KR20200066465A - A microorganism diagnostic kit for sensing mercury and the preparation method thereof - Google Patents

Abstract

The present invention relates to a microorganism diagnostic kit for sensing mercury with high-sensitivity and high-selectivity, and a method for manufacturing the same. Particularly, the present invention relates to a gene construct comprising a promoter of a zntA gene that specifically binds with mercury, a fluorescent protein expression vector based on the promoter gene, a transformant comprising the expression vector, a method for manufacturing a microorganism diagnostic kit for sensing mercury ions using the transformant, and a diagnostic kit manufactured by the method.

Description

{A microorganism diagnostic kit for sensing mercury and the preparation method thereof}

The present invention relates to a high sensitivity and high selectivity mercury sensing microbial diagnostic kit and method for manufacturing the same, specifically a gene construct including a promoter of a zntA gene that specifically binds mercury, fluorescence based on the promoter gene The present invention relates to a protein expression vector, a transformant comprising the expression vector, a method for manufacturing a mercury sensing microorganism diagnostic kit using the transformant, and a diagnostic kit prepared by the method.

Along with modern industrial development, humans are exposed to various heavy metal poisoning risks due to environmental pollution caused by industrial wastewater and waste. In particular, mercury is a fatal substance that can cause paralysis of the limbs when severe disorders in the nervous system, such as speech disorders and movement disorders.

Mercury is a very toxic component present at 0.08 ppm on the Earth's surface. 2000 to 6000 tons of mercury are released through volcanic activity, and 2000 to 3000 tons of mercury are released through coal burning, gold production and industrial waste. Once the mercury element is released, it can be converted into organic matter by bacteria in lakes and seas, and can be converted into minerals in the liver. In the environment, mercury exists in three forms: elemental, inorganic and organic, all of which are very toxic.

Specifically, mercury quickly crosses the blood brain barrier and accumulates in the brain, disturbing the central nervous system as well as the endocrine system, damaging DNA and damaging mitosis. Minamata disease in Japan is an example of how deadly mercury is to the human body.

Accordingly, there is a need to develop a mercury ion sensing method capable of quickly and easily sensing mercury ions with high sensitivity and selectivity for practical application in biological systems and environmental fields.

The present inventors include a promoter gene specifically binding mercury, a fluorescence protein expression vector based on the promoter gene, and the expression vector to develop a highly sensitive and highly selective mercury ion sensing microbial diagnostic kit and method for manufacturing the same The present invention was completed by developing a transformant, a manufacturing method of a high sensitivity and high selectivity mercury ion sensing microbial diagnostic kit using the transformant, and a diagnostic kit prepared by the manufacturing method.

One object of the present invention is to provide a gene construct (gene construct), including a reporter (reporter) gene linked operably to the promoter of the promoter and the gene of zntA zntA gene.

Another object of the present invention is to provide an expression vector comprising the gene construct.

Another object of the present invention is to provide a recombinant transformant in which the expression vector is transformed into a host cell.

Another object of the present invention is to provide a mercury sensing diagnostic kit comprising the recombinant transformant.

Another object of the present invention is to provide a method of manufacturing a mercury sensing diagnostic kit comprising the following steps:

(1) preparing an expression vector containing a gene construct comprising a promoter of the zntA gene and a reporter gene operably linked to the promoter of the zntA gene; And

(2) A step of manufacturing a mercury sensing diagnostic kit comprising a recombinant transformant transforming the expression vector of step (1) into a host cell.

Another object of the present invention is to provide a method for sensing mercury in a sample comprising the following steps:

(1) exposing the test sample to a mercury sensing diagnostic kit; And

(2) measuring the expression level of the reporter gene by the operation of the promoter of the zntA gene through the diagnostic kit of step (2).

Specifically, it is as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not limited by the specific descriptions described below.

In addition, one of ordinary skill in the art can recognize or identify a number of equivalents to certain aspects of the invention described herein using only routine experimentation. In addition, such equivalents are intended to be included in the present invention.

One aspect of the present invention for achieving the above object provides a gene construct comprising a zntA gene promoter and a reporter gene operably linked to a promoter of the zntA gene.

The " zntA gene" of the present invention is a Zinc/cadmium/lead-transporting P-type ATPase, which is a gene that acts as a metal pump that spreads toxic metals from bacteria out of cells.

