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Protocatechuic acid regulated on-off system as well as regulation method and application thereof

A technology of protocatechuic acid and switch system, which is applied in the field of switch system regulated by protocatechuic acid and its regulation and application, and can solve the problems of poor curative effect and increased pain of patients

Active Publication Date: 2019-03-12
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main treatment methods mainly include surgical treatment such as gastric reconstruction, drug treatment such as insulin injection, oral hypoglycemic drugs, exercise treatment and diet control treatment, etc., but these treatment methods have problems such as poor efficacy and increased patient pain, so there is an urgent need Develop some new methods to improve the dilemma faced by diabetes treatment

Method used

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  • Protocatechuic acid regulated on-off system as well as regulation method and application thereof
  • Protocatechuic acid regulated on-off system as well as regulation method and application thereof
  • Protocatechuic acid regulated on-off system as well as regulation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1, construction of a switch system regulated by protocatechuic acid

[0078] This example includes the construction method of the plasmid vector involved in the switch system regulated by protocatechuic acid, but does not limit the protection scope of the present invention. See attached table 1 for the detailed design plan and steps.

Embodiment 2

[0079] Example 2, the optimization study of the "on" system regulated by protocatechuic acid in HEK-293T cells, that is, using P SV40 The expression vectors that initiate the expression of KRAB-PcaV were optimized in combination with 10 different response elements.

[0080] The first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.

[0081] The second step is cell seeding. The day before transfection, HEK-293T cells were seeded in a 24-well plate at a volume of 5×10^4 cells per well, and 500 μl of DMEM medium containing 10% FBS was added to each well.

[0082] The third step is plasmid transfection. The transfection system in this example can be divided into 10 groups, including pJY18 and pJY1, pJY18 and pJY2, pJY18 and pJY3, pJY18 and pJY4, pJY18 and pJY5, pJY18 and pJY13, pJY18 and pJY14, pJY18 and pJY15, pJY18 and pJY16, pJY18 and pJY17. The total amount of each group of plasmids above was 200ng, premixed with the transfectio...

Embodiment 3

[0086] Example 3, the optimization study of the "on" system regulated by protocatechuic acid in HEK-293T cells, that is, using P hEF1α The expression vectors that initiate the expression of KRAB-PcaV were optimized in combination with 10 different response elements.

[0087] The first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.

[0088] The second step is cell inoculation (the specific steps are the same as in Example 2).

[0089] The third step is plasmid transfection. The transfection system in this example can be divided into 10 groups, including pJY29 and pJY1, pJY29 and pJY2, pJY29 and pJY3, pJY29 and pJY4, pJY29 and pJY5, pJY29 and pJY13, pJY29 and pJY14, pJY29 and pJY15, pJY29 and pJY16, pJY29 and pJY17. The total amount of each group of plasmids above was 200ng, premixed with the transfection reagent PEI (mass ratio of plasmid to PEI: 1:3) at a ratio of 1:1, and dissolved in 50ul serum-free and antibiotic-free DMEM. ...

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Abstract

The invention discloses a multifunctional gene expression platform regulated by green tea metabolite protocatechuic acid. The platform relates to an on-off system regulated by protocatechuic acid, i.e. the expression is initiated or closed by the protocatechuic acid regulation gene. The on-off system comprises a protocatechuic acid regulated 'on' system and a protocatechuic acid regulated 'off' system. The invention also provides a eukaryotic expression vector comprising the on-off system, a mammalian cell line and a microcapsule. The invention also discloses an application of the multifunctional platform capable of the accurate expression release of controllable insulin or glucagon-like peptide in treating diabetes; and the gene expression regulation platform can be used for establishinga complicated biological computer as well as regulating the gene edition and gene expression activation or inhibition mediated by a CRISPR / Cas9 or CRISPR / dCas9 system. The multifunctional gene expression platform provides a useful novel gene expression regulation tool for the gene treatment and cell treatment.

Description

technical field [0001] The invention relates to the field of synthetic biology, gene and cell therapy, and combines mammalian synthetic biology with disease treatment, biological computer and gene editing technology, and specifically relates to a multifunctional gene expression platform regulated by green tea metabolite protocatechuic acid and Applied research on diabetes treatment, biocomputer, and regulation of gene editing and gene transcription mediated by the CRISPR / Cas9 system. Background technique [0002] As an emerging research field, synthetic biology is committed to the purposeful design, transformation and assembly of basic biological components that exist in nature, such as nucleic acids, peptides, proteins, etc., through artificial means to form biological modules with specific functions. , so as to realize the functional transformation of cells and organisms. In the past few decades, synthetic biology has experienced a rapid development stage, from the activi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90A61K48/00A61P3/10
CPCA61K48/005C12N15/85C12N15/907C12N2830/005C12N2830/002C12N2800/107Y02A50/30
Inventor 叶海峰尹剑丽杨林凤董凯丽薛帅
Owner EAST CHINA NORMAL UNIV
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