Preparation method of immune cells for specific modification of inflammations
An immune cell, specific technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, blood/immune system cells, etc.
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[0035] The preparation method of the immune cell specifically modifying inflammation of the present invention comprises the following steps:
[0036] S100, packaging preparation of RET lentivirus: clone the RET gene fragment into a lentiviral expression vector and then package it to obtain a lentivirus containing the RET gene, namely RET-LV;
[0037] S200. Preparation of RET-modified immune cells: Infect and culture the lentivirus containing the RET gene together with human immune cells in a complete medium containing polybrene to obtain RET gene-modified immune cells, namely RET-immune cells .
[0038] The protein encoded by the RET (receptor tyrosine kinase, receptor tyrosine kinase) gene in the above step 100 is a receptor tyrosine kinase. The fragment length of RET gene (NCBI Ref Seq: NM_020975.4) is 3345 base pairs. On July 13, 2016, Veiga-Fernandes stated in the "Nature" that RET protein can regulate the signaling pathway in mucosal inflammation. Mice lacking RET are ...
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[0050] The preparation of peripheral blood mononuclear cells (RET-PBMC) specifically modified inflammation comprises the following steps:
[0051] 1. Packaging preparation of lentivirus (RET-LV) containing RET gene
[0052] 1.1 Amplification of RET gene fragment
[0053] The fragment length of the target gene RET (NCBI Ref Seq: NM_020975.4) is 3345 base pairs. Using the cDNA of large intestine epidermal cells containing the RET gene as a template, PCR amplification was carried out with the designed primers RET-F and RET-R. The PCR reaction system is as follows: DNA template 20ng, 100nM RET-F, 100nM RET-R, Taq polymerase 0.5μl, buffer 5μl, 10mM dNTP, double distilled water to 50μl. The PCR reaction conditions are as follows: 5 minutes at 95 degrees, (30 seconds at 94 degrees, 30 seconds at 57 degrees, 45 seconds at 72 degrees), 2-34 cycles, 10 minutes at 72 degrees, and storage at 4 degrees. The primer sequence is:
[0054] RET-F:5'-ATGGCGAAGGCGACGTCCGGT-3'
[0055] RET-R:5...
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