CN108164585A - For measure the polypeptide probe of transpeptidase A activity to, measure transpeptidase A activity method and the two application - Google Patents

Abstract

The invention provides a pair of polypeptide probes for measuring the activity of transpeptidase A, a method for measuring the activity of transpeptidase A and the application of the two, which relate to the technical field of biological detection. A active polypeptide probe pair, including the first polypeptide probe and the second polypeptide probe with a reaction region, a complementary region and a dye-binding region, has the advantages of strong specificity, good accuracy, and high sensitivity, without the need for Modification or labeling can achieve the purpose of measuring transpeptidase A activity. The method for measuring the activity of transpeptidase A provided by the present invention comprises mixing and incubating the above-mentioned polypeptide probe pair for measuring the activity of transpeptidase A with the sample to be tested in the transpeptidation reaction buffer, and then using a double arsenic dye To label the reaction product after incubation, the activity of transpeptidase A is determined by the fluorescence level of the complex formed by the combination of the reaction product after incubation and the double arsenic dye, and the operation is simple and convenient.

Description

Polypeptide probe pair for measuring transpeptidase A activity, method for measuring transpeptidase A activity law and its application

technical field

The invention relates to the technical field of biological detection, in particular to a pair of polypeptide probes for measuring the activity of transpeptidase A, a method for measuring the activity of transpeptidase A and the application of the two.

Background technique

Bacterial surface proteins play an important role in the infection process of pathogenic bacteria (such as adhesion, immune escape, biofilm formation), and are indispensable pathogenic factors that determine the severity of pathogenic bacterial infection (Nat.Rev. Microbiol.,2014,12,49 -62). In Gram-positive bacteria, transpeptidation reactions catalyzed by transpeptidases are required for proper anchoring of surface proteins to the cell wall. Usually, surface proteins are synthesized in the form of precursors, the N-terminus of the precursor protein contains a secretion signal peptide, the C-terminus contains an LPXTG (X represents any amino acid) domain, a hydrophobic region and a positively charged end of the residue. The signal peptide at the N-terminus will guide the precursor protein to be transported out of the cell through the secretory pathway. During the secretion process, the signal peptide at the N-terminus will be cleaved, and the hydrophobic region and positively charged tail at the C-terminus will retain the surface protein precursor in the cell membrane. Subsequently, transpeptidase bound to the cell membrane will cleave the covalent bond between threonine and glycine in the LPXTG amino acid sequence to form a thioesterase intermediate, which will be replaced by the free amino group on the type II lipid Nucleophilic attack covalently binds surface proteins to peptidoglycan cross-bridges, and lipid-linked surface proteins are further integrated into the cell wall through transglycosidation and transpeptidation. Through this mode of action, transpeptidases anchor more than 20 virulence factor proteins to the bacterial cell wall. Deletion of the transpeptidase gene or loss of transpeptidase activity will result in the inability of localization of various surface proteins to the surface of bacteria, and will reduce the ability of infection and adhesion of bacteria. However, transpeptidase is not necessary for the survival and growth of bacteria. Inhibiting the activity of transpeptidase will not bring survival pressure to pathogenic bacteria and cause drug resistance. Therefore, transpeptidase has become an ideal drug target for the development of new anti-infective drugs.

Currently, there are several ways to measure transpeptidase activity, the most commonly used is the fluorescence quenching activity assay, which requires the modification of a fluorophore at one end of the LPXTG motif and a quencher at the other end. Due to the fluorescence resonance energy transfer between the fluorescent group and the quencher group, the signal of the fluorescent group will be quenched, and the cleavage of the substrate by the transpeptidase will separate the quencher group and the fluorescent group to restore the fluorescent properties of the fluorophore. For the fluorescence quenching activity detection method, there are two main problems: one is that the substrate needs to be labeled, and the other is that only the cleavage activity of the transpeptidase can be measured, but its transpeptide ability cannot be measured. In addition, fibronectin-binding method, HPLC-MS detection method, and fluorescence assay based on yeast surface display have also been used to measure the activity of transpeptidase. The fibronectin-binding method has the disadvantages of cumbersome operation and poor detection specificity. HPLC-MS detection requires expensive instruments and professional operations. Although the fluorescence assay based on yeast surface display can be used for high-throughput screening of inhibitors targeting transpeptidases, the detection sensitivity is limited. This greatly limits the development process of "non-antibiotic" drugs.

