Novel SynNotch synthetic acceptor and coding gene thereof

A technology for encoding genes and receptors, which is applied in the field of new SynNotch synthetic receptors and its encoding genes, can solve the problems of limiting the use of SynNotch, and achieve the effects of efficient activation, important economic value and significance

Active Publication Date: 2019-01-11
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This will limit the use of SynNotch, especially when it is required to have a high expression level of downstream genes or factors

Method used

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  • Novel SynNotch synthetic acceptor and coding gene thereof
  • Novel SynNotch synthetic acceptor and coding gene thereof
  • Novel SynNotch synthetic acceptor and coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 SynNotch synthetic receptor design containing the zebrafish Notch core domain

[0025] The structure of the synNotch receptor includes an extracellular domain, a Notch transmembrane core domain and an intracellular domain, wherein the extracellular domain can be replaced by a single-chain antibody or a nanobody, and the intracellular domain can be replaced by a transcriptional activation or transcriptional repression domain. The present invention uses antiCD19 single-chain antibody as the extracellular domain part, GAL4VP64 as the intracellular domain part, and replaces the mouse Notch transmembrane core domain with the zebrafish Notch transmembrane core domain that can be recognized and cleaved by protease to obtain the modified synNotch receptor , the structure of the synNotch receptor designed and transformed by the present invention is as follows figure 2 shown. figure 2 Structures of synNotch receptors containing murine or zebrafish Notch transmembrane...

Embodiment 2

[0026] Example 2 Construction of expression plasmid vector containing zebrafish Notch transmembrane core domain

[0027] The flow chart of the construction of the expression plasmid vector (pHR-PGK-antiCD19-zebra synNotch-Gal4VP64) containing the core domain of zebrafish Notch is shown in image 3 As shown, it specifically includes the following steps:

[0028] (1) Use Trizol kit to extract zebrafish embryo RNA, reverse it into cDNA, and use PCR primer pair (upstream primer F: 5'-GGCTGCTCCTCCAAGCCATG-3' and downstream primer R: 5'-ACGCTTGCGGCGGGCAATCA-3') to expand the zebrafish Fish Notch transmembrane core domain, the nucleotide sequence of the zebrafish Notch transmembrane core domain is shown in SEQ ID NO.1, and the corresponding encoded amino acid sequence is shown in SEQ ID NO.2;

[0029](2) Reconstruct the pHR-PGK-antiCD19-oligo (containing BamH I / Xho I)-Gal4VP64 plasmid (obtained from pHR-PGK-antiCD19-synNotch-Gal4VP64 (addgene #79125), specifically: cutting the core ...

Embodiment 3

[0031] Example 3 The synNotch system containing the zebrafish Notch transmembrane core domain was tested and verified on cells

[0032] The synNotch system containing the zebrafish Notch transmembrane core domain (pHR-PGK-antiCD19-zebrasynNotch-Gal4VP64) such as figure 1 shown. figure 1 It shows that the specific process of the synNotch system to improve cell recognition of specific extracellular signals is: when the anti-CD19 antibody single chain of the extracellular domain of the synNotch receptor binds to the CD19 antigen of the transmission cell (sendercell), the Notch transmembrane core domain is cleaved by protease , GAL4VP64 is released into the cell, GAL4 can specifically recognize and bind to the GAL4 binding site, and the VP64 transcriptional activation element will activate the transcription of the downstream mcherry gene and promote the expression of Mcherry fluorescent protein.

[0033] In the present invention, K562 cells are used as transport cells, U2OS cells...

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Abstract

The invention discloses a novel SynNotch synthetic acceptor and a coding gene thereof. The novel SynNotch synthetic acceptor contains anti-CD19scFV, a zebra fish Notch core segment and a GAL-VP64 segment, the amino acid sequence of the novel SynNotch synthetic acceptor is represented by SEQ ID NO.5, and the corresponding nucleotide sequence of the coding gene is represented by SEQ ID NO.4. According to a SynNotch system modified by virtue of the novel SynNotch synthetic acceptor, a downstream gene can be relatively efficiently expressed. Compared with an original SynNotch acceptor system, theaverage fluorescence intensity for regulating and controlling the expression of a Mcherry fluorescent protein of the modified SynNotch system can be increased by 4.9 times. According to the modified SynNotch acceptor, the application range of SynNotch can be expanded, a novel application prospect is provided for the cell modification, and the novel SynNotch synthetic acceptor has potential important economic values and significances.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel SynNotch synthetic receptor and its coding gene. Background technique [0002] With the rise of synthetic biology and cell modification, one of the basic goals pursued by the academic community is to enable cells to recognize specific extracellular signals and make specific responses. In the self-defined cell recognition response system, cells can respond accordingly after recognizing specific disease or injury signals in the body, so as to be treated or repaired. A successful example is that CAR-T therapy has been approved for clinical use in the treatment of blood tumors. There are many receptors on the surface of cells in the body. How to transmit extracellular signals into cells is one of the research hotspots for scientists to transform cells. Using different receptor types, the researchers designed two types of receptor systems, the receptor system that can recognize ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C12N15/12
CPCC07K14/705
Inventor 荣知立林瑛黄洪新杨鸿程
Owner SOUTHERN MEDICAL UNIVERSITY
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