KR20130063012A - Rna aptamers for e.coli o157:h7 - Google Patents

Abstract

PURPOSE: An RNA aptamer is provided to detect E.coli O157:H7 by specifically binding to E.coli O157:H7, to prevent E.coli O157:H7, and to prevent food spoilage. CONSTITUTION: An RNA aptamer has a base sequence selected from a group consisting of sequence numbers 1-4, which specifically binds to E.coli O157:H7. Urecil(U) and cytosine(C) of sequence number 1 or 2 have 2' hydroxyl group which is substituted with a fluorine group. A biosensor for detecting E.coli O157:H7 contains the RNA aptamer. A pharmaceutical composition for preventing or treating diseases selected from a group consisting of food poisoning, hemolytic uremic syndrome(HUS), and hemolytic anemia contains the RNA aptamer. A food additive composition contains the RNA aptamer.

Description

RNA APTAMERS FOR E.COLI O157: H7 for E. coli O157: H7 strain

The present invention E. coli O157 : H7 (E. coil O157 : H7 ) relates to an RNA aptamer specifically binding.

E. coil is Escherichia, the scientific name for Escherichia coli stands for coli, yikolrayi O157: H7 strain of one of the proteins H7 O-type O, one of the anti-serum with anti-O157 agglutination reaction causes the O (cells) and antigen, H antigen in the serum with the Escherichia coli surface It has an H (flagella) antigen that causes agglutination reaction.

E. coli O157 : H7 The strain was first discovered in the United States in 1982. It is a type of pathogenic Escherichia coli that inhabits the intestines of humans or animals and causes diarrhea or abdominal pain. It belongs to the intestinal hemorrhagic Escherichia coli and usually causes bloody diarrhea. This is because it is difficult to distinguish it because its shape and size are similar to those of general E. coli, and to distinguish it, it is possible to observe agglutination reaction with a certain antigen antibody in vitro or use serological method. protein O-antigen are located in geotyieoseo found to 157th of the many types of proteins O157 O antigen It is called.

E. coli O157 : H7 The bacterium is highly contagious and has an average incubation period of 3 to 8 days, which makes it difficult to determine the cause of food poisoning and is difficult to prevent.This bacteria, once invaded into the human body, causes abdominal pain, diarrhea and bloody stools. It produces a toxic protein compound called vero toxin, which, when spread through the body, causes hemolytic uremic disease that destroys red blood cells and attacks the kidneys, causing the kidneys to lose function. Gastric uremic disorders are secondary to the nervous system, respiratory system, and circulatory system, and eventually lead to death. Death rates range from 5% to 10% and are reported to be infectious at all ages. Cause.

E. coli O157 : H7 The bacterium is infected when live or meat, hamburgers, or vegetables contaminated with animal stools are cooked below 65 ° C, or when drinking water contaminated with animal stools. E. coli O157 : H7 It is also infected by patients infected with the bacterium. In 1996, a large amount of food poisoning occurred to elementary school children in Japan, causing social problems, and it is widely known as a dangerous substance of food poisoning in the United States. According to the US Centers for Disease Control, poor cooking when making hamburgers causes the bacteria to die and cause food poisoning, resulting in more than 20,000 cases of food poisoning per year, resulting in 200-500 deaths.

In Korea, the number of food poisoning cases was 109 in 2005 and recorded 5711 patients. In 2007, the number of patients recorded 510 cases and 9686 cases, which increased to 228 cases and 5999 patients in 2009. The trend is decreasing. However, when eating out is becoming more common in recent years and food poisoning is occurring in restaurants and group lunch centers, Ekolai O157 : H7. There is a need for the prevention and treatment of food poisoning against bacteria.

Conventional E. coli O157 : H7 For the study of strains, E. coli is used for immunological diagnosis using monoclonal antibody of Korean Patent Publication No. 1999-68868 (E. coli O157 : monoclonal antibody for H7 detection and use). O157 : H7 For use in a method that can quickly and accurately detect the contamination of bacteria and the Korean Patent Publication No. 1999-65107 (E. coli O157 : H7 using a polymerase chain reaction) Pathogenic Escherichia coli (E. coli O157 : H7 ) using polymerase chain reaction method (PCR) of the simultaneous detection of multiple genes of strains By detecting four specific genes (two verotoxins, barrier-associated pathogens, and enzyme-specific genes) possessed by the strain, there are some that are applicable not only to pathogenic E. coli testing of meat but also to animals and humans. It is not a method for detecting E. coli O157: H7 bacteria using aptamer).

