JP6037259B2 - Recombinant vector for transformation for detecting nitric oxide, and nitric oxide sensor cell using the same - Google Patents

Description

  The present invention relates to a recombinant vector for transformation for detecting nitric oxide, and a nitric oxide sensor cell using the same. In addition, the present invention provides a method for detecting or quantifying intracellular nitric oxide using these, a method for monitoring changes in nitric oxide concentration in cells due to stimulation, and a test cell producing It relates to a method for detecting or quantifying nitric oxide.

  Since Nitric Oxide (NO) was reported to be a vascular relaxation factor derived from the vascular endothelium in 1987, its role as a physiologically active substance has been found one after another. In addition, it has been clarified that it is an important biomolecule that plays various roles in the regulation of local cell functions and communication between cells in the immune system and central nervous system. Regarding diseases, NO is considered to be related to infection in the immune system, arteriosclerosis, stroke, hypertension in the cardiovascular system, dementia, Alzheimer's disease, etc. in the central nervous system.

  It is known that NO synthesized by NO synthase (NOS) using L-arginine as a substrate in the cell is released from the cell and invades neighboring cells to exert its action. In the immune system, the cardiovascular system, and the central nervous system, macrophages, vascular endothelial cells, neurons (postsynapses), and the like can each be NO donor cells.

  Looking particularly at the immune system, macrophages exert an immunostimulatory effect by producing NO and constitute a defense system against various foreign antigens. On the other hand, NO produced from arginine is generally a radical gas and thus has toxic effects such as cell damage and carcinogenicity. For example, in sepsis, macrophages produce a large amount of nitric oxide, and vasodilation caused thereby is considered to be the main cause of hypotension. Thus, it can be said that NO has good and bad duality with respect to the living body.

  Therefore, if the amount of NO in the cells in the living body can be precisely controlled, for example, when trying to treat various diseases such as infectious diseases by drug therapy or immunostimulation therapy, the occurrence of undesirable events such as side effects It is considered possible to maximize the therapeutic effect while minimizing the above. In order to precisely control the amount of NO in the cell, the amount of NO in the cell is measured in real time and with a wide dynamic range (ratio of minimum and maximum identifiable signals). Technology to do is indispensable.

  Conventionally, as a technique for measuring the amount of NO in a cell, for example, Non-Patent Document 1 discloses a technique that utilizes expression of a regulatory protein (NorR) that is activated depending on the NO concentration. Specifically, first, an operon obtained by fusing a regulatory gene norR encoding NorR and a promoter region whose transcription is regulated thereby upstream of the reporter gene β-galactosidase gene is cloned into a plasmid. A sensor plasmid is constructed. Next, the obtained sensor plasmid is transformed into E. coli that does not have β-galactosidase activity to obtain sensor cells. Then, the NO concentration is measured using the enzyme activity of β-galactosidase produced by the sensor cell obtained according to the exposed NO concentration as an index.

M. I. Hutchings et al., J. Bacteriol., Vol. 184, No. 16, pp. 4640-4643 (2002)

The detection and quantification of NO by the technique disclosed in Non-Patent Document 1 is performed by measuring the color developed by cleavage of the synthetic substrate with β-galactosidase using a spectrophotometer. For this reason, the spectrophotometer has a measurement limit due to color saturation and the like, so that there is a problem that measurement in a wide dynamic range is difficult. On the other hand, measurement of NO concentration with a small number of bacteria (10 ³ to 10 ⁴ ) existing in eukaryotic cells using a monitor strain would be below the detection limit of the spectrophotometer, which is also highly sensitive. Is very difficult to measure. In addition, the measurement of β-galactosidase activity requires the reaction of β-galactosidase in Escherichia coli produced from the plasmid-encoded β-galactosidase gene with a synthetic substrate. And many steps such as enzymatic reactions are essential. As a result, the necessity of these series of operations has a problem that the speed of the detection operation as the NO sensor is lowered. In addition, since most E. coli retain endogenous β-galactosidase activity, E. coli that can be used in the art is a strain that does not retain endogenous β-galactosidase activity, or genetically There is also a problem that the strain is limited to the deleted strain.

  Therefore, the present invention has an object to provide means capable of measuring NO concentration in living cells by a simpler method, with higher sensitivity, and with a wider dynamic range as compared with the prior art. To do.

  The present inventors have conducted intensive studies in view of the above problems. As a result, the inventors have found that the above problem can be solved by adopting a photoluminescent gene encoding a photoprotein as a reporter gene, and have completed the present invention.

  That is, according to one aspect of the present invention, a luminescent gene encoding a luminescent protein and a series of gene sets that induce expression of the luminescent gene, the gene set comprising a regulatory gene norR and the regulatory gene norR And a promoter site for inducing transcription of the luminescent gene by the protein NorR encoded by the above, a recombinant vector for transformation for detecting nitric oxide is provided.

  Here, the promoter site is preferably the promoter region of the nor operon. The luminescent gene is preferably a luxCDABE gene. Furthermore, the vector is preferably composed of plasmid pAcGFP1.

  Moreover, according to the other form of this invention, the nitric oxide sensor cell which consists of a transformant which transforms a host using the recombinant vector for transformation mentioned above is also provided.

  Here, the host is preferably Escherichia coli or Salmonella, particularly enterohemorrhagic Escherichia coli EHEC EDL933.

  Furthermore, according to still another aspect of the present invention, various methods using the nitric oxide sensor cell described above are provided. For example, a method is provided for detecting or quantifying intracellular nitric oxide comprising measuring a signal change in the nitric oxide sensor cell described above. Also provided is a method for monitoring changes in nitric oxide concentration in a cell due to stimulation, including applying stimulation to the above-described nitric oxide sensor cell and measuring a signal change before and after the stimulation. .

  Further, a test cell comprising: a test cell to be detected or quantified for production of nitric oxide; and the above-mentioned nitric oxide sensor cell are disposed in close proximity, and a signal change in the nitric oxide sensor cell is measured. A method for detecting or quantifying the produced nitric oxide is also provided. Here, the test cell is preferably a phagocytic cell, more preferably a macrophage, and the macrophage is more preferably derived from a living body suffering from an infectious disease. In another preferred embodiment, the test cell is an intestinal epithelial cell, and the host is Salmonella.

  According to the present invention, it is possible to provide means capable of measuring NO concentration in living cells with a higher sensitivity and a wider dynamic range by a simpler method than in the prior art.

It is explanatory drawing which shows the characteristic structure of the recombinant vector which concerns on this invention compared with the structure of the nor operon (norRVW) of E. coli, and the structure of the norR-LacZ fusion reporter disclosed in Non-Patent Document 1. In an Example, it is a graph which shows the result of having investigated the responsiveness with respect to NO molecule of the cell strain transformed with the recombinant vector (pRPL3) which concerns on this invention. FIG. 2A is a graph showing the result of measuring the relative light emission amount (RLU). FIG. 2B is a graph showing the results of calculating the emission intensity (RLU / CFU). In an Example, the result (change of luminescence intensity (RLU / CFU)) which investigated the responsiveness with respect to NO molecule | numerator of the NO sensor cell currently disclosed by the nonpatent literature 1 using (beta) -galactosidase gene as a reporter gene is shown. It is a graph. In an Example, it is a graph which shows the result of having investigated the change of NO level in the macrophage during enterohemorrhagic Escherichia coli (EHEC) infection. In the graph shown in FIG. 4, ■ represents the result of calculating the luminescence intensity (RLU / CFU) of RAW 264.7 cells (without L-NMMA addition), and □ represents the luminescence intensity of RAW 264.7 cells (with L-NMMA addition) ( RLU / CFU) represents the result of calculation, and ▲ represents the result of calculation of the luminescence intensity (RLU / CFU) of THP-1 cells. In Examples, for the purpose of investigating whether NO can be detected and quantified by NO sensor cells using Salmonella as a host, the luminescence intensity (RLU / CFU) of NO sensor cells of Salmonella hosts in contact with various concentrations of SNPs It is a graph which shows the result of having measured.

