Mesothelin high-expression cancer therapeutic vaccine based on recombinant attenuated Listeria ivanovii
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A technology for the treatment of Listeria and cancer, applied in the biological field, can solve problems such as defects and low immunogenicity, and achieve good tolerance
Inactive Publication Date: 2018-12-14
南京颂悦生物科技有限公司
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Problems solved by technology
The key to its success is to induce a sufficient immune response, but the current nucleic acid and protein peptide vaccines have low immunogenicity and obvious defects
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Embodiment 1
[0063] Example 1. Preparation of vaccine bacteria LiΔactAplcB-Meso
[0064] (1) Synthesis of Mesothelin Gene Fragment
[0065] Query the mesothelin protein gene from NCBI, then optimize it according to the codons of Listeria, add a HindⅢ restriction site upstream, add a XhoⅠ restriction site downstream, and finally obtain the DNA sequence directly by synthesis (provided by the company The cloning plasmid pUC57-Meso was obtained), (the sequence is shown in Sequence 1 of the Sequence Listing).
[0066] (2) Construction of targeting plasmid
[0067] ① Construction of intermediate plasmid pCW203-Meso
[0068] Plasmids pUC57-Meso and pCW203 were extracted according to the instructions of the plasmids, and eluted with 30 μL Elution Buffer.
[0069] pUC57-Meso and pCW203 were double-digested with HindⅢ and XhoⅠ, 20 μL of enzyme digestion system: pUC57-Meso or pCW2032 O Make up the system to 20 μL. Digest the plasmid in a water bath at 37°C for 1 hour, add 0.5 μL CIAP to the vector ...
Embodiment 2
[0106] Example 2: Evaluation of the therapeutic effect of vaccine strains on tumors with high expression of mesothelin
[0107] (1) CT26-MESO tumor cell culture and tumor model establishment
[0108] Use RPMI1640 Medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin at 37°C in 5% CO 2 Cultivate CT26-MESO cells in an incubator until the density reaches about 80%, rinse the cells with Hank’s solution for 2 to 3 times, digest with trypsin and resuspend with PBS for counting, and prepare the cells to a concentration of 5×10 6 / ml of cell suspension, 6-8 week-old BALB / c female mice were injected with 100 μl of cell suspension into the tail vein to establish a mouse tumor model and observe the survival rate.
[0109] (2) Immunotherapy of model mice
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Abstract
The invention relates to a mesothelin high-expression cancer therapeutic vaccine based on recombinant attenuated Listeria ivanovii. Attenuated Listeria ivanovii (called Li for short) is used as a vector, and the unique ability of Listeria to grow in a host phagocytic cell is the characteristic of a natural T cell immune activation adjuvant, and is used to effectively improve the specific immune response in a tumor microenvironment and break the immune tolerance of bodies, so persistent viral infection of the bodies is removed, and good therapeutic effects of clearing focus and restraining tumor development are achieved. The cervical cancer therapeutic vaccine makes up for the shortcoming of low immunogenicity of nucleic acid and protein peptide vaccines, and the attenuated Listeria reserves the intracellular growth and antigen presentation completion characteristics of an original strain, and has a high safety.
Description
technical field [0001] The invention belongs to the field of biotechnology, and relates to a treatment for cancer with high mesothelin expression based on recombinant attenuated Listeria ovis. [0002] therapeutic vaccines. Background technique [0003] Studies have found that mesothelin is a cell surface glycoprotein whose expression is usually limited to the mesothelium (peritoneum, pericardium, and pleura), and found that in mesothelioma, pancreatic cancer, lung cancer, and ovarian cancer, mesothelin protein is significantly High expression. (MesothelinA New Target for Immunotherapy, Raffit Hassan, Tapan Bera, and Ira Pastan doi: 10.1158 / 1078-0432. CCR-03-0801 Clin Cancer Res June 15, 2004 10; 3937) This feature makes mesothelin an attractive target for the use of recombinant Lys. A tool antigen for the treatment of tumors by special bacteria. [0004] Mesothelioma is a tumor that arises primarily on the lining of the chest or abdominal cavity and is associated with ex...
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