Aptamers are single-chain DNA or RNA that can specifically bind to various compounds, such as biomolecules such as proteins and amino acids or small organic chemicals such as environmental hormones, and evolve using oligonucleotide libraries called SELEX. By conventional methods, oligomers which have a high affinity and selectivity for specific chemical or biological molecules are separated and obtained.

Aptamers, which are recognized as replacements for antibodies, can be used as elements of biosensors that can recognize molecules in detection and analysis systems, just like antibodies. Oligonucleotide-based aptamers have several advantages over protein-based antibodies. First, the aptamer is obtained in vitro, and second, various organic and inorganic substances including toxic can be used as target molecules of the aptamer. When specific aptamers that specifically bind are identified and obtained, they can be reproduced with low cost and high consistency by automated oligomer synthesis. It is also relatively easy for aptamers to introduce useful functional groups such as fluorescent molecules or photoreactive groups, and the structure of aptamers can have a longer time than that of antibodies because of the reversible process of denaturation by heat. have.

 Among these aptamers, RNA aptamers select specific RNA molecules, namely aptamers, from any RNA library through repeated in vitro screening process. RNA libraries are superior to other biological or synthetic libraries for selecting high affinity aptamers. Thus there is a growing interest in the potential use of RNA aptamers as therapeutic agents, where high affinity RNA aptamers can be selected by the SELEX process. In addition, RNA aptamers have an advantage as inhibitors over small chemicals because they generally provide a broad binding site for the target protein. Pathological protein-protein interactions can be excellent targets for RNA aptamers because high affinity RNA binds to the target protein and interferes with the binding of other proteins in the complex. Moreover, RNA aptamers can be expressed as intramers in cells using RNA vector systems.

Due to the properties of aptamers that specifically bind to these targets, a lot of research has recently been conducted to use aptamers for research in drug development, drug delivery systems, and biosensors. Patents for Korean Patent Publication No. 2004-54412 disclose RNA aptamers that promote coagulation and fibrinolysis, while Korean Patent Publication No. 2002-7009983 is used to signal the presence of cognate ligands in solution. Aptamers containing reporter molecules are described.

But E. coli O157 : H7 RNA aptamers that specifically bind to the strain are not disclosed.

The present invention is E. coli O157 : H7 An object of the present invention is to provide a RNA degrading enzyme resistant RNA aptamer and its use.

The present invention is E. coli O157 : H7 RNA degrading enzyme resistant RNA aptamers that specifically bind to the strain and have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 4 where the 2 'hydroxyl group of uracil (U) and cytosine (C) is substituted with a fluorine group To provide.

In addition, the present invention provides a biosensor comprising the above RNA aptamer.

In addition, the present invention provides a pharmaceutical composition and food additive composition for the prevention and treatment of food poisoning, hemolytic uremic syndrome and hemolytic anemia comprising a stomach RNA aptamer.

RNA aptamer of the present invention is E. coli O157 : H7 as a major cause of food poisoning By specifically binding to strains E. coli O157 : H7 It is useful for the detection of strains.

RNA aptamer of the present invention is E. coli O157 : H7 By specifically binding to the strain and inhibiting its action, it is possible to prevent the corruption of food.

RNA aptamer of the present invention is E. coli O157 : H7 It can be usefully used in the pharmaceutical composition for the prevention and treatment of various diseases caused by the strain, namely food poisoning, hemolytic uremic syndrome and hemolytic anemia.