  A first aspect of the present invention is a recombinant vector for transformation comprising a photoluminescent gene encoding a photoprotein and a series of gene sets that induce the expression of the photoprotein. This transformation recombinant vector is used to detect nitric oxide (NO). In the recombinant vector for transformation, the gene set includes a regulatory gene norR and a promoter site that induces transcription of the luminescent gene by the protein NorR encoded by the regulatory gene norR (hereinafter referred to as “norR-norV promoter”). ").

  Hereinafter, first, the recombinant vector for transformation according to the first embodiment will be described in detail, and then the transformant obtained using the vector and the use of the transformant will be described in order.

<Recombinant vector for transformation>
First, the recombinant vector for transformation according to the present embodiment includes a photoluminescent gene encoding a photoprotein and a series of gene sets that induce the expression of the photoluminescent gene.

  In this embodiment, the photoluminescent gene contained in the recombinant vector may be any gene that encodes a photoprotein, and its specific form is not particularly limited. In a preferred embodiment, the luxCDABE gene is used as the luminescent gene. However, if possible, a luminescent gene such as a firefly luciferase gene or a Renilla luciferase gene may be used.

  In this embodiment, the recombinant vector includes a predetermined gene set. The gene set includes a regulatory gene norR. This regulatory gene norR encodes the regulatory protein NorR. The regulatory protein NorR regulates transcription of the nor operon by binding to the promoter region of the nor operon involved in NO removal.

  Furthermore, in this embodiment, the gene set also includes a predetermined promoter site (norR-norV promoter). The operation of the norR-norV promoter is regulated by the regulatory protein NorR expressed from the regulatory gene norR. The mechanism can be explained as regulating the expression of the luminescent gene (inducing transcription) by binding of the regulatory protein NorR in a state of NO binding to the norR-norV promoter. In FIG. 1, the characteristic configuration of the recombinant vector according to the present invention is compared with the configuration of the nor operon of E. coli (norRVW) and the configuration of the norR-LacZ fusion reporter disclosed in Non-Patent Document 1. Show.

  As described above, in a preferred embodiment of the recombinant vector of the present embodiment, the gene set for inducing the expression of the luminescent gene includes the norR gene and the nor operon promoter site (norR-norV promoter). Thereby, the whole gene set can function as one promoter.

  The gene set in the above embodiment is, for example, a primer set comprising a forward primer (P841) of SEQ ID NO: 1 and a reverse primer (P842) of SEQ ID NO: 2 using the genomic DNA of enterohemorrhagic E. coli EHEC EDL933 as a template. Can be amplified by the PCR method.

  On the other hand, the luxCDABE gene, which is a luminescent gene, is obtained by PCR using a primer set consisting of a forward primer (P685) of SEQ ID NO: 3 and a reverse primer (P705) of SEQ ID NO: 4, using, for example, the genomic DNA of Photorhabdus luminescens GTC00819 as a template. It can be amplified by the method.

  Then, the amplified luxCDABE gene fragment is digested with a predetermined restriction enzyme (NotI and NcoI in the examples described later), and inserted into a restriction enzyme site of a predetermined recombinant vector (for example, pAcGFP1). Thereby, it is possible to construct a LuxCDABE expression vector dependent on the promoter inherent in the used recombinant vector (lac promoter in the case of pAcGFP1 vector).