The promoter of the zntA gene of the present invention preferably has a nucleotide sequence that is amplified into a specific primer pair by a polymerase chain reaction, but is not limited thereto. The "promoter" refers to a DNA sequence region to which transcription regulators bind, and specifically, may be a zntA gene promoter of the E. coli strain used in one specific embodiment of the present invention, but is not limited thereto.

More specifically, the promoter of the zntA gene may include the nucleotide sequence of SEQ ID NO: 1. Not only the nucleotide sequence of SEQ ID NO: 3 of the present invention, but also substantially 80% or more, specifically 90% or more, more specifically 95% or more, particularly specifically 97% or more of the sequence As described above, any gene sequence encoding a zntA protein that is identical to or corresponding to each protein is included. In addition, if it is a base sequence having such homology, it is obvious that some of the sequences are deleted, modified, substituted or added to the base sequence within the scope of the present invention.

"Homology" of the present invention refers to the same degree of base or amino acid residues between sequences after aligning both sequences in a specific comparison region as much as possible in the amino acid sequence or nucleic acid sequence of a gene encoding a protein. . When the homology is sufficiently high, the expression product of the gene may have the same or similar activity. The percentage of sequence identity can be determined using known sequence comparison programs, such as BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), and the like.

"Operably linked" of the present invention means that the polynucleotide sequence is functionally linked with a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein. Operable linkages can be made using known genetic recombination techniques in the art, and site-specific DNA cleavage and linkage can be made using artisan cleavage and linkage enzymes, but are not limited thereto. In addition, the polynucleotide includes DNA and RNA encoding a target protein.

The "reporter gene" of the present invention is a gene capable of sensing an expression material of a gene, and it is connected to a vector to make it possible to visually confirm whether a transformed gene is expressed. In a specific embodiment of the present invention, if the reporter gene can be operably linked to be expressed by the promoter of the zntA gene, various known reporter genes are available, GFP, luciferase, alkaline phosphatase (alkaline) phosphatase), bioluminescence, β-galactosidase (β-galactosidase) may be any one of the reporter genes, specifically, luciferase, but is not limited thereto.

Another aspect of the present invention is to provide an expression vector comprising the gene construct. In addition, another aspect of the present invention is to provide a recombinant transformant in which the expression vector is transformed into a host cell.

By "expression vector" of the present invention is meant a gene construct comprising essential regulatory elements operably linked to the insert so that the insert is expressed in the cell. The expression vector can be prepared and purified using standard recombinant DNA technology. The type of the expression vector is not particularly limited as long as it functions to express a desired gene in various host cells of prokaryotic and eukaryotic cells and to produce a desired protein, while retaining a strong activity and a promoter and a strong expression, the target protein Vectors capable of mass production of are preferred. The expression vector preferably contains at least a promoter, an initiation codon, a gene encoding a desired protein, and a terminator codon, but is not limited thereto. In addition, DNA, which encodes a signal peptide, enhancer sequences, non-translated regions on the 5'and 3'sides of a desired gene, a selectable marker region, or a replicable unit may be appropriately included, but is not limited thereto. The expression vector may be a pBJY vector, but is not limited thereto.

HD Cloning Kit User Manual 참조)으로 luc 유전자 바로 위에 정제된 프로모터 DNA와 pBJY 벡터를 연결하여 zntA:pBJY 재조합 플라스미드를 제작하였다(도 3).In a specific embodiment of the present invention, In Fusion Method (In-Fusion

HD Cloning Kit User Manual) to prepare the zntA:pBJY recombinant plasmid by linking the purified promoter DNA directly above the luc gene and the pBJY vector (FIG. 3).

The term "transformant" of the present invention means a cell transformed such that the expression vector of the present invention is introduced into a host cell to express a gene encoding a target protein contained in the expression vector.

The "transformation" refers to a phenomenon in which the genotype of a host cell (prokaryotic and eukaryotic organisms, animal cells and plant cells) is changed by a plasmid or genome containing a gene introduced from the outside using a genetic manipulation technique. That is, it means that a vector containing a polynucleotide encoding a target protein is introduced into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can include both of them, whether they can be inserted into the host cell chromosome or located outside the chromosome, as long as it can be expressed in the host cell. The polynucleotide may be introduced into a host cell and expressed as long as it can be expressed in any form. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression. The expression cassette may include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto. The transformation method includes any method of introducing a nucleic acid into a cell, and can be performed by selecting a suitable standard technique as known in the art according to the host cell. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), EAE-dextran method, cationic liposome method, and lithium acetate -There is a DMSO method, but is not limited thereto. The method of transformation is not particularly limited, and may be performed by a method commonly used in the art.

