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Detoxifying function-enhancing gene fragment and modified HepG2 cell

A technology of detoxification function and gene fragments, which is applied in the field of genetic engineering to achieve the effect of improving detoxification ability

Active Publication Date: 2020-10-16
WUHAN TOGO MEDITECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the defects of existing HepG2 cells, and provide a HepG2 cell that enhances detoxification function gene fragments and transformation

Method used

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  • Detoxifying function-enhancing gene fragment and modified HepG2 cell
  • Detoxifying function-enhancing gene fragment and modified HepG2 cell
  • Detoxifying function-enhancing gene fragment and modified HepG2 cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0031] Embodiment: the scheme of HepG2 cell line transformation:

[0032] 1) The entire genome was assembled into pTBSCH2-OTC-ARG1-OATP1B1-Bst, and linearized. The sequence is shown in SEQNo.2.

[0033] 2) Digest HepG2 cells (ATCC, American Type Culture Collection Center) that night, and its sequence can be found in (NCBIGenBank), and plant them in T25 at a density of 40%-50%. After overnight attachment, the next morning Perform transfection.

[0034] 3) Transfection reagents and materials

[0035]

[0036] (plasmid:transposon=10:1)

[0037] Mix well, let it stand for 5min, add it to T25, and change the medium according to the state of the cells.

[0038] 4) Five days after the cells were transfected, they were digested and counted, and about 1000-2000 cells per well were seeded into a 96-well plate.

[0039] 5) After the cells have grown to a certain number, continue to seed the plate for screening. After the preliminary detection of negative cells (HepG2), screening ...

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Abstract

The invention discloses a detoxifying function-enhancing gene fragment and a modified HepG2 cell; the detoxifying function-enhancing gene fragment is composed of three gene fragments of OTC, ARG1 andOATP1B1 in sequence. The albumin expression of the HepG2 cell modified by the gene fragment is doubled; the expression of glutamic oxalacetic transaminase is improved by 10%; the secretion of key protein related to endogenous detoxifying capacity in the cytochrome P450 family is increased, and the detoxifying capacity is improved: CYP2C9 secretion is improved by 5 times, CYP2D6 secretion is improved by 50 times, CYP2A6 secretion is improved by 2 times, and CYP3A4 secretion is improved by 100 times.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a gene fragment for enhancing detoxification function and transformed HepG2 cells. Background technique [0002] HepG2 cells are a type of liver cancer cells. The cells secrete a variety of plasma proteins: albumin, α2-macroglobulin, plasminogen, transferrin, etc. [0003] Existing HepG2 cells have blood ammonia metabolism and detoxification defects when they are used for blood purification. Contents of the invention [0004] The purpose of the present invention is to overcome the defects of the existing HepG2 cells, and provide a gene segment for enhancing detoxification function and the transformed HepG2 cells. [0005] In order to achieve the above purpose, the present invention firstly provides a gene segment for enhancing detoxification function, which is composed of three gene segments of OTC, ARG1, and OATP1B1 in sequence, wherein the secreted proteins corresponding ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/85C12N15/65C12N5/10C12R1/91
CPCC12N9/1018C12N9/78C12N15/85C12N15/65C07K14/47C12Y201/03003C12Y305/03001C07K2319/00
Inventor 王柯丰明乾望威
Owner WUHAN TOGO MEDITECH CO LTD
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