  Then, the norR-norV promoter fragment obtained above was digested with restriction enzymes (NcoI and PvuII in the examples described later), and the expression vector obtained above was previously digested with the same restriction enzymes. It is possible to produce a recombinant vector for transformation according to the present invention by replacing the original promoter of the expression vector by cloning into the vector. Whether or not the obtained recombinant vector is correctly produced can be confirmed by techniques such as PCR screening, restriction enzyme digestion, and DNA sequencing.

  In addition, it is preferable that the obtained recombinant vector contains an antibiotic resistance gene. Many such antibiotic resistance genes are currently known (for example, gentamicin, kanamycin, ampicillin, tetracycline, etc.). Therefore, those skilled in the art can include a desired antibiotic resistance gene in a recombinant vector by referring to known knowledge. This antibiotic resistance gene is for selecting a desired transformant. Therefore, in some cases, genes other than antibiotic resistance genes may be included in the recombinant vector as long as they can be used for selection.

<Nitric oxide (NO) sensor cell>
A second aspect of the invention relates to a nitric oxide (NO) sensor cell. This NO sensor cell consists of a transformant obtained by transforming a host using the above-described recombinant vector for transformation.

  There is no restriction | limiting in particular about a host, What is possible is just what can be transformed with the above-mentioned recombinant vector and can express a luminescent gene. The host is preferably Escherichia coli or Salmonella. These bacteria are most suitable for practicing the present invention because of simple culture conditions and convenient operation. In Examples described later, E. coli (JM109 strain), enterohemorrhagic E. coli (EHEC EDL933), Salmonella (Salmonella enterica serovar Typhimurium MI1 strain) and the like were used as hosts. In addition to these, bacteria that can be used as a host include, for example, Shigella, broadly pathogenic Escherichia coli, Enterobacteriaceae such as Yersinia and Klebsiella, Vibrio cholerae and Vibrio parahaemolyticus. However, it is not limited to these.

  Here, the detection and quantification of NO by the technique disclosed in Non-Patent Document 1 described above is performed by measuring the color developed by cleavage of the synthetic substrate by β-galactosidase with a spectrophotometer. Since most Escherichia coli and Salmonella have endogenous β-galactosidase activity, the bacteria that can be used in the above-mentioned prior art include strains that do not have endogenous β-galactosidase activity, or There was a problem that a genetically deleted strain had to be selected.

  On the other hand, according to the study by the present inventors, even when E. coli or Salmonella having β-galactosidase activity is used as a host for producing a NO sensor cell by applying the recombinant vector according to the present invention. It has been confirmed that there is no problem.

  Transformation of a host using a recombinant vector can be easily carried out by those skilled in the art with reference to known knowledge about conventional methods. In addition, selection of transformed hosts can also be performed using various conventionally known techniques. For example, when the recombinant vector contains an antibiotic resistance gene, only a transformed host can be selected by culturing using a medium containing the corresponding antibiotic. In some cases, an appropriate strain may be further selected from the strains selected in this manner, depending on the presence or absence of a luminescence reaction (ie, expression of a luminescent gene) with respect to NO exposure.

<Uses of NO sensor cells>
The transformant (NO sensor cell) thus obtained has a property that its luminescence intensity increases when NO is sensed. Therefore, the NO sensor cell according to the present invention can be used for various applications as follows.

  For example, when NO sensor cells are used, NO present in the cells can be detected and quantified. According to the present invention, it is possible to provide means capable of measuring NO concentration in living cells with a simpler method, with higher sensitivity, and with a wider dynamic range as compared with the prior art. .

  For example, after measuring a signal from a NO sensor cell emitted under normal conditions, a specific stimulus is applied, and the signal change (that is, the difference in signal before and after the stimulus is applied) is measured. It becomes possible to monitor the influence of stimulation on changes in intracellular NO concentration. Then, by continuously performing such measurement, it is possible to observe the temporal influence of the stimulus, that is, the temporal change in the NO concentration due to the stimulus. In addition, there is no restriction | limiting in particular about the irritation | stimulation provided in this method, Biochemical irritation | stimulation, such as a hormone and an endocrine disruptor, and physical irritation | stimulation, such as electricity, radiation, and heat, may be sufficient.