Therefore, there is an urgent need to develop some simple, efficient and sensitive methods to measure the activity of transpeptidases and accelerate the development of new non-antibiotic anti-infective drugs.

In view of this, the present invention is proposed.

Contents of the invention

The first object of the present invention is to provide a pair of polypeptide probes for measuring the activity of transpeptidase A, so as to alleviate the need for labeling, Technical problems of cumbersome preparation and limited sensitivity.

The second object of the present invention is to provide a method for measuring the activity of transpeptidase A, so as to alleviate the problems that the method for measuring the activity of transpeptidase A existing in the prior art is cumbersome to operate, has low sensitivity, and needs professional technicians to realize. technical problem.

The third object of the present invention is to provide the application of the above-mentioned polypeptide probe pair for determining the activity of transpeptidase A or the method for determining the activity of transpeptidase A in determining the potential pathogenicity of Gram-positive bacteria.

The present invention provides a pair of polypeptide probes for measuring the activity of transpeptidase A, wherein the pair of polypeptide probes includes a first polypeptide probe and a second polypeptide probe;

The first polypeptide probe and the second polypeptide probe respectively include a reaction zone, a complementary zone and a dye binding zone;

The reaction region is used to participate in the transpeptidation reaction mediated by transpeptidase A, so that the first polypeptide probe and the second polypeptide probe are combined into a chimeric polypeptide; the complementary region is used to make the chimeric polypeptide The complex polypeptide self-assembles into a hairpin structure; the dye-binding region is used to bind the dye.

Further, the complementary region is a polypeptide fragment that can form a hairpin structure.

Further, the amino acid sequence of the reaction region of the first polypeptide probe is LPXTGX, the amino acid sequence of the complementary region of the first polypeptide probe is PSQPTYPG, and the dye binding region of the first polypeptide probe is The amino acid sequence is CC;

Wherein, X in the amino acid sequence LPXTGX of the reaction region of the first polypeptide probe is any amino acid except cysteine;

Preferably, the amino acid sequence of the reaction region of the first polypeptide probe is LPATGG.

Further, the amino acid sequence of the reaction region of the second polypeptide probe is GG, the amino acid sequence of the complementary region of the second polypeptide probe is VEDLIRFYDNLQQYLNV, and the dye binding region of the second polypeptide probe is The amino acid sequence is CC.

Further, the first polypeptide probe has the amino acid sequence shown in SEQ ID NO.1, and the second polypeptide probe has the amino acid sequence shown in SEQ ID NO.2.

Further, the dye is bisarsenic dye;

Preferably, the diarsenic dye is FlAsH-EDT ₂ , ReAsH-EDT ₂ , F2FlAsH or F4FlAsH.

The present invention also provides a method for measuring the activity of transpeptidase A, comprising:

Mix and incubate the above-mentioned polypeptide probe pair for determining the activity of transpeptidase A with the sample to be tested in the transpeptidation reaction buffer to obtain a reaction product, and the fluorescence of the complex formed by combining the reaction product with a double arsenic dye Level to determine the activity of transpeptidase A.

Further, the buffer for the transpeptidation reaction contains a reducing agent, preferably a disulfide bond reducing agent, more preferably DTT.

Further, the transpeptide reaction buffer includes: Tris·HCl 40-60mmol/L, NaCl 120-180mmol/L, CaCl ₂ 1-10mmol/L and DTT 0.5-1.5mmol/L;

Preferably, the transpeptide reaction buffer consists of the following components: Tris·HCl 45-55mmol/L, NaCl 130-170mmol/L, CaCl ₂ 2-7mmol/L and DTT 0.7-1.3mmol/L.

