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Combined regulation and control system and application thereof

A technology of joint regulation and regulation system, applied in the field of protein expression, can solve the problems of joint use, no system given, no combination of two regulation systems, etc.

Pending Publication Date: 2020-01-24
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing technology, there is no existing technology that combines the two control systems, and there is no technical enlightenment on whether the two systems can be used in combination, and what effect the combined use of the two systems will bring

Method used

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  • Combined regulation and control system and application thereof
  • Combined regulation and control system and application thereof
  • Combined regulation and control system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Constructing a Plasmodium berghei strain of a combined regulatory system to regulate the reporter gene GFP

[0075] Insert the desired rTRAD gene at the BamHI restriction site of the pl0018 vector, insert TetO7-Pmin-DHFR-GFPm3 and TetO7-Pmin-GFPm3-DHFR at the KpnI site to construct pl0018-TetO7-Pmin-DHFR-GFPm3-3'CAM -rTRAD and pl0018-TetO7-Pmin-GFPm3-DHFR-3'CAM-rTRAD two vectors, the specifically constructed vectors are as follows Figure 1(A)-Figure 1(B) shown.

[0076] The constructed pl0018-TetO7-Pmin-DHFR-GFPm3-3'CAM-rTRAD and pl0018-TetO7-Pmin-GFPm3-DHFR-3'CAM-rTRAD vectors were transformed into Escherichia coli, positive transformants were selected for identification, and the plasmids were linearized After purification and enrichment, Plasmodium berghei P.bANKA was electroporated. To identify the extracted genome of Plasmodium after electroporation, the identification primers are as follows:

[0077] Identification of pl0018-TetO7-Pmin-DHFR-GFPm3-3'CAM-...

Embodiment 2

[0084] Example 2 verifies that the joint regulation system can normally induce the expression of the target protein

[0085] The combined regulatory Plasmodium strains constructed are administered in the following manner:

[0086] TMP administration: TMP 1mg / mL concentration 100mg TMP dissolved in 2mL DMSO, dissolved in 100ml water, changed every three days.

[0087] Dox administration: intraperitoneal administration to mice: Dox (0.1 g / kg, 2 mg for 20 g mice, 200 μL of Doxycycline (Dox) stock solution for intraperitoneal administration), once every 24 hours.

[0088] pl0018-TetO7-Pmin-DHFR-GFPm3-3’CAM-rTRAD is called Tet-On / DDD-GFP for short, and pl0018-TetO7-Pmin-GFPm3-DHFR-3’CAM-rTRAD is called Tet-On / GFP-DDD for short.

[0089] Fluorescent microscope observations were carried out on the worm strains of the non-administration group (blank) and the administration group (TMP / Dox) and the wild-type P.bNAKA worm strain of the control, and the results were as follows: image 3...

Embodiment 3

[0090] Example 3 verifies that the combined control system has the lowest background compared with the Tet-On and DDD control systems alone

[0091] Tet-On / DDD-GFP strains (joint regulation), DDD-GFP strains (single DDD regulation), Tet-On-GFP strains (single Tet-On regulation) and wild-type control P.bANKA strain Compare, carry out fluorescent microscope to detect the fluorescence of target protein GFP to each worm strain after administering respectively and not administering, the result is as follows Figure 4 shown, from Figure 4(A) - Figure 4 (D) The results show that the combined regulation system has the lowest background level, which is consistent with the wild type. Both DDD and Tet-On regulation alone have a higher background level, and the combined regulation system is effective.

[0092] In summary, combining the Tet-On regulatory system and the DDD regulatory system to obtain a combined Tet-On / DDD regulatory system can effectively reduce the background level of T...

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Abstract

The invention relates to the field of protein expression, in particular to a combined regulation and control system and application thereof. The system comprises a Tet-On regulation and control systemand a DDD (DHFR degradation domain) regulation and control system, and the Tet-On regulation and control system and the DDD regulation and control system are subjected to fusion expression on one carrier. The combined regulation and control system can realize a purpose of lowering a background generated when only one regulation and control system is used, and therefore, the combined regulation and control system has a lower background purpose protein expression quantity when the combined regulation and control system does not need to express target protein.

Description

technical field [0001] The present invention relates to the field of protein expression, in particular to a combined regulation system and its application, specifically the construction of the combined regulation system based on Tet-On and DDD regulation systems and its application in expressing target proteins. Background technique [0002] The Tet system is a mature regulatory system established by the tetracycline operon to regulate the expression of foreign genes in eukaryotic cells. It has the characteristics of high efficiency and non-toxicity. The first transactivated Tet-off system was established. Later, by modifying the four amino acids encoded by the tetR protein gene in the Tet-off system, Glu at position 71 was mutated to Lys, and Asp at position 95 was mutated to Asn. , Leu at position 101 is mutated to Ser, and Gly at position 102 is mutated to Asp to obtain a Tet-on system that can be positively induced. When tetracycline or doxycycline is added to the enviro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
CPCC12N15/85C12N2830/003C12N2840/002
Inventor 陶铸梁兴祥李晓芬卢俊南李姣姣赵思婷陈小平秦莉
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI