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Novel Synnotch Synthetic Receptor and Its Encoding Gene

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

Active Publication Date: 2021-12-28
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 Receptor and Its Encoding Gene
  • Novel Synnotch Synthetic Receptor and Its Encoding Gene
  • Novel Synnotch Synthetic Receptor and Its Encoding Gene

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. In the present invention, the antiCD19 single-chain antibody is used as the extracellular domain part, GAL4VP64 is used as the intracellular domain part, and the Notch transmembrane core domain of the mouse is replaced with the zebrafish Notch transmembrane core domain that can be recognized and cut by protease, so as 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 ...

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 receptor and its coding gene. The SynNotch synthetic receptor comprises anti-CD19scFV, zebrafish Notch core fragment and GAL-VP64 fragment, its amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence of the corresponding coding gene is shown in SEQ ID NO.4 . The modified synNotch receptor system of the present invention can more efficiently activate the expression of downstream genes. Compared with the original synNotch receptor system, the average fluorescence intensity of regulating the expression of Mcherry fluorescent protein can be improved by 4.9 times. The modified synNotch receptor of the invention can expand the application range of the synNotch, provides a new application prospect for cell transformation, and has potentially important economic value and significance.

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 Patents(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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