1 shows E. coli using the SELEX method 0157 : Schematic diagram for discovering RNA aptamer specific for H7 strain.
2 shows E. coli O157 : H7 in SERNA aptamer clones 6 It shows the result of performing real-time PCR to confirm the binding to the strain.
Figure 3 shows the nucleotide sequence of 6 times SERNA aptamer clones.
4 shows E. coli O157 : H7 in SERNA aptamer clones 6 The result of performing ELISA to select the aptamer binding to the strain is shown.
FIG. 5 shows E. coli O157 : H7 in clones ＃ 13, ＃ 28,, 40, ＃ 49 RNA aptamers selected by ELISA Real-time PCR was performed to select RNA aptamers that bind to the strain with high affinity.
Figure 6 shows the structure of the cloned # 40 RNA aptamer selected by real-time PCR and the structure and location of the miniaturized aptamer.
Figure 7 shows the results confirmed by binding assay (specific binding) of the selected clone ＃ 40 RNA aptamer and miniaturized aptamer and E. coli O157: H7 strain.
Figure 8 shows the results confirmed by ELISA binding of the selected clone ＃ 40 RNA aptamer and miniaturized aptamer and E. coli O157: H7 strain LPS.

The present invention is E. coli O157 : H7 It binds specifically to and has a base sequence selected from the group consisting of SEQ ID NOs: 1 to 4 in which the 2 'hydroxyl group of uracil (U) and cytosine (C) is substituted with a fluorine group, and thus, mains such as food poisoning. yikolrayi causing O157: useful in the detection of the H7 strains, and yikolrayi O157: by specifically binding to H7 strains by inhibiting the action it is possible to prevent food spoilage of, the group consisting of food poisoning, hemolytic uremic syndrome, and hemolytic anemia It relates to RNA aptamer useful for the prevention and treatment of diseases selected from.

Hereinafter, the present invention will be described in more detail.

The RNA aptamer of the present invention specifically binds to E. coli O157 : H7 . The RNA aptamer of the present invention preferably has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 4, but is not limited thereto and adds an arbitrary nucleotide sequence to the base sequence selected from the group consisting of SEQ ID NOs: 1 to 4. Even if it is included as long as it can specifically bind to E. coli O157 : H7 can be considered to be substantially the same as the RNA aptamer of the present invention.

In the nucleotide sequences of SEQ ID NOs: 1 to 4, uracil (U) and cytosine (C) are those in which the 2 ′ hydroxyl group is substituted with a fluorine group. This modification is carried out to produce RNA resistant to RNA degrading enzymes.

Yikolrayi O157 in foods beverages because it binds specifically to H7 Purpose: This is one of RNA aepta major strain causing dimmer poisoning of the invention yikolrayi O157 may determine that it contains the H7 strains, the patient yikolrayi O157: H7 It can function as a sensor to check whether a number of diseases caused by food poisoning, hemolytic uremic syndrome and hemolytic anemia.

In addition, since the RNA aptamer of the present invention can specifically bind to E. coli O157 : H7 and decrease its function, the pharmaceutical composition comprising the same has various diseases caused by E. coli O157 : H7 , that is, food poisoning, hemolytic uremic syndrome and hemolytic It can be effective in preventing and treating anemia.

In addition, the use of the RNA aptamer of the present invention in food additives prevents the propagation of E. coli O157: H7 .

Hereinafter, preferred embodiments are provided to help the understanding of the present invention, but it is obvious to those skilled in the art that various changes and modifications can be made within the scope and spirit of the invention only to illustrate the present invention. It goes without saying that it belongs to the following claims.

Example  One

Example

One. RNA Of app tamer  excavation

To prepare the RNA library required to perform the SELEX process, a DNA library was prepared by PCR with the 5'-primer and 3'-primer below using a single oligonucleotide containing 60 bases as a template. The 5-primer contains a T7 RNA polymerase promoter portion for synthesizing RNA.

5'-GGGTAATACGACTCACTATAGGGATACCAGCTTATTCAAT-3 '(5'-primer)

5'-AGATTGCACTTACTATCT-3 '(3'-primer)

PCR was performed by mixing 0.25 μM 5′-primer, 0.25 μM 3′-primer, 10 × PCR buffer, 200 μM dNTP mixture, DNA tap polymerase (Finzyme) 3 units. As a PCR cycle, 10 cycles were repeated under conditions of 95 ° C. 30 seconds, 55 ° C. 30 seconds, and 72 ° C. 30 seconds, and finally, various DNA libraries were prepared at 72 ° C. for 7 minutes.

As a template, a DNA library having various base sequences was prepared using a T7 RNA polymerase (Epicentre Technologies) as a template, followed by in vitro transcription, to prepare an RNA library having various base sequences.