  Further, NO released from cells can be detected and quantified using NO sensor cells. This can be achieved by placing a cell (hereinafter also referred to as “test cell”) in which NO release is to be detected / quantified in close proximity to the NO sensor cell and measuring a signal change in the NO sensor cell. That is, this method is enabled by the principle that NO released from the test cell enters the NO sensor cell, thereby causing a signal change in the NO sensor cell.

  In this case, examples of the test cell include phagocytic cells. When phagocytic cells are used as test cells, NO sensor cells are taken into phagocytic cells by the phagocytosis. For this reason, there exists an advantage that operation for introducing a NO sensor cell into a test cell is unnecessary. Examples of such phagocytic cells include, but are not limited to, macrophages, neutrophils, monocytes, dendritic cells and the like. In particular, since macrophages derived from living organisms affected by infectious diseases are used as test cells, the amount of intracellular NO in infected macrophages can be monitored non-destructively in real time. preferable.

  In another preferred embodiment, epithelial cells such as intestinal epithelial cells are used as test cells, and Salmonella is used as a host. By producing NO sensor cells using Salmonella, which is a cell-invading bacterium, as a host, in addition to monitoring NO concentration in phagocytic cells such as macrophages and neutrophils, intestinal epithelial cells without phagocytosis It is also possible to monitor the NO concentration in many eukaryotic cells.

  In various applications described above, measurement of a signal (luminescence) from the NO sensor cell can be appropriately performed by referring to conventionally known knowledge in a technique using a luminescent gene. For example, the relative luminescence (RLU) can be measured using a luminometer. By using such a method, detection and quantification can be performed in a dynamic range (7 digits or more) with high sensitivity and wide linearity that could not be considered in the prior art. This indicates that it is possible to measure both microbial cells that emit light very strongly and microbial cells that emit light very weakly. Thus, it is very advantageous when measuring a large number of specimens that the same operation can be performed without diluting the sample. Moreover, the NO sensor cell according to the present invention can continuously emit light independently without adding a synthetic substrate. For this reason, it is possible to continuously monitor changes in the NO concentration in any environment where NO sensor cells exist. As a result, continuous measurement in a eukaryotic cell environment, which could not be measured by the conventional technique, has become possible. In addition, the advantage of this method that it is not necessary to add a relatively expensive synthetic substrate is not only quick and easy to operate, but also reduces the cost of measurement. Therefore, the present invention is compared with the prior art. It is understood that it is extremely superior.

  EXAMPLES Hereinafter, although this invention is explained in full detail using an Example, this invention is not limited to a following example.

≪Construction of recombinant vector for transformation≫
A region containing the regulatory gene norR and the norR-norV promoter that is regulated by norR adjacent to norV was amplified by PCR using the genomic DNA of enterohemorrhagic E. coli EHEC EDL933 as a template to obtain a promoter fragment. At this time, the primers shown in Table 1 below were used.

  On the other hand, using the genomic DNA of Photorhabdus luminescens GTC00819 (obtained from National Bioresource Project (Gifu University)) as a template, a region containing the luxCDABE gene, which is a luminescent gene, was amplified by PCR to obtain a luxCDABE gene fragment. At this time, the primers shown in Table 2 below were used.

  The amplified luxCDABE gene fragment was digested with restriction enzymes (NotI and NcoI) and then inserted into the NotI-NcoI site of the pAcGFP1 vector (Clontech) to construct a lac promoter-dependent LuxCDABE expression plasmid (placlux8) .

