KR20100079579A - Manufacturing method of bactriophage in which molecular recognition material is arranged and using method of the same for detecting extremely small quantities and mass analysis device using the same - Google Patents

Abstract

PURPOSE: A method for preparing bacteriophage in which molecular recognition material is aligned is provided to use in mass spectrometer which is able to specifically detect a small amount of materials. CONSTITUTION: A method for preparing bacteriophage in which molecular recognition material is aligned comprises: a step of inserting a gene spacer corresponding to thrombine amino acid cutting sequence and signal amplification peptide to obtain a recombinant phagemid; a step of inserting a gene fragment corresponding to Z domain peptide to modified fd-SN-tet GGGGS linker to obtain a recombinant phage; and a step of preparing a phage having thrombin amino acid cutting sequence and a peptide for signal amplification. The body part of the bacteriophage is p8 surface protein and head part is p3 surface protein.

Description

Method for producing a bacteriophage in which a molecular cognitive substance capable of reacting specifically to a detection substance is displayed, a method for utilizing the bacteriophage prepared by the method for detecting a trace detectable substance, and a mass spectrometer using the bacteriophage { Manufacturing method of bactriophage in which molecular recognition material is arranged and using method of the same for detecting extremely small quantities and mass analysis device using the same}

The present invention provides a method for producing a bacteriophage in which a molecular cognitive substance that can be specifically reflected on a detection material and a method for utilizing the bacteriophage prepared by the manufacturing method to detect traces and a mass using the bacteriophage It relates to an analysis device. More specifically, the present invention is a molecular cognitive substance that can display a large amount of high signal output peptide on the body (p8 surface protein) of the bacteriophage and specifically react to the detection substance on the head (p3 surface protein). Method for producing the island-like filamentous bacteriophage displayed, the bacteriophage produced by the production method and the method using the bacteriophage to selectively and efficiently analyze when a trace amount of the detection substance is present in the complex mixture and the It relates to a mass spectrometer using bacteriophage.

The phage display technique was initiated in 1985 by Dr. George Smith for the first time suggesting the idea (Smith GR. 1985 Science. 228 (4705): 1315-1317). The idea originated from the principle that filamentous bacteriophages can display the desired peptide or protein outside of its surface protein by modifying it with gene combining techniques because the entire genome is not long. The filamentous bacteriophage has five types of surface proteins, such as 3, 6, 7, 8 and 9, of which proteins 3 and 8 are widely used for this display.

First, a sequence corresponding to a material (protein or peptide) of a type suitable for the application (protein or peptide) is added to the front of gene 3 or 8 in the phage genome in a reasonable size, and the recombinant phage gene group produced therefrom. When the bacteria are injected into the bacteria and grown, they can obtain a group of bacteriophages displaying the added substances on the surface of the surface protein 3 or 8 times. Screening is used to identify which individuals in this group capture the target material of interest, with high selectivity and high affinity. The use of this technique has led to the development of molecular recognition materials that can specifically recognize specific targets.

Currently, the method used for diagnosing diseases is mainly based on immune response diagnosis. In general, this diagnostic method using an antigen-antibody reaction uses spectroscopic signals such as fluorescence or luminescence, so it is often difficult to measure several different markers at the same time. In addition, spectroscopic signals may have a high degree of interference between the signals, which may result in inaccurate results. Diagnosis using mass spectrometry has been proposed as a way to supplement or replace these shortcomings. Mass spectrometry is an accurate method of measuring the intrinsic mass of a substance, which has the advantage of being able to precisely measure even small differences and simultaneously measuring individual substances even when they are mixed. This feature complements the shortcomings of spectroscopic diagnostic methods currently used.

The problem with mass spectrometry, which has many advantages, is that it still falls short of the sensitivity of the spectroscopic method. In other words, there is a need for signal amplification to detect traces of substances, and thus to have at least the same level of sensitivity as that of spectroscopic methods.

The present invention was developed to introduce a new direction of approach to achieve the necessity of such technology development, and to promote the development of high sensitivity diagnostic method using mass spectrometry.

In the present invention, the protein 8 of the bacteriophage was displayed with the thrombin cleavage sequence and the high signal output peptide connected to each other during mass spectrometry, and the protein 3 displayed the molecular cognitive substance that can directly or indirectly recognize the target substance.

The present invention has been made to solve the problems described above, an object of the present invention is to provide a method for producing a bacteriophage on which a molecular recognition material that can be specifically reflected on the detection material.