In this case, by the transcription of a template synthesized in vitro using 2 'deoxy 2' fluorine CTP and UTP (Epicentre Technologies) and normal GTP, ATP and T7 RNA polymerase to prepare RNA resistant to RNA degrading enzyme An RNA was produced in which the fluorine group was modified every two times with pyrimidine groups.

Specifically, 20 μl of DNA library, 5X transcription buffer, 5 mM DTT, 5 mM ATP, GTP, 2'-F CTP, 2'-F UTP, T7 RNA polymerase, DEPC-H2O and reacted for 6 hours at 37 ° C. I was. RNA library was extracted from 7M urea-8% polyacrylamide gel after treatment with DNaseI (Epicentre Technologies) at 37 ° C. for 30 minutes to remove the DNA used as template.

The following RNA library sequences obtained comprise N60 representing 60 nucleotides incorporated into the same mole of A, G, C and U positions respectively.

5 'ATA CCA GCT TAT TCA AT (N60) AGA TAG TAA GTG CAA TCT 3'

1 shows E. coli O157 : H7 using SELEX method Schematic diagram showing the excavation of RNA aptamer specific to the strain is shown below. O157 : Details of the discovery process of RNA aptamers specific for the H7 strain.

Using the counter SELEX technique, a 300 picomol RNA library was used as a starting material and 3X10 ⁶ K12 for 10 minutes at room temperature. strain cells were reacted in 200 μL binding buffer (30 mM TrisHCl, pH 7.5, 150 mM NaCl, 1.5 mM MgCl ₂ , 2 mM dithiothreitol, and 1% BSA), resulting in K12 strain Non-specific RNA reacted with was precipitated and removed by centrifugation. The supernatant was then transferred to a new tube and reacted with 1 × 10 ⁶ O157 : H7 strains at room temperature for 10 minutes. Then centrifugation O157 : After removing the RNA that did not bind with H7 pellet was washed 5 times with 400ul binding buffer. Thereafter, 200ul of Elution buffer (10mM EDTA) was added thereto, followed by heat treatment at 95 ° C. for 5 minutes to extract the bound RNA. The RNA thus obtained was subjected to six selections by repeating reverse transcription-gene amplification and in vitro transcription. After making six selections, the amplified DNA was cloned and 43 nucleotide sequences were analyzed.

Figure 3 shows the nucleotide sequence of 6 SERNA (RNA obtained after 6 selection) aptamer clones analyzed through the above process.

2. Excavated Of the aptamers (SERNA aptamer clone 6) Ekolai O157 : H7 Coupling test with strain (real time PCR Use)

2 shows SERNA aptamer pool 6 and E. coli. O157 : specific binding to the H7 strain was confirmed by real-time PCR.

Hereinafter, the RT PCR and the real-time PCR process performed using the RNA aptamer and the initial RNA library where the SELEX process was performed six times will be described in detail.

RNA aptamers and initial RNA libraries subjected to SELEX 6 procedures were 6 picomolar and 5X10 ⁶ O157 : H7 and K12, respectively. After reacting each strain , RNA was bound to each other. After RNA was obtained through phenol extract / ethanol deposition process, DEPC-H2O was added to dissolve RNA, 200 nM 3 ′ primer was added thereto, heated at 65 ° C. for 5 minutes, and RNA and 3 ′ primer were combined at room temperature for 10 minutes.

After adding 1 mM dNTP, 5X RTase buffer, 200 units M-MLV RTase (Finzzyme) for 1 hour at 42 ° C, RTase was inactivated at 95 ° C for 5 minutes. Only one third of the back-transferred single DNA was used as a PCR template.

At this time, real-time PCR (Rotor Gene RG-6000) was used to measure the amount of generated DNA under the following conditions: cDNA, 100 nM 5 'primer, 100 nM 3' primer, 10 μl PCR buffer 10 μl, 500 μM dNTP , 7X of 5X RTase buffer, 100X Syber green, 2 unit tap polymerase (Finnzyme) was mixed and maintained at 95 ° C for 5 minutes, followed by 40 cycles of 95 ° C 30 seconds, 58 ° C 30 seconds, 72 ° C 30 seconds. The amount of RNA obtained is measured repeatedly.

