KR101657881B1 - The peptide probes high specific and high selective for target biomarker, and the biochip for clinical prediction of acute kidney injury and chronic kidney disease - Google Patents

Abstract

The present invention relates to a peptide probe useful for early diagnosis of acute renal injury and chronic renal disease, and a biochip using the same. More specifically, the present invention relates to a molecular recognition peptide probe capable of highly specific and highly selective binding to a target protein NGAL used for testing acute renal damage or kidney disease, and a biochip using the same.
In addition, this method can be used for the diagnosis of acute kidney injury (AKI) or chronic kidney disease (CKD) including the response to treatment of kidney disease. And can be used to observe progress and process.

Description

TECHNICAL FIELD The present invention relates to a peptide probe capable of high specificity and high selective binding to a target biomarker, and a biochip for early diagnosis of acute and chronic renal disease using the peptide probe and a biochip for the same, chronic kidney disease}

The present invention relates to a peptide probe useful for early diagnosis of acute and chronic renal diseases, and a biochip using the same. More specifically, the present invention relates to a molecular recognition peptide probe capable of highly specific and highly selective binding to a target protein NGAL used for testing acute renal damage or kidney disease, and a biochip using the same.

This enables efficient early diagnosis of kidney damage or kidney disease. This method can also be used to monitor the progression and course of acute kidney injury (AKI) or chronic kidney disease (CKD), including responses to treatment of kidney disease.

The peptide probe of the present invention can be selectively fixed on a variety of substrates including gold, which is widely used in biochips, without chemical surface treatment, thereby simplifying the biochip manufacturing process, The manufacturing process is simplified as a whole, and a great improvement in productivity and economy can be expected.

The kidneys are located on both sides of the vertebrae in the waist of the human body. The body also functions as a main function of filtering out the metabolites and wastes in the body and discharging them into the urine. In addition to maintaining the body water content, electrolyte and acidity in a narrow range, And an endocrine function that maintains blood pressure, corrects anemia and produces and activates various hormones important for calcium and phosphorus metabolism.

Renal diseases which cause weakening of kidney function include glomerulonephritis, chronic renal failure, acute renal failure, nephrotic syndrome, pyelonephritis, kidney stones, and kidney cancer. Acute kidney injury (AKI) and chronic kidney disease (CKD) can be classified according to the progress of the deterioration of kidney function.

In the case of chronic kidney disease, the reduction of kidney function occurs slowly over several months and progresses to end-stage renal disease, which is usually unrecoverable, progressive and often requires dialysis therapy or kidney transplantation. On the other hand, acute renal damage is usually due to dehydration or hypotension, nephrotoxic substances or drugs, urinary tract obstruction, or the like, as renal function rapidly worsens within days or weeks. It is usually a conservative treatment that removes the causes of dehydration or renal failure, but it can restore the renal function. However, depending on the severity of the underlying disease, some can progress to chronic renal failure.

Despite the advances of modern medicine, many patients admitted to hospital are suffering from the decrease of renal function. Especially, patients with high severity of disease are often in need of renal replacement therapy because of decreased renal function. The prevalence of acute renal injury has been reported in about 5% of inpatients and in about 30-50% of patients admitted to the ICU, and this prevalence is steadily increasing despite the development of new therapies (Lameire et al., Lancet, 2005; Devarajan, Contrib Nephrol, 2007).

There are many possible causes of high acute renal injury mortality, but the lack of appropriate methods for early diagnosis of acute renal failure may be the main reason for missed treatment. The traditional method of evaluating kidney function is to indirectly reflect the degree of renal function by measuring serum creatinine. However, since serum creatinine is affected by individual body weight, age, gender, muscle mass, protein intake, and medication, there is a disadvantage in that it does not reflect changes in kidney function in real time. In other words, serum creatinine is elevated only if the renal function is lowered by more than 50%, so that diagnostic methods using serum creatinine have limitations for early diagnosis of acute kidney damage (Belcher et al., Am J Kidney Dis, 2011; Endre and Westhuyzen, Nephrology, 2008). Luckily, with the development and application of innovative genomics such as functional genomics and proteomics, several gene products have been identified with biomarkers.

