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Recombinant multi-stimulus molecular cell, preparation method and application in NKT cell amplification thereof

A NKT cell, multi-stimulatory technology, applied in recombinant DNA technology, animal cells, vertebrate cells, etc., can solve the problems of high cost, poor repeatability, complex preparation process, etc., and achieve the effect of low cost, high efficiency and easy preparation

Active Publication Date: 2014-07-23
COBAXER BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] The purpose of the present invention is to provide a recombinant multi-stimulatory molecular cell and its preparation method, which solves the problems of complex preparation process, high cost and poor repeatability of recombinant multi-stimulatory molecular cell in the prior art

Method used

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  • Recombinant multi-stimulus molecular cell, preparation method and application in NKT cell amplification thereof
  • Recombinant multi-stimulus molecular cell, preparation method and application in NKT cell amplification thereof
  • Recombinant multi-stimulus molecular cell, preparation method and application in NKT cell amplification thereof

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Embodiment Construction

[0052] The present invention will be described in detail below in conjunction with the examples, but they are not further limiting the present invention.

[0053] 1. Preparation of ME64 cells: Obtain CD64 cDNA, perform PCR amplification on CD64 cDNA, obtain the PCR amplification product of CD64 cDNA, then construct prokaryotic expression vector pGEMT-CD64, and further construct its eukaryotic expression vector pcDNA3.1(+) -CD64, the recombinant plasmid pcDNA3.1(+)-CD64 was transfected into MEG-01 cells, G418 screening, and FACS sorting to obtain ME64 cells; specifically:

[0054] (1) Acquisition of CD64 cDNA and amplification of target fragments: Take U937 cells in logarithmic phase, routinely extract RNA with Trizol, use Oligod(T)n as a primer, reverse transcribe mRNA to generate cDNA; and then amplify cDNA by PCR , to obtain the PCR amplification product of CD64cDNA, the fragment size is about 1000pb, roughly consistent with the experimental design of 949 bp; as figure 1 As...

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Abstract

The invention discloses a recombinant multi-stimulus molecular cell ME64 / CD137L / mIL-18, a preparation method and application in NKT (natural killer T) cell in-vitro amplification thereof. The preparation method comprises: taking an MEG-01 cell as a carrier, transfecting a CD64 eukaryotic expression vector, and conducting screening and sorting to obtain an ME64 cell; then preparing an eukaryotic expression vector expressing both CD137L and mIL-18; and transfecting the ME64 cell with the vector, thus obtaining the ME64 / CD137L / mIL-18 cell. Being easy for large-scale preparation, the cell has the advantages of low cost and good repeatability, and can stably express foreign protein. The cell can stimulate mass amplification of the NKT cell when it is used for NKT cell in-vitro amplification, and can increase proliferation frequency of the NKT cell and tumor cell killing toxicity.

Description

technical field [0001] The invention belongs to the field of biotechnology and disease immunotherapy, and specifically relates to a recombinant multi-stimulatory molecular cell, a preparation method thereof and an application in NKT cell expansion in vitro. Background technique [0002] The principle of preparation of recombinant multi-stimulatory molecule cells is to simulate the dual signaling mechanism of T cell activation. It is constructed by displaying antigen peptide-MHC molecular complexes and co-stimulatory molecules, adhesion molecule ligands or antibodies on the surface of a certain carrier. It can effectively Activate and expand antigen-specific T cells with high efficiency and good feasibility. [0003] At present, the carriers used to prepare recombinant multi-stimulatory molecular cells mainly include magnetic beads, cells and liposomes. Although the source of recombinant multi-stimulatory molecular cells is constructed using magnetic beads as a carrier, the s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N5/0783
Inventor 申重阳朱姣莲陈洁
Owner COBAXER BIOTECH
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