Real-time PCR is a technology that monitors and interprets the increase in PCR amplification products in real time. As the amount of initial DNA increases, the amount of amplification products quickly reaches a detectable amount, and thus the amplification curve rises steeply.As a result of real-time PCR using a standard sample diluted in stages, the amount of initial DNA is amplified side by side at regular intervals. A curve can be obtained. In this case, setting the minimum threshold that causes a reaction in a proper place calculates the point where the minimum threshold that causes a reaction meets the amplification curve (CT value), which is obtained when the PCR amplification product is amplified by a certain amount. The number of cycles means that the greater the initial DNA amount, the faster the amount of amplification products can be detected. Therefore, the lower the CT value compared to the control group, the higher the initial DNA amount.

Looking at the average CT value of Figure 2 SERNA pool 6 + O157 : H7 , library SERNA pool + O157 : H7 , library RNA pool + K12 strain , SERNA pool 6 + K12 be seen that the CT values increase in the order of the strain which there yikolrayi the RNA pool obtained after the SELEX process, a single 6 O157: H7 This means that there is a large amount of RNA aptamer specifically binding to the strain, which means that the SERNA pool 6 is E. coli. O157 : it can be confirmed that the specific binding to the H7 strain.

3. Excavated Of app tamer  Selection

3-1. Excavated Of app tamer O157 : H7 Screening by measuring binding with strains ( ELISA  Streptavidin coated plate Use)

Figure 4 shows each of the RNA aptamer clones and E. coli discovered through 6 SELEX O157 : The result of ELISA to confirm the binding to the H7 strain. Among the RNA aptamer clones discovered, clones ＃ 13 RNA aptamer, ＃ 31 RNA aptamer, ＃ 40 RNA aptamer, ＃ 49 RNA aptamer Ekolai It was confirmed that the binding to the O157 : H7 strain.

The ELISA process performed below will be described in detail.

An A16 tail was applied to the 3 'end of the excavated RNA aptamer and nonspecific RNAs. 30 picomolar of A16 tailed RNA and 10 picomolar of dT (16) -biotin were reacted at 10 占 DEPC-H 2 O at room temperature for 30 minutes. Bound hybrid RNA was immobilized on the plate for 30 minutes in a streptavidin-coated 96well plate. After incubation for 1 hour at room temperature with blocking buffer (PBS with 5% BSA / yeast tRNA (10ug) and then O157 : H7 strain of 5X10 ⁶ 100μL binding buffer (0.05% Tween-20, 1.5mM MgCl ₂ , 1XPBS) After releasing to each well and treated each well for 20 minutes at room temperature 0157 : H7 strain was bound. After washing three times with binding buffer (0.05% Tween-20, 1.5mM MgCl ₂ , 1XPBS), the primary antibody (100ng / well) against O157 : H7 strain was reacted at room temperature for 1 hour. After washing three times with buffer solution (0.1% Tween-20, 1.5mM MgCl ₂ , 1XPBS), 100 μl binding buffer (0.05% Tween-20, 1.5mM MgCl ₂ , HRP-conjugated secondary antibody) was added. 1 XPBS) for 1 hour. After rinsing three times with buffer solution, 100 μl of QuantaBlu peroxidase substrate solution (Pierce) was released, and fluorescence was measured at a wavelength of Excitation 325 nm and Emssion 420 nm with a fluorometer (Fluoroskan, Thermo Scientific).

3-2. ELISA Selected via RNA Aptamer  Clones O157 : H7 Measurement of binding to strains (real time PCR Use)

Also, Ekolai O157 : To identify clones that bind with a high affinity for the H7 strain, a library pool, clone ＃ 28 RNA aptamer and ELISA as a control showed a 2-3-fold increase compared to the control ＃ 13 RNA aptamer, ＃ 31 Real-time PCR was performed on RNA aptamers, # 40 RNA aptamers, and # 49 RNA aptamers. Since the real-time PCR performed is the same as the known method, a detailed description thereof will be omitted.

Referring to FIG. 5, clones ＃ 13 RNA aptamers, ＃ 31 RNA aptamers, ＃ 40 RNA aptamers, ＃ 49 RNA aptamers and library pools, clones ＃ 28 clones ＃ 40 RNA aptamers that were subjected to real-time PCR were performed. Lee Icolai O157 : H7 strain was confirmed to bind with a high affinity.