Another object of the present invention is to provide a bacteriophage prepared by the method for producing a bacteriophage in which a molecular recognition material that can be specifically reflected on a detection substance is displayed.

It is still another object of the present invention to provide a method of using a bacteriophage on which a molecular cognitive substance that can be specifically reflected on a detection substance is displayed to detect a trace amount.

Still another object of the present invention is to provide a mass spectrometer using bacteriophages in which molecular recognition materials are displayed which can be specifically reflected on a detection material.

The present invention provides a recombinant phagemid by inserting a gene spacer corresponding to a thrombin amino acid cleavage sequence and a signal amplifying peptide into a modified p8 phagemid to achieve the object as described above; (B) preparing a recombinant phage by inserting a gene segment corresponding to the modified Z domain peptide into the modified fd-SN-tet GGGGS linker; And (C) preparing a page having a modified Z domain attached to p3 and a thrombin amino acid cleavage sequence and a signal amplifying peptide at p8. A method for producing a bacteriophage on which molecular recognition materials are displayed is provided.

In another aspect, the present invention provides a bacteriophage in which a molecular recognition material is displayed which can be specifically reflected on a detection material, which is prepared by the above method.

The bacteriophage preferably displays a large amount of high signal output peptide on the body and a molecular cognitive material capable of reacting specifically with a detection material on the head.

The body is a p8 surface protein, the head is preferably a p3 surface protein.

It is preferable that the p8 surface protein is displayed with the thrombin cleavage sequence and the high signal output peptide connected to each other during mass spectrometry.

The bacteriophage is preferably an island-like bacteriophage.

In addition, the present invention provides a method for utilizing the bacteriophage to detect traces in order to achieve the object as described above.

In addition, the present invention provides a mass spectrometer that detects a trace detectable object using the bacteriophage in order to achieve the object as described above.

The bacteriophages of the present invention are displayed with molecular recognition materials that can be specifically reflected on the detection material. Therefore, the bacteriophage of the present invention can be utilized in a mass spectrometer that can specifically detect trace substances. One phage is attached to one antigen, but the signal is amplified because more than 150 peptides are suspended in one phage. Therefore, it is possible to effectively detect trace substances.

The present invention consists of three parts. The first part is the process of recombining the thrombin cleavage amino acid sequence and signal amplification peptide sequence to protein No. 8, which forms the trunk of an islet bacteriophage, using genetic recombination to reconstruct the phage genome and inject it into bacteria. The second part is the process of separating and purifying a large amount of bacteriophage displaying the thrombin cleavage amino acid sequence and the signal amplifying peptide on the body by culturing the bacteria containing the reconstituted bacteriophage genome. The third part is a process of analyzing the detection substance present in the trace amount in the sample present in the protein mixture phase using purified recombinant bacteriophage.

It will be described below in more detail through a specific experiment.

Experiment 1 A recombinant phagemid was prepared by inserting a gene spacer corresponding to a thrombin amino acid cleavage sequence and a signal amplifying peptide into the modified p8 phagemid shown in FIG. 2.

1) Preparation of phagemid fragments digested with NcoI and NotI and dephosphatylated

As shown in FIG. 2, 8 μg of modified pS1607 LVPRGS linker phagemid was added to 100 μl of total reaction solution and mixed well with 2 μl of restriction enzyme NcoI and 2 μl of restriction enzyme NotI, followed by 37 ° C. for 5 hours. After incubation at and purified, 2μl dephosphatase was added thereto, followed by incubation at 37 ° C. for 1 hour, followed by electrophoresis on agagel to separate DNA by heat extraction.

2) Preparation of spacers with thrombin cleavage site and peptide sequence for high signal output

As shown in FIG. 3, 50 μl of the forward primers (1for, 2for, 3for, 4for, 5for) and the reverse primers (1back, 2back, 3back, 4back, 5back) forming the spacers were respectively 50 μl into 5 μl total reaction solution. After incubation with 37 μl of polynucleic acid kinase and incubated at 37 ° C. for 1 hour, the two were mixed in equal amounts (1for and 1back, 2for and 2back, 3for and 3back, 4for and 4back, 5for and 5back) at 100 ° C. After heating in a water bath for 10 minutes, and then cooled to room temperature over 90 minutes to fuse with each other finally to obtain a spacer (1, 2, 3, 4, 5).

3) Coupling reaction between spacer and cleaved phagemid fragment

2 μl and 1 μl of the fused phagemid fragment cut with restriction enzyme were added to 20 μl of the total reaction solution, and 1 μl of ligase (ligase) was added thereto, followed by mixing for 18 hours at 16 ° C. It was incubated.

