCSF (Genbank number: NM_001003245) gene, with BamHI and XhoI restriction sites at both ends of the synthesized gene, and then digest with BamHI and XhoI. The system is as follows: 5 μg of GMCSF plasmid, digestion buffer 4 μL, BamHI 1 μL, XhoI 1 μL, add water to a total volume of 40 μL, and let stand at 37°C for 12 hours. Take out the EP tube, add 4.4 μL of 10× sample buffer, run electrophoresis on 1% agarose gel, recover the GMCSF gene fragment after electrophoresis, and set aside.
[0045] Digest the expression vector pLentis-CMV-MCS-IRES-PURO, and the system is as follows: plasmid 2 μg, enzyme digestion buffer 3 μL, BamHI 1 μL, XhoI 1 μL, add water to a total volume of 30 μL, and stand at 37°C for 12 hours. Take out the EP tube, add 3.3 μL of 10× sample buffer, run electrophoresis on 1% agarose gel, recover the carrier fragment after electrophoresis, and set aside.
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Embodiment 3I
[0049] Example 3 Construction of IL2 expressing cells
[0050] Synthesize the coding region of the canine IL2 (Genbank number: NM_001003305) gene, with BamHI and XhoI restriction sites at both ends of the synthesized gene, and then digest with BamHI and XhoI. The system is as follows: IL2 plasmid 5 μg, digestion buffer 4 μL, BamHI 1 μL, XhoI 1 μL, add water to a total volume of 40 μL, and let stand at 37°C for 12 hours. Take out the EP tube, add 4.4 μL of 10× sample buffer, and perform electrophoresis on 1% agarose gel. After electrophoresis, the IL2 gene fragment is recovered for use.
[0051] Digest the expression vector pLentis-CMV-MCS-IRES-PURO, and the system is as follows: plasmid 2 μg, enzyme digestion buffer 3 μL, BamHI 1 μL, XhoI 1 μL, add water to a total volume of 30 μL, and stand at 37°C for 12 hours. Take out the EP tube, add 3.3 μL of 10× sample buffer, run electrophoresis on 1% agarose gel, recover the carrier fragment after electrophoresis, and set aside.
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