That is, E. coli among SERNA aptamers discovered Clone # 40 that specifically binds to O157 : H7 strain was selected.

4. cutting RNA Aptamer Production

As we have seen above, E. coli O157 : The clone ＃ 40 aptamer that specifically binds to the H7 strain was screened.In order to optimize the selected clone ＃ 40 RNA aptamer, the full length RNA aptamer was cut out stepwise to reduce the size of 64mer, 38mer, and 28mer. Aptamers were prepared by in vitro transcription using T7 polymerase. Figure 6 shows the nucleotide sequence and structure of the selected clone # 40 RNA aptamer and the nucleotide sequence and structure of the aptamers downsized to three sizes.

Hereinafter, a method of manufacturing a miniaturized aptamer into 64mer, 38mer, and 28mer will be described in detail.

The 5mer and 3 'primers were used for the 64mer clone ＃ 40LS RNA aptamer, respectively, and the 5' and 3 'primers were used for the 38mer clone ＃ 40L aptamer, respectively, and the 28mer clone ＃ 40S was used. Aptamers were amplified by PCR using 5 'primers and 3' primers, respectively.

The sequence of these primers is as follows.

5'-GGTAATACGACTCACTATAGGGTCTTCCTGGACTGTCGAAAA-3 '(64' clone 5 'primer of 5LS RNA aptamer)

5'-TTTTTTTTTTTTTTTTTACTATCTATAAACCAAATACG-3 '(64' clone 3 'primer of 40LS RNA aptamer)

5'-GGTAATACGACTCACTATAGGGTCTTCCTGGACTGTCGAAAA-3 '(38' clone 5 'primer of 40L RNA aptamer)

5'-TTTTTTTTTTTTTTTTCCTCCCGATACTGAATTTTCGACAGTC-3 '(38mer clone 3' primer of 40L RNA aptamer)

5'-GGTAATACGACTCACTATAGGGTACGTATTTGGTT-3 '(28mer clone 5' primer of 40S RNA aptamer)

5'-TTTTTTTTTTTTTTTTTACTATCTATAAACCAAATACG-3 '(28mer clone 3' primer of 40S RNA aptamer)

Specifically, PCR was performed by mixing 0.25 μM 5′-primer, 0.25 μM 3′-primer, 5X PCR buffer, 200 μM dNTP mixture, Phusion DNA tap polymerase (Finzzyme) 0.4 unit, and the PCR cycle was 95 ° C 30 seconds. After repeating 25 cycles under the conditions of 55 ° C. 30 sec, 72 ° C. 30 sec, a DNA template was prepared at 72 ° C. for 7 minutes. The amplified double-stranded DNA template was subjected to in vitro transcription using a T7 RNA polymerase, wherein pyrimidine nucleotides were substituted with 2'-fluorine groups. Each of the prepared RNA aptamers was purified by electrophoresis on urea polyacrylamide gel.

5. Selected RNA Aptamer  And his cutting Of app tamer Ekolai O157 : H7 Confirmation of binding specificity with strain

5-1. Clone ＃ 40 RNA Aptamer  And his Downsized Of app tamer O157 : H7 Measurement of binding to strains Streptavidin - HRP )

Figure 7 shows clone # 40 RNA aptamer and aptamer downsized to three sizes In order to confirm the specific binding to the E. coli O157: H7 strain, streptavidin-HRP for biotin was measured to confirm the binding degree by measuring fluorescence.

Referring to FIG. 7, only E. coli was treated with streptavidin-HRP used as a control and treated with library RNA. O157: H7, but rarely associated with strains, clones and clones # 40 RNA aptamer # 40LS RNA aptamer clone # 40L RNA aptamer clone # If the 40S RNA aptamer is K12 E. coli without binding to strain strain It was confirmed that specifically binding to O157 : H7 strain.

Hereafter, E. coli using clone ＃ 40 RNA aptamer and miniaturized aptamer streptavidin-HRP O157 : Details of the measurement of the degree of binding to the H7 strain.