The incubation results were purified and injected into DH10beta bacterial cells prepared by electroshock method. The reaction conditions of the electroshock method were 12.5 kV / cm, 200 Ω, and 25 mF. After the electric shock work, the bacteria were tested on each agar medium plate containing ampicillin, and the surviving clones were selected. Bacteria were selected.

Selected bacteria were grown in 2xTY medium containing ampicillin, purified recombinant DNA therein, and sequenced using pS1607 Seq-Hoon primer (5'- TGC ACG CAT CCT CGC ATT ATC -3 '). It was confirmed whether the spacer entered the recombinant phagemid shown in FIG. 4.

<Experiment 2> Recombinant phage was made by inserting the gene fragment corresponding to the modified Z domain peptide into the modified fd-SN-tet GGGGS linker.

1) Preparation of Phosphate Sections Cleaved with SfiI and NotI and Dephosphatase-treated

6.5 μg of modified fd-SN-tet GGGGS linker phage was added to 100 μl of total reaction solution, mixed well with 2.5 μl of restriction enzyme SfiI, incubated at 50 ° C. for 16 hours, and purified. After addition of the restriction enzyme NotI of the reaction, the reaction was incubated at 37 ° C. for 4 hours, followed by purification. After adding 2 μl of dephosphatase, the reaction was incubated at 37 ° C. for 1 hour, followed by electrophoresis on agagel, followed by heating extraction. DNA was isolated.

2) Preparation of a fragment containing the modified Z domain peptide sequence

A modified Z domain peptide sequence as shown in FIG. 6 was inserted into 5.3 μg of the fd-SN-tet GGGGS linker, and the recombinant phage was added to 100 μl of total reaction solution and mixed well with 2.5 μl of restriction enzyme SfiI. After incubation at 50 ° C. for 16 hours, purification was carried out, and 2.5 μl of restriction enzyme NotI was added thereto, followed by incubation at 37 ° C. for 4 hours, followed by purification. Was separated.

3) Linkage of the fragment with the modified Z domain peptide sequence to the cleaved phage fragment

2 μl and 0.4 μl of the phage fragment cut with restriction enzyme were added to 20 μl of the total reaction solution, and 1 μl of ligase (ligase) was added thereto, followed by mixing at room temperature for 16 hours at 16 ° C. Reaction.

The incubation result was purified and injected into DH10beta bacterial cells prepared by electroshock method. The reaction conditions of the electroshock method were 12.5 kV / cm, 200 Ω, and 25 mF.

After the electric shock work, the bacteria were applied to the tetracycline-containing agar medium plate, and the surviving clones were selected to select the bacteria that had been properly recombined with the phage.

As shown in FIG. 7, the selected bacteria were grown in 2 × TY medium containing tetracycline and purified therein, and the recombinant DNA was purified therefrom and fd-tet VIII Hoon primer (5′-TCT TTA GTC CTC AAA GCC TCC − Sequencing was performed using 3 ') to confirm that the correctly modified Z domain sequence was introduced into the recombinant phage.

<Experiment 3> The recombinant phage was purified in Experiment 2, and the phagemid recombined in Experiment 1 was put into K91BluKan cells to make starved cells. Purified phage in Experiment 2 was infected with starved cells to amplify and purify the desired phage for 16 hours.

1) In the experiment 2, the selected bacterium identified as containing the recombinant phage inserted into the gene fragment corresponding to the modified Z domain peptide was grown in 2xTY medium containing 1 mM IPTG and tetracycline, and then the supernatant was centrifuge. Transfer to a new tube and remove bacteria.

Transfer 1/5 volumes of PEG / NaCl to the supernatant in a new tube, mix well, and place on ice for 1 hour in a cold room. Remove the supernatant by centrifuge and invert the tube and dry for 5 minutes. Dissolve the phage pellet in PBS buffer and once again transfer the supernatant with dissolved phage to a new tube by centrifuge. After repeating this process once again, dialysis is carried out in PBS buffer and the purification of the pie is completed using a 0.45 μm filter.

2) In experiment 1, phagemid recombined to insert a gene corresponding to a thrombin amino acid cleavage sequence and a signal amplifying peptide into p8 phagemid was injected into K91BluKan bacterial cells prepared by electroshock method. The reaction conditions of the electroshock method were 12,5kV / cm, 200Ω, and 25mF.

After the shock, the bacteria were applied to the agar medium plate containing ampicillin and kanamycin, and the surviving clones were selected to select the bacteria that had been properly recombined with the phage.