NGAL (Neutrophil gelatinase-associated lipocalin) is a 25 kDa glycoprotein that binds to the epithelium of the neutrophil or renal tubule. It is a rapidly increasing biomarker when acute renal damage occurs for various reasons. Currently, it is mainly used to diagnose kidney dysfunction early and to determine the prognosis of kidney transplant patients.

In March 2008, a team of researchers from the Cincinnati Children's Hospital Medical Center published a report in the American Journal of Clinical Nephrology that measurement of biological markers, called NGAL, (Bennett et al., Clin J Am Soc Nephrol., 2008). In 196 patients with congenital cardiac anomalies who underwent surgery, the urinary "NGAL" was measured. In 51% of the patients, severe renal damage was observed after surgery. Among them, creatinine was measured 2 ~ 3 days after the increase , Whereas urinary 'NGAL' increased only in a few hours in children with such sequelae. Especially in some patients, the creatinine test was normal, but NGAL was 15 times higher than normal within 2 hours and 25 times higher in 4 hours.

The results of this study are useful in predicting the early diagnosis of acute renal impairment in the urine, and in estimating the risk of death, need for dialysis, and how long it will take to recover renal function in such risk groups . In conclusion, urinary NGAL is a potent biologic marker that makes early predictable acute renal damage more likely than serum creatinine currently used as a standard.

The Messina team in Italy reported that the risk of kidney disease and its rapid progression is very high when the blood NGAL is> 435 ng / mL and urine is> 231 ng / mL (D'Anna et al., 2009) Bolignano et al., 2010; Bolignano et al., Kidney Blood Press Res., 2009; Bolignano et al., 2009; , Rejuvenation Res., 2009; Bolignano et al., Clin. Am Soc Nephrol., 2009). Therefore, the measurement of blood and urinary NGAL makes it possible to predict the risk of chronic kidney disease regardless of the patient 's age. Recently, diabetic nephropathy has also been recognized as an early marker of diabetic nephropathy. As many research papers have been published in Korea, it has been approved as a non-payment item (CZ247) of Ministry of Health and Welfare notification on April 24,

Various tests have been developed to measure the concentration of NGAL in the blood and urine by being recognized as a biologic marker of acute renal damage, which is excellent. As a representative NGAL test method, a new marker test for the diagnosis of acute renal failure patients was developed in 2010. The ABBOTT Urinary NGAL Test, which was approved by the FDA, and the Triage Meter using fluorescence immunoassay were used for EDTA whole blood or plasma And the method of testing the concentration of NGAL in blood plasma, serum and urine specimens using the TriageⓡNGAL Test for rapid measurement of NGAL and the Human NGAL Rapid ELISA Kit (BioPortoⓡ Diagnostics, Denmark).

Despite these developments, there is a need for a diagnostic system that can be analyzed more accurately and in a short time using a simpler, smaller amount of sample (blood and urine).

Accordingly, the present inventors have conducted intensive studies to develop a biochip capable of early diagnosis of acute renal injury and chronic renal disease by using an increase in the expression amount of biomarker protein in the blood and urine of a patient, A peptide probe capable of specific and selective binding to each protein was discovered and prepared using a standard biomarker protein (NGAL) (Patent No. 10-2007-0108158) that was predictable and patent-verified And to develop a biochip capable of early diagnosis of acute renal injury and chronic kidney disease.

The present inventors have succeeded in replacing the enzyme-linked immunosorbent assay (ELISA), which has traditionally been used for early diagnosis of diseases, through an antigen-antibody reaction, a sandwich method, etc., And to enable a biochip capable of improving sensitivity and specificity through an economical process.

As a result, biopanning using M13 bacteriophage, which is one of the molecular evolutionary methods, was carried out to find and construct a peptide probe composed of 12 amino acids having a low molecular weight capable of selective binding to a target protein and utilizing the peptide probe Thereby making it possible to manufacture a biochip capable of early prediction of acute and chronic renal diseases.