The term "host cell" of the present invention refers to a cell that is a target for introducing a gene of a target protein from the outside. In one embodiment of the present invention, the host cell may be a microorganism of the genus Escherichia, and more specifically, S Escherichia coli may be, and more specifically, Escherichia coli K-12 strain, but is not limited thereto.

Another aspect of the present invention is to provide a diagnostic kit for mercury sensing that includes the recombinant transformant.

The "mercury sensing diagnostic kit" is a kit designed to specifically sense mercury, and in a specific embodiment of the present invention, it was confirmed that the diagnostic kit gradually increases the luminous intensity as the mercury concentration increases. , It was confirmed to have a sensitivity of 0.5 to 5ppm, it was confirmed to have a high sensitivity to mercury (Fig. 5 and Fig. 6).

Another aspect of the present invention is to provide a method of manufacturing a mercury sensing diagnostic kit comprising the following steps:

(1) preparing an expression vector containing a gene construct comprising a promoter of the zntA gene and a reporter gene operably linked to the promoter of the zntA gene; And

(2) A step of manufacturing a mercury sensing diagnostic kit comprising a recombinant transformant transforming the expression vector of step (1) into a host cell.

The " zntA gene", "promoter", "reporter gene", "expression vector", "host cell", "transformant" and "mercury sensing diagnostic kit" are as described above.

Another aspect of the present invention is to provide a method for sensing mercury in a sample comprising the following steps:

(1) exposing the test sample to a mercury sensing diagnostic kit; And

(2) measuring the expression level of the reporter gene by the operation of the promoter of the zntA gene through the diagnostic kit of step (1).

The mercury to be analyzed can be measured using a diagnostic kit when adsorbed on a surface or dissolved in a liquid medium. A diagnostic kit can be used to sense mercury attached to particles or dissolved in an aqueous sample, but it is generally desirable to measure in a liquid phase. Moisture can be used so that the diagnostic kit including the gene construct can function.

In a specific embodiment of the present invention, it was confirmed that the expression of the zntA gene of Escherichia coli is specifically high in mercury, and the purified promoter DNA and pBJY vector are linked by using PCR primers specific to the zntA gene. A zntA:pBJY recombinant plasmid was prepared, and the recombinant plasmid was transformed into Escherichia coli wild type strain K-12 to prepare a mercury sensing transformant.

In another specific embodiment of the present invention, the diagnostic kit confirmed that zntA gene expression increases depending on the mercury concentration according to quantitative analysis of luminescence, and the diagnostic kit gradually increases the luminous intensity as the mercury concentration increases. It was confirmed that (Fig. 5 and Fig. 6).

In addition, in another specific embodiment of the present invention, it was confirmed that the mercury sensing diagnostic kit has a sensitivity of 0.5 to 5 ppm, and thus has a high sensitivity to mercury (FIG. 6).

Therefore, since the mercury sensing diagnostic kit of the present invention is very specific and highly sensitive to mercury, the transformant transformed with the gene construct containing the zntA gene promoter having the nucleotide sequence of SEQ ID NO: 1 of the present invention has high sensitivity. / Can be used as a specific mercury sensing diagnostic kit that can be sensed.

The mercury sensing diagnostic kit including the promoter and reporter gene luc (luciferase) gene of the zntA gene of the present invention has excellent specificity and sensitivity, and thus can more sensitively and accurately sense mercury.

1 is a diagram showing a recombinant plasmid in which the mercury sensing promoter and reporter gene are linked to the expression vector pBJY.
2 is a diagram showing the sequence of the zntA gene promoter.
3 is a diagram showing the structure of the zntA:pBJY recombinant plasmid.
4 is a schematic diagram showing a process of generating fluorescence by mercury in the mercury sensing recombinant E. coli ( Escherichia coli ).
5 is a diagram quantitatively showing the expression pattern according to the concentration of mercury.
6 is a view showing the degree of light emission according to the concentration of mercury.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example 1: zntA Preparation of a diagnostic kit for mercury sensing that includes a gene promoter and a luciferase gene

To prepare a diagnostic kit for mercury sensing that includes the promoter of the zntA gene and the luciferase gene ( luc ), a recombinant plasmid containing the promoter of the zntA gene was first prepared (FIG. 1 ), and this was transformed into a host cell ( Fig. 4).