  Subsequently, the digestion of the promoter fragment obtained above with restriction enzymes (NcoI and PvuII) is cloned into the expression plasmid obtained above (placlux8) previously digested with NcoI-PvuII. The plasmid (pRPL3), a recombinant vector for transformation according to the present invention, was obtained by replacing the lac promoter of placlux8. In this plasmid (pRPL3), it was confirmed by PCR screening, restriction enzyme digestion, and DNA sequencing that the norR-norV promoter fragment was located upstream of the luxCDABE gene of Photorhabdus luminescens lacking the promoter.

≪Preparation of NO sensor cells and measurement of luminescence intensity≫
For the purpose of investigating the responsiveness of the cell line transformed with the recombinant vector (pRPL3) constructed above to the NO molecule, the specific luminescence intensity of the transformed cell line (NO sensor cell) was determined by the following method. (RLU / CFU) was measured. In addition, the value of this luminescence intensity is a value obtained by dividing the value of the measured relative luminescence (Relative Light Unit; RLU) by the value of the colony forming unit (CFU) of bacteria. It can be an index representing the amount of light emission per unit amount.

  First, Escherichia coli JM109 strain having no β-galactosidase activity was used as a host, and transformed with the recombinant vector (pRPL3) constructed above by electroporation, and transformed cell strain (NO sensor cell; JM109 ( pRPL3)) was obtained.

  Subsequently, the obtained NO sensor cells were cultured overnight in LB medium, and then diluted 1: 100 with LB medium, and various concentrations (0 mM (control), 0.001 mM, 0.01 mM,. Sodium nitroprusside (SNP) was added to 1 mM and 1 mM), and the cells were further cultured at 37 ° C. for 6 hours under anaerobic conditions.

  Then, relative luminescence (RLU) was measured about 0.1 mL culture solution using GLOMAX 20/20 luminometer (Promega). The results are shown in FIG. In addition, the number of viable cells in the same culture was measured as a colony forming unit (CFU) by the colony counting method, and the luminescence intensity (RLU / CFU) was calculated. The results are shown in FIG. In the experiment described in this example, all the values shown in the figure and the like are average values (± standard error) of values obtained independently and repeated at least three times.

  As shown in FIG. 2 (B), it was confirmed that the value of luminescence intensity (RLU / CFU) changes depending on the amount of the norR gene product, so that the NO sensor by activation of the norV promoter by SNP. The cellular response was shown to be borne by the NorR protein. As described above, since the NO sensor cell according to this example can detect and quantify NO in a wide dynamic range, a quantitative assay in a wide concentration range of NO molecules in proliferating cells becomes possible.

  In addition, it has been confirmed by the present inventors that the emission intensity under anaerobic conditions increases in a concentration-dependent manner even when PROLI NONOate, which is an NO donor having a shorter half-life than SNP, is added. Further, when C-PTIO, which is a specific scavenger molecule of NO, is added at the same time, the emission intensity (RLU / CFU) is significantly reduced compared to the case where PROLI NONOate is added alone, the NO according to the present invention. Sensor cells do not respond to nitrate and hydrogen peroxide, but respond to nitrite, but the addition of C-PTIO reduces the level of LuxCDABE protein expression by nitrite to the same level as the control , Has been confirmed. From these, it is judged that the main effector of the NO sensor cell constructed in this example is NO.

≪Measurement of NO concentration by NO sensor cells using β-galactosidase gene as a reporter gene according to the prior art≫
As a comparative example of the present invention, the NO concentration was measured by the following method using NO sensor cells disclosed in Non-Patent Document 1 using a β-galactosidase gene as a reporter gene.

  Specifically, E. coli JM109 strain was used as a host and transformed by a recombinant vector (pRPZ2) constructed using the same method as in Non-Patent Document 1 by electroporation, and transformed cell strain (NO Sensor cell; JM109 (pRPZ2)) was obtained.

  Subsequently, the obtained NO sensor cells were cultured overnight in LB medium, and then diluted 1: 100 with LB medium, and various concentrations (0 mM (control), 0.001 mM, 0.01 mM,. Sodium nitroprusside (SNP) was added to 1 mM and 1 mM), and the cells were further cultured at 37 ° C. for 6 hours under anaerobic conditions.