The main object of the present invention is to find a peptide probe capable of molecular recognition through high specific binding with a high specificity to a target protein, To construct a peptide library which basically contains amino acids to be immobilized on a biochip.

According to an aspect of the present invention, there is provided a biochip for early diagnosis of acute renal injury and chronic renal disease including a peptide probe targeting a biomarker protein NGAL.

At this time, the peptide probe for the biomarker protein NGAL is characterized in that it is composed of a sequence comprising amino acid sequence listing 3 (MQIQISSRSMQT).

In addition, the amino acid of the peptide probe is characterized by being a D-type, an L-type peptide mimetic, or a non-natural amino acid including a peptide monomer.

Also, the peptide probe is characterized by being a dimer, trimer, or multimer containing various functional groups at the N-terminus or C-terminus.

In addition, the N-terminal functional group of the peptide probe includes free amine, acetylation, biotin or flurophore,

The C-terminal functional group is characterized by containing free acid, amidation, biotin or flurophore.

The present invention is also characterized in that it is a peptide probe for early diagnosis of acute renal injury and chronic renal disease comprising a sequence comprising amino acid sequence listing 3 (MQIQISSRSMQT).

The present invention as described above will be further described.

The present inventors have uncovered a peptide probe for early diagnosis of acute renal injury and chronic kidney disease through a fusion technique including a biopanning process using M13 bacteriophage, of ligands by exponential enrichment).

The peptide probe of the present invention is prepared by liquid-phase synthesis or solid-phase synthesis including basically attachment, protection, coupling, deprotection and cleavage processes.

The peptide probes prepared by the above method include at least 12 amino acids (D-type, L-type, peptide mimetics including peptoids, unnatural amino acids) composed of different amino acids.

In addition, the N-terminal or C-terminal of the peptide probe having at least 12 amino acids prepared by the above method may contain various functional groups.

The functional group of the peptide probe prepared by the above method may include free amine, acetylation, biotin, various flurophores, and the C terminus may include free acid, amidation, biotin, and various flurophores.

The above-mentioned peptide probe can be specifically fixed on various chips (gold, silver, magnetic beads, silica, graphene, carbon nanotube, etc.) usable as a biochip. However, the features of the present invention are related to the above-described probe, and manufacturing of such a probe by a biochip is a part that can be performed according to known technology.

The reaction between protein and protein, the reaction between the protein and the reactive ligand, the reaction between the target ligand and the reaction protein, or the reaction between the target ligand and the reactive ligand using the biochip equipped with the peptide probe for early diagnosis of acute renal injury and chronic kidney disease of the present invention Can be detected. The reactive ligands may be low molecular weight biomaterials, peptides, carbohydrates, aptamers, fatty acids or lipids which react with proteins or peptides.

The reaction protein may be an enzyme or an antibody.

The target ligand may also be a low molecular weight compound, nucleic acid, peptide, carbohydrate, aptamer, fatty acid or lipid which reacts with a protein or peptide.

In addition, the above reaction detection can be performed using a label or a non-standard support.

The biochip using the peptide probe for early diagnosis of acute renal injury and chronic renal disease according to the present invention is capable of highly specific and selective binding to the NGAL protein in the blood and urine of a patient, and the amount (increase or decrease) of each target biomarker protein Can be accurately detected. Therefore, early diagnosis of acute renal injury and chronic renal disease can be made easily and prompt treatment is possible, which is expected to contribute to increase the survival rate of patients with acute renal failure and chronic renal disease, which are increasing every year.

FIG. 1 shows the result of ELISA for comparing the binding strength against the target protein NGAL using the selected four bacteriophages.
FIG. 2 shows the result of measuring the binding force of the selected bacteriophage (NR5 # 22) according to the NGAL concentration as a target protein.
FIG. 3 shows the result of measuring the binding capacity to the NGAL protein according to the concentration of the selected phage (NR5 # 22).
FIG. 4 shows the result of measuring the binding force of the selected bacteriophage (NR5 # 22) according to serum addition.

