A dna fragment for uric acid-induced expression of target protein, related promoter and application

A DNA molecule and fragment technology, applied in DNA/RNA fragments, recombinant DNA technology, applications, etc., can solve problems such as inapplicability to recombinant proteins and weak transcription ability

Active Publication Date: 2016-07-06
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The lactose promoter lac itself has weak transcription ability and is not suitable for large-scale expression of recombinant proteins

Method used

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  • A dna fragment for uric acid-induced expression of target protein, related promoter and application
  • A dna fragment for uric acid-induced expression of target protein, related promoter and application
  • A dna fragment for uric acid-induced expression of target protein, related promoter and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, promoter A and the acquisition of DNA fragments used for uric acid induction

[0035] The DNA shown in sequence 1 in the following examples is the plasmid SHY, the DNA shown in sequence 2 is the control plasmid HY, the only difference between the nucleotide sequence sequence 1 of the SHY plasmid and the nucleotide sequence sequence 2 of the control plasmid HY is It consists in replacing the lac promoter at the 143-200th nucleotide from the 5' end of sequence 2 with the promoter A shown at the 143-200th nucleotide from the 5' end of sequence 1 in the sequence listing.

[0036] The specific construction method of the above plasmid is as follows:

[0037] 1. Acquisition of uric acid regulatory protein HucR and its coding gene

[0038] Genomic DNA of Deinococcus radiodurans (ATCC13939) was extracted and used as a template to perform PCR amplification with 5'-gcacatatgatgagcgcgcgcatggataac-3'(forward) and 5'-agagagctcttaaacaccctgttcgaggc-3'(reverse) as primers...

Embodiment 2

[0062] Example 2. Application of promoter A and DNA fragments for uric acid induction in the expression of target genes induced by uric acid

[0063] The recombinant vector SHY and control plasmid HY obtained in Example 1 were transformed into Escherichia coli DH10B respectively to obtain recombinant bacteria DH10B / SHY containing SHY and recombinant bacteria DH10B / HY containing HY (the plasmids were extracted and sequenced to verify correctness).

[0064] Pick a single colony of recombinant bacteria DH10B / SHY and recombinant bacteria DH10B / HY, add ampicillin antibiotic resistance LB medium (tryptone 10g / L, NaCl10g / L, yeast extract 5g / L, add antibiotic ampicillin to 100μg / mL) at 37°C for overnight (12h) on a shaker until the bacterial concentration reaches saturation (OD600 is 3.5) to obtain seed liquid;

[0065] The seed solution was inserted into uric acid medium of different concentrations according to the inoculum amount of 1% for induction for 12 hours, and the culture con...

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Abstract

The invention discloses a DNA fragment for uric acid-induced target protein expression, a related promoter and application. The present invention provides a DNA molecule whose nucleic acid sequence is the 143-200 nucleotides from the 5' end of sequence 1 in the sequence listing. DNA fragments containing the above DNA molecules are also within the scope of the present invention. The experiments of the present invention prove that the present invention obtains a new promoter by transforming the -35 and -10 regions of the lac promoter of Escherichia coli, which can utilize uric acid under the cooperation of the uric acid regulatory protein HucR and the uric acid transporter YgfU Induce the expression of the target protein. Therefore, the promoter and related expression system of the present invention can be used to express the target protein under the induction of uric acid, and can also be used in a biological uric acid detection system.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA fragment for uric acid-induced expression of a target protein, a related promoter and application thereof. Background technique [0002] Recombinant protein expression system is an important research content of modern biotechnology, and has been widely used in food, pharmaceutical, detergent production and other fields. Among them, the bacterial heterologous expression system has significant advantages such as short bacterial growth time, high cell growth density, cheap substrate, clear genetic background, and suitable for engineering strain transformation, especially the E. coli expression system, which has become the most widely used One of the expression systems. [0003] Recombinant expression of heterologous proteins usually requires molecular cloning and other techniques to place the coding sequence of the target protein behind a promoter with strong transcription abilit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/31C12N15/63C12N5/10C12N1/21C12N15/70G01N33/68C12R1/19C12R1/01
Inventor 梁朝宁熊丹丹唐双焱
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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