An A16 tail was applied to the 3 'end of selected RNA aptamers and nonspecific RNAs. 5 picomolar of A16 tailed RNA and 10 picomolar of dT (16) -biotin were reacted at 10 占 DEPC-H 2 O at room temperature for 30 minutes. The bound hybrid RNA was reacted with O157 : H7 strain of ⁵ × 10 ⁶ in 200 μl binding buffer (30 mM TrisHCl, pH 7.5, 150 mM NaCl, 1.5 mM MgCl ₂ , 2 mM dithiothreitol, and 1% BSA) and precipitated. Washed three times with 400ul binding buffer. The pellet was released in 100 µl of streptavidin-HPR solution diluted 1: 1000 in 1X PBS, and then streptavidin-HRP and hybrid RNA were bound at room temperature for 30 minutes. After washing twice with 200 μl 1XPBS / MgCl 2, the pellet was released in 100 μl QuantaBlu peroxidase substrate solution (Pierce), and fluorescence was measured at a wavelength of Excitation 325 nm and Emssion 420 nm with a Fluorometer (Fluoroskan, Thermo Scientific).

5-2. Clone ＃ 40 RNA Aptamer  And his Downsized Of app tamer O157 : H7 Strain and K12  strain of LPS Combined measurement with

As discussed in FIG 7 clone # 40 and clone # 40LS RNA aptamer RNA aptamer, RNA aptamer clone # 40L, clone # 40S For RNA aptamer K12 E. coli without binding to strain strain O157 : binds specifically to H7 strain, to see if it binds to LPS, a label recognized as an external antigen LPS was extracted from O157 : H7 strain and K12 strain , and binding to clone 클 40 RNA aptamer and miniaturized aptamer was measured by ELISA.

Figure 8 shows the above results and the same as the results shown in the direct binding via streptavidin-HRP, LPS extracted from each strain, coated on a plate clone in the experiment of clone # 40 RNA aptamer and a small app Tamers K12 Compared with the LPS of the strain almost no binding to the LPS of the E. coli O157 : H7 strain was confirmed that the specific binding.

Below, O157 : H7 strain and K12 The process of producing LPS-coated plates of strains and clone ＃ 40 RNA aptamers and miniaturized aptamers were performed by O157 : H7 strain and K12. The experimental procedure for measuring binding to strain LPS is described in detail.

The LPS for the experiment was 1X10 ⁹ O157 : H7 Strain and K12 The strain was isolated using LPS extraction kit (Intron Biotechnology). The separated LPS was diluted in PBS at 100 ng / well and dispensed in 100 ul into 96 well plates (TPP), followed by reaction in a 37 ° C. incubator, followed by reaction at 4 ° C. for 16 hours. First, LPS-coated plates were incubated for 1 hour at room temperature with blocking buffer (PBS with 5% BSA / yeast tRNA (10 ug). Then, A16 tail was performed at the 3 'end of the selected RNA aptamer and nonspecific RNAs. 30 picomolar of A16 tailed RNA and 10 picomolar of dT (16) -biotin were reacted for 30 minutes in 10 DE DEPC-H2O at room temperature The combined hybrid RNA was reacted for 30 minutes in each LPS-coated 96well plate. After washing three times with binding buffer (0.05% Tween-20, 1.5mM MgCl ₂ , 1XPBS), the hybrid RNA conjugated with streptavidin-HRP and LPS was bound for 30 minutes at room temperature. 100 μl QuantaBlu peroxidase substrate solution (Pierce) was released and fluorescence was measured at a wavelength of Excitation 325 nm and Emssion 420 nm with a Fluorometer (Fluoroskan, Thermo Scientific).

E. coli of the present invention The aptamer nucleotide sequence that can bind to O157 : H7 strain with specific affinity was the same as the nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1-4.

Claims (5)

Ekolai O157 : an RNA aptamer having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 4 that specifically binds to H7 . The RNA aptamer according to claim 1, wherein uracil (U) and cytosine (C) of SEQ ID NOs: 1 to 4 are substituted with fluorine 2 'hydroxyl group. E. coli comprising the RNA aptamer of claim 1 or 2 O157 : H7 Detection biosensor. A pharmaceutical composition for preventing or treating a disease selected from the group consisting of food poisoning, hemolytic uremic syndrome and hemolytic anemia comprising the RNA aptamer of claim 1. Food additive composition comprising the RNA aptamer of claim 1 or 2.

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