Application of cGAMP and derivatives thereof to preparation of anti-tumor vaccines
A derivative and anti-tumor technology, applied in the field of preparation of anti-tumor vaccines, can solve problems such as inability to effectively induce anti-tumor immune responses, low immunogenicity, and lack of immune cofactors
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Embodiment 1
[0015] Example 1: Preparation of GS-CSF tumor cell vaccine
[0016] The mouse cell colony factor GM-CSF gene (gene ID: 12981) was synthesized from Nanjing GenScript Biotechnology Co., Ltd. pLenti and packaged viral vectors were purchased from Life Technology. Colorectal cancer cells MC38, melanoma cells B16, prostate cancer TRAMP-C2 cells, Hepa 1-6 liver cancer cells, Lewis lung cancer cells, and MDA-MB-231 breast cancer cells were purchased from ATCC in the United States. The GM-CSF gene was cloned into the pLenti vector, and co-transfected with the packaging plasmid into 293e cells, and the supernatant containing the packaging virus was collected. For the method of preparing GM-CSF lentivirus, refer to the Life Technology lentivirus preparation manual. The prepared lentiviruses were used to infect colorectal cancer cells MC38, melanoma cells B16, prostate cancer TRAMP-C2 cells, Hepa 1-6 liver cancer cells, Lewis lung cancer cells, MDA-MB-231 breast cancer cells, and add res...
Embodiment 2
[0017] Embodiment 2: cGAMP preparation
[0018] cGAMP (cyclization-GMP-AMP) synthetase cGAS and cGAMP are catalyzed and synthesized by cyclization cGMP-AMP dinucleotide synthase (cGAS) under activation conditions after binding to DNA according to literature method. The purity is above 98%. (Ping wei Li, et al., Immunity, 2013, 39(6), 1019-1031.) Adenosine (guanosine) 5'-α thio(selenium) phosphorotriphosphate was synthesized according to literature method (Caton-Williams Julianne et al., Science China, Chemistry, 2012, 55(1), 80-89; Boyle Nicholas A., Nucleosides, Nucleotides and Nucleic Acids, 2005, 24, 1651-1664.). According to the method for preparing cGAMP, the prepared recombinant cGAS enzyme is used to catalyze the preparation of sulfur (seleno)phosphoryl cyclic dinucleotide cGAMP.
Embodiment 3
[0019] Example Three: Establishment of Mouse Tumor Model and Vaccine Injection
[0020] 1. Establishment of tumor-bearing mouse model
[0021] C57BL / 6 female mice, 6-8 weeks old, were purchased from Shanghai Slack Experimental Animal Co., Ltd. The tumor cells used in tumor model mice are tumor cells that do not express GM-CSF, including: colorectal cancer cell MC38, melanoma cell B16, prostate cancer cell TRAMP-C2, liver cancer cell Hepa 1-6, lung cancer cell Lewis, breast cancer cell Cell MDA-MB-231. Cell culture and passage were carried out according to the corresponding cell culture methods, and were used in the establishment of tumor mouse models in the logarithmic phase. Collect the cells in the logarithmic phase of the mouse tumor cells, make a cell suspension with a concentration of (1-5×10e6) / ml per milliliter, inject 0.2 ml of the cell suspension into the axilla of the right forelimb of the mouse (the number of cells is 2-10 ×10e5 per mouse), the tumor grew to a di...
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