Recombinant lentiviral expression vector, reconstitution cell and application of reconstitution cell to culturing natural killer cell

A technology of recombinant lentivirus and lentiviral vector, which is applied in the field of cell engineering, can solve the problems of inability to effectively stimulate and improve NK cell tumor killing, limit cell proliferation trend and killing ability, and low level of cell activation, so as to avoid NK cell apoptosis , increase the expression level, and prolong the effect of proliferation time

Pending Publication Date: 2019-02-22
BEIJING DCTY BIOTECH CO LTD
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Problems solved by technology

[0003] The Chinese invention patent with the application number CN201710096536.1 discloses a method of efficiently expanding NK cells using K562 engineered cells with high expression of membrane proteins CD19, CD137L, CD86, CD64 and transmembrane protein IL-21 combined with human IL-2 mutants method, although this method solves the problem of NK cells specifically recognizing CD19+ cells, it cannot effectiv

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  • Recombinant lentiviral expression vector, reconstitution cell and application of reconstitution cell to culturing natural killer cell
  • Recombinant lentiviral expression vector, reconstitution cell and application of reconstitution cell to culturing natural killer cell
  • Recombinant lentiviral expression vector, reconstitution cell and application of reconstitution cell to culturing natural killer cell

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Embodiment 1

[0038] 1. MLG (Membrane Ligand GrowthBooster) trophoblast construction:

[0039] The human leukemia cell line K562 (American Type Culture Collection; Rockville, MD) was cultured in RPMI-1640 with 10% FBS. Viral packaging was performed by co-constructing "membrane-anchored" IL21 (IL-21 fused with CD8), 41BBL, and MICA in a lentiviral vector. The virus packaging process includes: transiently transfecting pLenti-MLG lentiviral backbone plasmid and lentiviral packaging helper plasmid with liposome or polymer transfection reagent (such as PEI 25K, Polyscience; Transdux, SBI, etc.) by suspending 293T expression system, The cells then express the lentivirus and secrete it in the cell culture supernatant. Concentrate and harvest the virus supernatant to obtain high-titer virus, then add Transdux (SBI) according to the concentration instructed by the supplier, and infect K562 cells to construct K562-mbl21-41BBL-MICA trophoblasts. The vector is resistant, and high-expression cells are...

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Abstract

The invention provides a recombinant lentiviral expression vector, and belongs to the technical field of cell engineering. The recombinant lentiviral expression vector comprises a 21mb gene, a 41BBL gene and an MICA gene which are sequentially connected in series; and the 21mb gene is obtained by sequentially connecting a signal peptide gene, an IL21 gene, a CD8 Hinge gene and a CD8 transmembranegene in series. After the provided recombinant lentiviral expression vector transfects a host cell, the host cell expresses the MICA and the 41BBL, and the IL21 is fused and expressed (but not secreted in a culture environment) on a cytomembrane of the host cell. After the provided recombinant lentiviral expression vector transfects the host cell, an obtained reconstitution cell promotes the highexpression of the NKG2D, avoids excessive activation of an NK cell and prolongs the proliferation time of the NK cell in the process of induction culture of the NK cell.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a recombinant lentiviral expression vector, recombinant cells and their application in natural killer cell culture. Background technique [0002] Natural killer cells (NK) are important immune cells in the body, not only related to anti-tumor, anti-viral infection and immune regulation, but also involved in the occurrence of hypersensitivity and autoimmune diseases in some cases, and can recognize Target cells, killing mediators. The exact source of natural killer cells is not very clear, generally believed to be derived directly from the bone marrow. In view of the wide application of natural killer cells, providing an efficient and stable method for inducing natural killer cells has broad application prospects. [0003] The Chinese invention patent with the application number CN201710096536.1 discloses a method of efficiently expanding NK cells using K562...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12N5/0783
CPCC12N5/0646C12N5/0694C12N15/86C12N2501/2302C12N2501/998C12N2510/00C12N2740/15043
Inventor 焦顺昌张嵘袁翰
Owner BEIJING DCTY BIOTECH CO LTD
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