The present inventors used the biomarker protein (NGAL) as a target for the early diagnosis of acute renal damage and chronic renal disease which show a rapid expression amount in the blood and urine of a patient, A new peptide probe was uncovered through biopanning with M13 bacteriophage.

The novel peptide probes discovered through the above method basically include 12 amino acids, and the probe may be a duplex, a trimer, or a multimer having various functional groups at the N-terminus or C-terminus.

In the present invention, the novel peptide probes can be produced in the form of recombinant proteins in host cells. The host cell can be E. coli, and various tags of the target protein in the recombinant protein form can be bound to the N-terminus and the C-terminus.

The bio-material interaction produced by the above method and using the bio-chip in which the fusion protein of the target protein-probe is immobilized can be detected. The fixation may be performed through a specific immobilization method, a microchannel, or a spotting method using a microarray. The detection can be performed via CV, EIS, QCM, SPR or SPR imaging. That is, it can be used for early diagnosis of acute renal damage and chronic kidney disease through the interaction of target protein-probe and target DNA-probe using a common assay method in a biosensor system.

Hereinafter, the present invention will be described in more detail by way of examples. It is to be understood, however, that these examples are for illustrative purposes only and that the scope of the present invention is not limited by these examples.

Example 1 : Target protein (NGAL) and biotinlyation

The target protein used in the present invention was purchased from Randox life sciences as a recombinant protein of human neutrophil gelatinase-associated lipocalin (NGAL) and subjected to biotinylation using Thermo Scientific's EZ-Link Sulfo-NHS-Biotinylation Kit The concentration was calculated by the Bradford assay and stored in a -80 ° C freezer until use.

Example 2: Detection of novel peptide library capable of highly specific and selective binding to NGAL using M13 bacteriophage display

The bacteriophage random peptide library used in the present invention is Ph.D.-12 (New England BioLab), which is a gene terminator that produces pIII, a kind of coat protein in the genome of M13 bacteriophage Is composed of recombinant bacteriophage expressing hundreds of millions of different peptides obtained by artificially inserting gene sequences to express peptides of 12 random amino acid sequences and then infecting E. coli.

(Streptavidin High Binding Capacity Coated 96-Well Plates (Thermo scientific, Cat. No. 15501) were used for panning.

First, biotinylated NGAL protein and M13 bacteriophage are allowed to react at room temperature for 1 hour to form a pre-complex. Then, streptavidin-coated plate was washed with 0.1% Tween 20 in TBS (Tris buffered saline) three times. Pre-complex was added to the microwell and stirred at room temperature for 10 minutes at 150 rpm. The unbound bacteriophage was washed with 0.1% Tween 20 in TBS (Tris buffered saline). Finally, the phage bound specifically to NGAL was eluted in 100 μL of 0.2 M Glycin-HCl (pH 2.2) solution and neutralization 15 [mu] L of Tris-HCl (pH 9.0) was added. Through the above procedure, four bacteriophage candidates that bind well to NGAL were first selected and the amino acid sequence was confirmed by DNA sequencing (Table 1).

Example 3: Measurement of binding force of candidate bacteriophage containing a novel peptide probe capable of highly specific and highly selective binding to NGAL using enzyme immunoassay (ELISA)

Amplification was performed in order to use four bacteriophage candidates selected through 5 biopanning for ELISA experiments. Each phage stock was inoculated into LB medium with previously cultured E. coli ER2738 and cultured at 37 DEG C and 150 rpm for 4-5 hours. The supernatant was recovered by centrifugation, and then 20% PEG / 2.5M NaCl was added and left on ice for 1 hour. After centrifuging again, the precipitate was dissolved in TBS (Tris buffered saline) and subjected to phage titration to calculate the concentration of each phage (PFU / mL: Plaque forming units / mL).

To compare the binding strength of each of the four phages and NGAL, ELISA was performed as follows. First, 100 μl of biotinylated NGAL (NGAL concentration: 30.8 μg / ml) and each of four phages are reacted at a concentration of 10 ¹¹ PFU / ml for 1 hour at room temperature to form a pre-complex. The plate coated with streptavidin was then washed three times with 0.1% Tween 20 in TBS (Tris buffered saline), pre-complex was added to the microwell, and the mixture was stirred at room temperature for 15 minutes at 150 rpm. After washing with 0.1% Tween 20 in TBS (Tris buffered saline), OD values were measured and compared at 405 nm using M13-HRP antibody and ABTS. As a result, NR5 # 22 among the four phages showed the greatest binding force to NGAL (FIG. 1).

