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Thiostrepton-gentamicin resistance gene system as well as resistance expression box and recombinant plasmid containing same

A technology of thiostrepton and resistance gene, applied in the field of genetic engineering, can solve the problems of restriction, low solubility, high toxicity, etc., and achieve the effect of avoiding polar effect

Inactive Publication Date: 2017-05-10
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

THS, a thiazole antibiotic, was first isolated and identified from Streptomyces azureus in 1954. It is mainly used to treat mastitis in animals and also to treat some diseases in dogs. However, its Uses are limited due to low solubility and high toxicity

Method used

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  • Thiostrepton-gentamicin resistance gene system as well as resistance expression box and recombinant plasmid containing same
  • Thiostrepton-gentamicin resistance gene system as well as resistance expression box and recombinant plasmid containing same
  • Thiostrepton-gentamicin resistance gene system as well as resistance expression box and recombinant plasmid containing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of plasmid p60GTE

[0037] The schematic diagram of the structure of plasmid p60GTE is shown in figure 1 , see restriction site map figure 2 , which contains the above-mentioned tsr-aacC1 sex gene. Depend on figure 1 It can be seen that the plasmid p60GTE contains in a clockwise direction: Escherichia coli origin of replication (ori E), tsr-aacC1 resistance gene, promoter hsp60 (for starting the thiostrepton-gentamycin resistance gene, i.e. tsr -aacC1), mainly used for plasmid screening.

[0038] The plasmid p60GTE contains a thiostrepton-gentamycin resistance gene (tsr-aacC1), as described in SEQ ID NO: 1, the 2760-3145th sequence on the gene is a promoter sequence (hsp60), Used to activate the expression of tsr-aacC1 gene. The hsp60 sequence connected to both ends of the tsr-aacC1 gene sequence is shown in SEQ ID NO:6.

[0039] In addition, a Not I restriction site, an XbaI restriction site, a HindIII restriction site, and an XhoI restric...

Embodiment 2

[0049] Example 2 Construction and Application of Integrated Plasmid p60GTI

[0050] The schematic diagram of the structure of the integrated plasmid p60GTI is shown in Figure 4 , Plasmid p60GTI contains in clockwise order: LacZ promoter and Lac Z operator (can be used for blue-white screening and expression of the entire plasmid backbone behind), M13 reverse primer (for PCR amplification identification), dif2- hsp60-aacC1-tsr-dif1 resistance expression cassette, phage integration site attP, integrase gene Int (can express integrase, so that the plasmid can be integrated into the mycobacterium genome through the attP site), M13 forward primer (for PCR amplification identification), Escherichia coli origin of replication (f1), ampicillin resistance gene promoter, ampicillin resistance gene Amp R (for plasmid selection), mycobacterial origin of replication ori M. The two ends of the dif2-hsp60-aacC1-tsr-dif1 resistance expression cassette are respectively connected with the Hin...

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Abstract

The invention discloses an independent thiostrepton (tsr)-gentamicin resistance gene (aacC1) system with a nucleotide sequence as shown in SEQ ID NO:1, a resistance expression box and a recombinant plasmid containing the system, as well as a novel recombinant plasmid used for effectively constructing a recombinant mycobacterium without a resistance maker. According to the invention, the condition that thiostrepton THS can be used as a resistance selection marker of mycobacterium is illuminated for the first time, and the working concentration of thiostrepton THS is far lower than those of other selection markers used in the mycobacterium. The resistance expression box is characterized in that a dif sequence is added at two ends of a tsr-aacC1 gene, and can automatically dissociate in the mycobacterium. After the tsr-aacC1 gene is lost, a damaged gene still can express a shortened small peptide without influencing the translation of a downstream gene, thereby effectively avoiding a polar effect. Multiple restriction enzyme cutting sites are further added at two ends of the resistance expression box, so that the resistance expression box can be inserted into a specific sequence, and other sequences can be conveniently and directionally added on two sides of the specific sequence.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a resistance expression cassette for efficiently constructing recombinant mycobacteria without resistance markers and an application thereof. Background technique [0002] At present, in the process of genetic manipulation of mycobacteria, the resistance marker genes that can be used for screening are very limited. The main reason is that mycobacteria are naturally resistant to a variety of antibiotics, and they require low-frequency resistance, that is, more stable drugs. as a resistance marker gene. The combined use of multiple resistance markers makes the genetic manipulation of mycobacteria require a variety of genetic modifications, such as the stable maintenance of multiple plasmids and the inactivation of multiple genes, so the feasibility is quite difficult. [0003] The aminoglycoside phosphotransferase (APH) gene, which is resistant to kanamycin and highl...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/65
CPCC12N15/74C12N15/65C12N2800/101
Inventor 张天宇马格文如·朱丽俄斯刘燕王邦兴曹元元黄少波玛卡芬·盖乐张洋郭锦涛柴然吉比·筹多拉瑞刘洋
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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