  Thereafter, the relative luminescence (RLU) of 0.1 mL of the culture solution was measured using a GLOMAX 20/20 luminometer (Promega), and the viable cell count of the same culture solution was determined by the colony counting method (colony forming unit ( CFU), and the emission intensity (RLU / CFU) was calculated. The results are shown in FIG.

  As shown in FIG. 3, when the NO concentration was measured using the technique disclosed in Non-Patent Document 1, measurement with a wide dynamic range could not be performed.

≪Quantification of NO level in macrophages during EHEC infection≫
In order to investigate whether or not NO levels in macrophage cells are increased by contact with enterohemorrhagic Escherichia coli (EHEC), the luminescence intensity (RLU) of NO sensor cells in macrophage cells infected with EHEC by the following method / CFU). EHEC EDL933 is known to be able to survive in macrophage cells for at least 24 hours.

Specifically, first, a macrophage cell line (RAW 264.7) was seeded at 5 × 10 ⁵ cells / well in a 24-well tissue culture dish and cultured at 37 ° C. for 12 hours. Similarly, a human monocyte-derived cell line (THP-1) was seeded at 5 × 10 ⁵ cells / well in a 24-well tissue culture dish, and 10 ⁻⁷ M phorbol 12-myristate 13-acetate (PMA) was added. After the addition, the cells were cultured at 37 ° C. for 48 hours to differentiate the THP-1 cell line into macrophage-like cells. THP-1 cells have been reported to produce lower NO production than RAW 264.7 cells.

  The EHEC-derived NO sensor cells prepared above were added at a multiplicity of infection (m.o.i.) of 10 to each of the culture systems established above (all were monolayer culture systems).

Next, the plate was lightly centrifuged to actually cause infection, and then incubated at 37 ° C. for 20 minutes (the end point of this incubation was defined as “0 hour”). Media was collected, cells were washed, fresh DMEM medium (containing 10% FBS and 100 μg / mL gentamicin) for the RAW 264.7 cell line, and fresh RPMI-1640 medium (10% for the THP-1 cell line). FBS and 100 μg / mL gentamicin) were added respectively to kill extracellular bacteria. After 2 hours, the medium was collected, the cells were washed, and the medium was changed to contain 12 μg / mL gentamicin. At this time, 1 mM L-NMMA ( ^NG -monomethyl-L-arginine), which is a specific inhibitor of NO synthase in macrophages, was divided into a group containing gentamicin and a group not containing it.

  Subsequently, the cells of each dish were lysed by addition of PBS containing 0.1% deoxycholic acid or further incubated. For the lysed cells, the relative luminescence (RLU) was measured using a luminometer and the viable cell count was counted by counting colony forming units (CFU) by the same method as described above. The results of calculating the emission intensity (RLU / CFU) based on these measured values are shown as a graph in FIG. In the graph shown in FIG. 4, ■ represents the result of RAW 264.7 cells (without L-NMMA added), □ represents the results of RAW 264.7 cells (with L-NMMA added), and ▲ represents the results of THP-1 cells. Represents the result.

  As shown in FIG. 4, the luminescence intensity (RLU / CFU) of RAW264.7 cells began to increase immediately after infection and reached a high level (0.5) 8 hours after infection. On the other hand, the luminescence intensity (RLU / CFU) of RAW264.7 cells treated with L-NMMA was significantly lower than that of untreated RAW264.7 cells at 6 hours and 8 hours after infection. The luminescence intensity (RLU / CFU) in PMA-treated THP-1 cells slowly increased after infection, as in RAW264.7 cells.

  From the above, it is shown that the recombinant vector and the NO sensor cell according to the present invention can specifically recognize NO molecules in macrophage cells during bacterial infection and emit light in response thereto. Therefore, it is suggested that the recombinant vector and NO sensor cell according to the present invention can be useful tools for detecting and quantifying NO in macrophage cells during bacterial infection.