As a result of comparing the binding force of NR5 # 22 with the change of NGAL concentration by the same ELISA method as above, it was confirmed that the binding force of NR5 # 22 increased in proportion to the increase of NGAL concentration. However, when the concentration of NGAL was 7.6 ug / ml or more, it was not significantly increased (FIG. 2).

The same NGAL protein-coated plate was treated with the NR5 # 22 phage concentration by the same ELISA method as described above, and the binding strength was compared. As a result, it was observed that the binding force was increased in proportion to the concentration (FIG. This result shows that the selected phage NR5 # 22 specifically binds to NGAL, and the sequence "MQIQISSRSMQT" of the 12 peptides of NR5 # 22 is used for peptide-derived acute renal injury and chronic kidney disease diagnosis kit .

As shown above, the peptide probe discovered by the present inventor can highly precisely and selectively bind to the NGAL protein in the patient's blood and urine, so that the amount (increase or decrease) of the target biomarker protein can be accurately detected , Thereby enabling early diagnosis of acute renal injury and chronic renal disease.

Example 4: Effect of serum addition on the binding force of bacteriophage candidates containing a novel peptide probe capable of high specificity and selective binding to NGAL

The following experiment was carried out to investigate the change of high specific binding capacity to NGAL in the condition that the selected bacteriophage NR5 # 22 was mixed with serum sample (FBS: Fetal bovine serum) and NGAL. First, streptavidin-coated plates were rinsed three times with 0.1% Tween 20 in TBS (Tris buffered saline), and 100 μL of biotinylated NGAL (NGAL concentration: 7.6 ug / ml) was added to the microwell for 16 hours at 4 ° C. And reacts. Then, the plate coated with biotinylated NGAL was washed three times with washing buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1 g BSA, 0.05% Tween 20) FBS (serum 1%, 2%, 10%) was added to the microwell and stirred at 150 rpm for 1 hour at room temperature. After washing three times with washing buffer, OD values were measured and compared at 405 nm using M13-HRP antibody and ABTS. As a result, the binding of bacteriophage (NR5 # 22) containing a novel peptide probe capable of high specific and highly selective binding to NGAL due to serum addition did not change significantly compared to the case without serum addition (FIG. 4).

In addition, the binding force was measured under the condition of adding 1, 2, and 10% of serum. As a result, the binding force was slightly decreased at a high concentration of 10%, but not significantly at a concentration of 1 or 2% . This suggests that using the NR5 # 22 peptide probe, it is possible to detect a very small amount of NGAL protein contained in the blood of patients with a small amount of kidney disease.

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Claims (6)

delete A peptide probe targeting the biomarker protein NGAL,
Wherein the peptide probe for the biomarker protein NGAL comprises a sequence comprising amino acid sequence listing 3 (MQIQISSRSMQT). 3. The biochip for early diagnosis of acute renal damage and chronic kidney disease. 3. The method of claim 2,
Wherein the amino acid of the peptide probe is any one of peptide mimetics including D-type, L-type, peptido monomer, or non-natural amino acid. The method of claim 3,
Wherein the peptide probe is a dimer, a trimer, or a multimer containing various functional groups at the N-terminus or the C-terminus, for the early diagnosis of acute renal damage and chronic renal disease. 5. The method of claim 4, wherein the N-terminal functional group of the peptide probe comprises free amine, acetylation, biotin or flurophore,
Wherein the C-terminal functional group comprises free acid, amidation, biotin, or flurophore; and a biochip for early diagnosis of acute renal injury and chronic renal disease. A peptide probe for the early diagnosis of acute renal injury and chronic renal disease comprising a sequence comprising amino acid sequence listing 3 (MQIQISSRSMQT).

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