In addition, the present invention also provides the application of the above-mentioned polypeptide probe pair for determining transpeptidase A activity or the method for determining transpeptidase A activity in determining the potential pathogenicity of Gram-positive bacteria.

The pair of polypeptide probes for measuring the activity of transpeptidase A provided by the present invention comprises a first polypeptide probe and a second polypeptide probe having a reaction region, a complementary region and a dye binding region. The reaction region of the polypeptide probe pair provided by the present invention can specifically participate in the transpeptidation reaction mediated by transpeptidase A, which ensures that the polypeptide probe pair has the advantages of strong specificity, good accuracy and high sensitivity. The transpeptidation reaction mediated by transpeptidase A will cause the first polypeptide probe and the second polypeptide probe to form a new chimeric polypeptide, and the complementary region can make the newly formed chimeric polypeptide generate itself through intramolecular complementarity. turn back to form a specific spatial conformation, the spatial conformation can bind the dye to make the dye emit fluorescence, and the unconnected first polypeptide probe and the second polypeptide probe cannot bind the dye to make the dye emit fluorescence, so that the present invention provides The polypeptide probe pair can achieve the purpose of measuring the activity of transpeptidase A, is simple to operate, and saves manpower and material resources. Moreover, using the polypeptide molecule as the detection probe has good biocompatibility with the assay target transpeptidase A.

The method for determining the activity of transpeptidase A provided by the present invention comprises mixing and incubating the above-mentioned polypeptide probe pair for determining the activity of transpeptidase A with the sample to be tested in a transpeptidation reaction buffer to obtain a reaction product, which is obtained by The activity of transpeptidase A is determined by the fluorescence level of the complex formed by the reaction product combined with the dual arsenic dye. Since the dual arsenic dye will only emit fluorescence when it is combined with a chimeric polypeptide, it will not combine with the first polypeptide probe and the second polypeptide probe to emit fluorescence, so the method for measuring the activity of transpeptidase A provided by the present invention The method does not require separation or cleaning steps during the entire detection process, and is simple and convenient to operate without requiring expensive instruments or professional technicians to operate.

The potential pathogenicity of Gram-positive bacteria can be determined by using the polypeptide probe pair for determining the activity of transpeptidase A provided by the present invention or the method for determining the activity of transpeptidase A, which has high sensitivity, strong specificity, and simple operation .

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without creative work.

Figure 1A is a mass spectrometry analysis result diagram of CC-aPP-CC formed by transpeptidase A catalyzing aPP-N and aPP-C provided by Example 2 of the present invention;

Fig. 1B is the fluorescence emission result of the complex formed after the double arsenic dye FlAsH-EDT ₂ provided in Example 2 of the present invention combines with the mixture of aPP-N and aPP-C, CC-aPP-CC and CC-aPP ^P -CC picture;

Fig. 2A is an imaging result diagram of fluorescent signals generated by the combination of transpeptidase A, T4 DNA ligase and ubiquitin ligase catalyzed by aPP-N and aPP-C to form aPP-N and aPP-C provided in Example 3 of the present invention;

Fig. 2B is a graph showing the results of fluorescent signals generated by combining transpeptidase A at different concentrations in Example 3 of the present invention and its catalysis of aPP-N and aPP-C to form CC-aPP-CC combined with dual arsenic dyes;

Fig. 3 A is the molecular structural formula of the curcumin provided by the embodiment of the present invention 4;

Fig. 3 B is the result figure between the concentration of curcumin provided by the embodiment of the present invention 4 and the inhibition rate of transpeptidase A activity;

Fig. 4A is a graph showing the result of the enhanced fluorescent signal caused by the action of the Staphylococcus aureus lysate provided in Example 5 of the present invention on the polypeptide probe pair;

Fig. 4B is a graph showing the results of the quantitative determination of curcumin's inhibition of Staphylococcus aureus endotranspeptidase A activity by the polypeptide probe pair provided in Example 5 of the present invention.

