Chimeric receptor targeting human membrane-bound and soluble NKG2D ligands, nucleic acid molecule, immune effector cell and application thereof
A technology of immune effector cells and chimeric receptors, applied in the field of biomedicine or bioengineering, can solve the problems of poor efficacy and disadvantages of CAR therapy
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Embodiment 1
[0063] Rational design and construction of chimeric receptors targeting human membrane-bound and soluble NKG2D ligands in this example:
[0064] The NKG2D-CAR fusion gene fragment was designed according to the following coding sequence: extracellular signal peptide, Flag tag, NKG2D ligand-binding domain based on NKG2D molecule, hinge region, transmembrane region, intracellular co-stimulatory signal domain and intracellular Signal transduction domain. figure 1 It is a structural schematic diagram and a vector schematic diagram of the chimeric receptor (NKG2D-CAR) targeting human membrane-bound and soluble NKG2D ligands constructed in Example 1 of the present invention. figure 1 a is a schematic diagram of the structure of NKG2D-CAR in the present invention, wherein the signal peptide part is preferably CD8α molecule, the transmembrane domain part is preferably CD28 molecule, and the signal transduction domain is CD28, CD137 and CD3ζ signal molecules as examples. figure 1 b ...
Embodiment 2
[0069] In this example, the synthesis of nucleic acid molecules encoding chimeric receptors targeting human membrane-bound and soluble NKG2D ligands and the construction of viral vectors (ie, the synthesis of NKG2D-CAR fusion genes and the construction of expression vectors):
[0070] Firstly, the nucleotide sequence (SEQ ID NO: 16), Nanjing GenScript Biotechnology Co., Ltd. provides gene synthesis technical services.
[0071] Cloning the synthetic fusion gene fragment into the pCDH-CMV-MCS-P2A-copGFP-T2A-Puro lentiviral vector, such as figure 1 shown. Restriction endonucleases Xba I and EcoR I were used to digest the lentiviral vector and the gene fragment (see Table 1), respectively, to obtain the linearized lentiviral vector and the NKG2D-CAR gene fragment after digestion, and T4DNA Ligase system, incubated at 16°C for 3h (see Table 2). Then transform Escherichia coli Stbl3 competent cells and smear the medium plate containing ampicillin, pick multiple clone colonies fo...
Embodiment 3
[0078] Packaging, concentration and titer determination of viral vector (ie NKG2D-CAR lentiviral expression vector) in this example:
[0079] (1) Packaging of NKG2D-CAR lentiviral expression vector
[0080] Take 8×10 6 HEK293T cells (purchased from ATCC, product number CRL-1573) were inoculated in 175cm 2 In a cell culture flask, culture overnight at 37°C. When the cell density reaches 75%-85%, replace 25 mL of fresh penicillin- and streptoxan containing 6% FBS (BI, product number 04-001-1ACS) 1 hour in advance. Mycin-based DMEM medium (Hyclone, product number SH30022.01), continued to be placed in the incubator for cultivation, and virus packaging was carried out after 1 hour.
[0081]Use calcium phosphate kit (Beiyuntian, article number is C0508) for packaging, packaging steps: the recombinant NKG2D-CAR expression plasmid (pCDH-NKG2D-CAR) extracted in Example 2, and helper plasmids pSPAX2, pMD2.G according to 4 : 3:1 ratio mix, add to CaCl 2 solution (provided by the kit...
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