≪NO detection and quantification by NO sensor cells using Salmonella as a host≫
In order to investigate whether or not the NO level in Salmonella cells increases upon contact with SNP, the luminescence intensity (RLU / CFU) of the NO sensor cells of Salmonella host in contact with various concentrations of SNP was determined by the following method. It was measured.

  Specifically, first, Salmonella enterica serovar Typhimurium MI1 strain was transformed with the recombinant vector pRPL3 by the same method as described above. Subsequently, the obtained transformant (NO sensor cell) was cultured overnight in LB medium, and then diluted 1: 100 with LB medium, and various concentrations (0 mM (control), 0.001 mM, 0) were added thereto. Sodium nitroprusside (SNP) was added to a concentration of 0.01 mM, 0.1 mM, and 1 mM, and the cells were further cultured at 37 ° C. for 6 hours under anaerobic conditions.

  Thereafter, the luminescence intensity (RLU / CFU) was calculated for 0.1 mL of the culture solution by the same method as described above. The results are shown in FIG. From the results shown in FIG. 5, even when Salmonella was used as a host, it was possible to detect and quantify NO as in the case of using enterohemorrhagic Escherichia coli EHEC EDL933. Therefore, by producing the NO sensor cell according to the present invention using Salmonella, which is a cell-invading bacterium, as a host, various kinds of true cells such as epithelial cells having no phagocytosis (eg, intestinal epithelial cells) can be obtained. It is suggested that monitoring of NO concentration in nuclear cells may be possible.

[SEQ ID NO: 1]
The nucleotide sequence of the forward primer for amplifying the region containing the norR gene and the norR-norV promoter.
[SEQ ID NO: 2]
This represents the base sequence of the reverse primer for amplifying the region containing the norR gene and the norR-norV promoter.
[SEQ ID NO: 3]
Represents the base sequence of the forward primer for amplifying the luxCDABE gene.
[SEQ ID NO: 4]
Represents the base sequence of the reverse primer for amplifying the luxCDABE gene.

Claims (9)

A photogenic gene that encodes a photoprotein and is a luxCDABE gene ;
A series of gene sets that induce the expression of the luminescent gene;
Including
Said gene set is to detect a regulatory gene Norr, to induce transcription of the light-emitting gene by a protein NorR encoded by the regulatory gene Norr, a promoter site is a promoter site nor operon, the the including nitric oxide A nitric oxide sensor cell comprising a transformant obtained by transforming a host which is Escherichia coli or Salmonella using a recombinant vector for transformation. The nitric oxide sensor cell according to claim 1 , wherein the host is enterohemorrhagic E. coli EHEC EDL933. A method for detecting or quantifying intracellular nitric oxide, comprising measuring a signal change in a nitric oxide sensor cell according to claim 1 or 2 . A method for monitoring changes in nitric oxide concentration in cells due to stimulation, wherein stimulation is applied to the nitric oxide sensor cell according to claim 1 or 2, and signal change before and after the stimulation is measured. Monitoring method. A method for detecting or quantifying nitric oxide produced by a test cell, the test cell for detecting or quantifying nitric oxide production, and the nitric oxide sensor cell according to claim 1 or 2 , And measuring the signal change in the nitric oxide sensor cell, a method for detecting or quantifying cell-produced nitric oxide. The detection or quantification method according to claim 5 , wherein the test cell is a phagocytic cell. The detection or quantification method according to claim 5 or 6 , wherein the phagocytic cell is a macrophage. The detection or quantification method according to claim 7 , wherein the macrophages are derived from a living body suffering from an infectious disease. The detection or quantification method according to claim 5 , wherein the test cell is an intestinal epithelial cell, and the nitric oxide sensor cell is derived from Salmonella.