After growing selected bacteria, 100 μl of bacteria were placed in a terrific broth and grown until 1/10 diluted bacteria were grown between OD ₆₀₀ and 0.1 to 0.2 to produce starved cells containing phagemid.

3) Amplify the desired phage for 16 hours by infecting the phage prepared in Experiment 2 with the thus produced starved cells. By centrifuge, the supernatant is transferred to a new tube and starved cells are removed.

Transfer 1/5 volumes of PEG / NaCl to the supernatant in a new tube, mix well, and place on ice for 1 hour in a cold room. Remove the supernatant by centrifuge and invert the tube and allow to dry for 5 minutes. Dissolve the phage pellet in PBS buffer and once again transfer the supernatant with the phage dissolved into a new tube by centrifuge. After repeating this process once again, dialysis is carried out in PBS buffer and purified using a 0.45 μm filter.

Finally, p3 has a modified Z domain, and p8 has a phage containing a thrombin amino acid cleavage sequence and a signal amplification peptide. The purified recombinant bacteriophage thus obtained is present in a very small amount in a sample mixed with a protein. It can be used to analyze the detection material, such a method can be employed in a precise mass spectrometer.

The principle of detection of trace substances is explained. Proteomics samples contain a mixture of proteins. It is the purpose of this patent to detect very small amounts of them and to see if they differ from the control. Recognition of trace substances can be the path by which a small antibody displayed on phage surface protein 3 directly captures the substance, or the z-domain protein that catches the antibody is displayed on the phage surface protein 3 and then it catches the antibody. Indirect pathways in which the antibody recaptures traces are possible. Traces in the sample, on the other hand, must be immobilized. For example, one may hang an antibody that specifically reacts with the tracer on a certain wall, treat the sample mixture on it, and then incubate it. When the phage is processed on the immobilized trace material, the phage is displayed on the protein 3, and the phage material is attached to the trace material again. As a result, the phage that is attached to the trace material is immobilized. Then, the protease is processed to cut out the body of the phage, that is, the peptide displayed on protein 8 (150copy / phage). If the trace material (antigen) is in the sample, the peptide on the protein 8 is cut out. By measuring the mass spectroscopy of this peptide, trace substances can be detected and mass spectrometry can be performed. One phage is attached to one antigen, but the signal is amplified because more than 150 peptides are hung in each phage.

The present invention mainly targets the detection, but may be a different type of receptor depending on what the trace substance in the sample is. For example, if it is an EGF, the receptor may be an anti-EGF antibody but may be an EGFR receptor (EGFR). Displaying this is accomplished by adding this receptor gene to the phage gene using genetic recombination techniques. For example, a small antibody (ScFv, VH) or receptor gene can be inserted in front of the phage 3 gene to achieve this. Thus, a protein that can capture a receptor (Z-domain: captures the Fc domain of an antibody, streptavidin like S-tag: Biotin) is displayed on protein 3, rather than directly displaying the receptor on phage 3 protein. Either by catching the antibody or by catching an antibody that is conjugated with streptavidin can be employed.

On the other hand, the method of measuring the content of the detection material and the method of processing the molecular cognitive material when the content of the signal to determine the strength of the signal, as described above by inferring the content of the detection material by quantifying the cut out peptide. The peptide used at this time is composed of peptides that are sensitive to MALDI-TOF.

The present invention can be used in various industrial fields such as biotechnology industry, pharmaceutical industry, diagnostic industry, ultra-precision mass spectrometry industry.

1 is a schematic diagram of a bacteriophage utilized for mass spectrometry using the bacteriophage display technique,

2 is a modified pS1607 LVPRGS linker nucleotide sequence according to the present invention,

Figure 3 is a forward & reverse primer and spacers for the spacer according to the present invention,

4 is a nucleotide sequence of a recombinant region of the spacer according to the present invention,

5 is a nucleotide sequence of fd-SN-tet according to the present invention,

6 is a modified Z domain nucleotide and peptide sequence according to the present invention, and

7 is a recombinant phage salt of a portion containing a modified Z domain according to the present invention

This is a sequence.