HD Cloning Kit User Manual 참조)으로 luc 유전자 바로 위에 정제된 프로모터 DNA가 놓일 수 있도록 하여 zntA:pBJY 재조합 플라스미드를 제작하였다(도 3). Specifically, the In Fusion method (In-Fusion

HD Cloning Kit User Manual) so that the purified promoter DNA can be placed directly on the luc gene to prepare a zntA:pBJY recombinant plasmid (FIG. 3).

The recombinant plasmid was transformed into Escherichia coli wild type to prepare a mercury sensing transformant.

Example 2: Confirmation of mercury specificity of the diagnostic kit

In order to confirm the mercury specificity of the diagnostic kit of the present invention, after culturing the transformant prepared in Example 1, the obtained culture solution was centrifuged to remove the supernatant, and then the cells were recovered. Cells were lysed by adding a buffer solution to the recovered cells, and luciferase activity in the lysed cells was measured.

As a result, it was confirmed that the promoter of the zntA gene responds very specifically only to mercury.

Example 3: Confirmation of the mercury sensitivity of the diagnostic kit

In order to confirm the sensitivity of the diagnostic kit to mercury of the present invention, the expression pattern according to the mercury concentration of the diagnostic kit was confirmed.

Specifically, the mercury sensing diagnostic kit containing the transformant prepared in Example 1 reached a sensitivity of 0.5 to 5 ppm specifically for mercury. In addition, it was confirmed that the luminescence intensity gradually increased with increasing mercury concentration (FIG. 6).

Through this, it was confirmed that the expression of zntA is increased even at a low mercury concentration, and thus has a high sensitivity to mercury.

Example 4. Confirmation of the expression pattern according to the mercury concentration of the diagnostic kit

In order to confirm the expression pattern according to the mercury concentration of the diagnostic kit of the present invention, the diagnostic kit was treated with mercury, and it was visually confirmed that the degree of fluorescence in recombinant E. coli was proportional to the concentration of mercury according to the expression level of zntA . (Figure 5).

Through this, it was confirmed that the expression of zntA in the diagnostic kit of the present invention increased depending on the mercury concentration.

As described above, the mercury sensing diagnostic kit including the promoter and reporter gene luc (luciferase) gene of the zntA gene of the present invention is excellent in specificity and sensitivity, so it can be more sensitive and accurately sensed. It is expected to be used.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts, rather than the above detailed description.

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Claims (16)

A gene construct comprising a promoter of the zntA gene and a reporter gene operably linked to the promoter of the zntA gene.
According to claim 1,
The promoter of the zntA gene has a nucleotide sequence of SEQ ID NO: 1, gene construct.
According to claim 1,
The reporter gene is a gene of proteins that induce fluorescence, a gene construct.
According to claim 3,
The reporter gene is a gene construct, which is a luc gene.
An expression vector comprising the genetic construct of claim 1.
A recombinant transformant in which the expression vector of claim 5 is transformed into a host cell.
The method of claim 6,
The host cell is Escherichia coli , a recombinant transformant.
A mercury sensing diagnostic kit comprising the recombinant transformant of claim 6 or 7.
The method of claim 8,
The mercury sensing diagnostic kit is to have a sensitivity of 0.5 to 5ppm, mercury sensing diagnostic kit.
(1) preparing an expression vector containing a gene construct comprising a promoter of the zntA gene and a reporter gene operably linked to the promoter of the zntA gene; And
(2) A method of manufacturing a mercury sensing diagnostic kit comprising the step of preparing a mercury sensing diagnostic kit comprising a recombinant transformant transforming the expression vector of step (1) into a host cell.
The method of claim 10, wherein the reporter gene of step (1) is a gene of a protein that induces fluorescence.
The method of claim 11, wherein the reporter gene of step (1) is a luc gene.
The method of claim 10, wherein the host cell of step (2) is Escherichia coli , a mercury sensing diagnostic kit.
(1) exposing the test sample to the mercury sensing diagnostic kit of claim 8; And
(2) The mercury sensing method in a sample comprising the step of measuring the expression level of the reporter gene by the operation of the promoter of the zntA gene through the diagnostic kit of step (1).
The method of claim 14, wherein the promoter of the zntA gene has a nucleotide sequence of SEQ ID NO: 1, a mercury sensing method.
The method of claim 14, wherein the reporter gene is a luc gene.

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