Claims (12)

(A) preparing a recombinant phagemid by inserting a gene spacer corresponding to a thrombin amino acid cleavage sequence and a signal amplifying peptide into the modified p8 phagemid; (B) preparing a recombinant phage by inserting a gene segment corresponding to the modified Z domain peptide into the modified fd-SN-tet GGGGS linker; And (C) p3 is attached to a modified Z domain, and p8 to prepare a phage having a thrombin amino acid cleavage sequence and a signal amplifying peptide; molecular recognition that can be specifically reflected on the detection material comprising a; Method for producing a bacteriophage on which the material is displayed. The method of claim 1, Step (A) is  (A-1) pS1607 LVPRGS linker phagemid mixed with restriction enzymes NcoI and restriction enzyme NotI, incubated at predetermined temperature and time conditions, purified after addition of dephosphatase and constant temperature at predetermined temperature and time conditions After the reaction, electrophoresis to separate and extract DNA;  (A-2) preparing a spacer having a thrombin cleavage site and a peptide sequence for high signal output;  (A-3) After mixing the ligated enzyme obtained in the step (A-1) and the fused spacer obtained in the step (A-2), the incubation reaction at a predetermined temperature and time conditions Making; And (A-4) injecting the resultant incubation reaction with the ligase into the bacterial cells by electroshock method; the bacteriophage on which the molecular recognition material is displayed, which can be specifically reflected on the detection material, comprising: Method of preparation. The method of claim 1, Step (B) is  (B-1) The modified fd-SN-tet GGGGS linker phage was mixed with restriction enzyme SfiI, incubated at a predetermined temperature and time condition, purified, and then dephosphatase was added to the predetermined temperature and time condition. After incubation, electrophoresis to separate and extract DNA; (B-2) inserting the modified Z domain peptide sequence into the fd-SN-tet GGGGS linker and mixing the restriction enzyme SfiI to the recombinant phage, and then incubated at a predetermined temperature and time conditions for purification. Adding a restriction enzyme NotI and then incubating at a predetermined temperature and time condition, followed by electrophoresis to separate and extract DNA; (B-3) incubating the fragment having the modified Z domain peptide sequence and the cleaved phage fragment with the linking enzyme and incubating at a predetermined temperature and time condition; (B-4) Bacteriophage in which the molecular recognition material that can be specifically reflected on the detection material, comprising the step of injecting the resultant reaction with the ligase to the bacterial cell by electric shock method Method of preparation. The method of claim 1, Step (C) is (C-1) Selected bacteria identified as having a phage recombined so that the gene fragment corresponding to the modified Z domain peptide in step (B) are grown in a medium containing IPTG and tetracycline and centrifuged Separating and removing bacteria and purifying bacteriophage; (C-2) preparing a bacterium containing a phagemid recombined to insert a gene corresponding to a thrombin amino acid cleavage sequence and a signal amplifying peptide in p8 phagemid in step (A) as a starved cell; (C-3) amplifying and purifying the bacteriophage by infecting the phage prepared in the step (B) to the starved cell; molecular recognition material that can specifically reflect the detection material comprising a Method for producing this displayed bacteriophage. 3. The method of claim 2, Step (A-2), After the addition of the polynucleic acid kinase to the forward primer (1for, 2for, 3for, 4for, 5for) and the reverse primer (1back, 2back, 3back, 4back, 5back) constituting the spacer, and incubated at a predetermined temperature and time conditions, Mix the mixture of any one of the forward primer and the reverse primer (1for, 1back), (2for, 2back), (3for, 3back), (4for, 4back), and (5for, 5back), and then set a predetermined temperature. And a bacterio having a molecular recognition material that can be specifically reflected on a detection material, wherein the heating is performed under a time condition and then fused by cooling to a predetermined temperature and time condition to obtain a fused spacer. Method of making phage. A bacteriophage in which a molecular cognitive substance, which can be specifically reflected on a detection substance, is produced by the manufacturing method of any one of claims 1 to 5. The method of claim 6, Molecular cognitive material that can be specifically reflected in the detection material, characterized in that the display of a large amount of high signal output peptide on the body and the molecular cognitive material that can react specifically to the detection material on the head portion Displayed bacteriophages. The method of claim 7, wherein The body is a p8 surface protein, wherein the head is a p3 surface protein is a bacteriophage in which a molecular recognition material is displayed that can be specifically reflected on the detection material. The method of claim 8, The p8 surface protein is a bacteriophage exhibited with a molecular recognition material that can be specifically reflected on the detection material, characterized in that the high signal output peptide is displayed while connecting the thrombin cleavage sequence and mass spectrometry. The method of claim 6, The bacteriophage A bacteriophage in which a molecular cognitive substance that can be specifically reflected on a detection substance, which is an island-like bacteriophage, is displayed. A method of utilizing the bacteriophage of claim 6 to detect traces. Mass spectrometer utilizing the bacteriophage of claim 6.

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