Detailed ways

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Apparently, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

In one aspect of the present invention, a pair of polypeptide probes for determining the activity of transpeptidase A is provided, including a first polypeptide probe and a second polypeptide probe;

Both the first polypeptide probe and the second polypeptide probe are used to participate in the transpeptidation reaction mediated by transpeptidase A, so that the first polypeptide probe and the second polypeptide probe are combined into a chimeric polypeptide reaction region, a complementary region for folding the chimeric polypeptide back on itself, and a dye binding region for binding a dye.

The reaction region of the polypeptide probe pair provided by the present invention can specifically participate in the transpeptidation reaction mediated by transpeptidase A, which ensures that the polypeptide probe pair has the advantages of strong specificity and good accuracy. The transpeptidation reaction mediated by transpeptidase A will cause the first polypeptide probe and the second polypeptide probe to form a new chimeric polypeptide, and the complementary region can make the newly formed chimeric polypeptide generate itself through intramolecular complementarity. Turn back to form a specific spatial conformation, which can bind the dye. Since the fluorescence level of the dye is extremely dependent on the specific spatial conformation formed by the chimeric polypeptide through intramolecular complementarity, the polypeptide probe pair provided by the present invention does not need to be modified. Or labeling treatment, the activity of transpeptidase A is measured by combining with the dye to generate fluorescence, which is simple to operate and saves manpower and material resources. On the contrary, in the absence of transpeptidase A, the first polypeptide probe and the second polypeptide probe cannot form the above-mentioned specific spatial conformation, so they cannot bind the dye, which further illustrates that the polypeptide probe pair provided by the present invention has higher specificity. Moreover, the use of polypeptide molecules as detection probes has better biocompatibility with assay targets.

In a preferred embodiment, the complementary region is a polypeptide fragment capable of forming a hairpin structure.

The hairpin structure is a hairpin-like structure formed on the basis of the primary structure of RNA and polypeptide through intramolecular complementarity or interaction. The polypeptide fragment that can form a hairpin structure can make the dye-binding regions in the chimeric polypeptide obtained by combining the first polypeptide probe and the second polypeptide probe approach each other, thereby forming a specific spatial conformation for binding the dye. The preferred sequences of polypeptide fragments capable of forming a hairpin structure are: PSQPTYPG and VEDLIRFYDNLQQYLNV.

In another preferred embodiment, the complementary region is a tryptophan zipper.

The tryptophan zipper is a short peptide containing multiple tryptophan residues, which spontaneously forms a β-hairpin structure. Tryptophan residues are discontinuously distributed on both sides of the hairpin structure, and the hydrophobic interaction between tryptophan-tryptophan pairs in the chain will greatly stabilize the β-hairpin structure. Selecting the tryptophan zipper as the complementary region can make the dye-binding regions in the chimeric polypeptide obtained by combining the first polypeptide probe and the second polypeptide probe close to each other, forming a very stable hairpin structure to bind the dye. The preferred tryptophan zipper complementary pair sequences are: GKKWTWTW and WTWTWQEG.

In a preferred embodiment, the amino acid sequence of the reaction region of the first polypeptide probe is LPXTGX, the amino acid sequence of the dye-binding region is CC, and the amino acid sequence of the complementary region is PSQPTYPG.

Wherein, X in the amino acid sequence LPXTGX of the reaction region of the first polypeptide probe can be any amino acid except cysteine, such as, but not limited to, A, G, T, L, etc.

Preferably, the amino acid sequence of the reaction region of the first polypeptide probe is LPATGG.

In a preferred embodiment, the amino acid sequence of the reaction region of the second polypeptide probe is GG, the amino acid sequence of the dye-binding region is CC, and the amino acid sequence of the complementary region is VEDLIRFYDNLQQYLNV.

The complementary region PSQPTYPG fragment of the first polypeptide probe and the complementary region VEDLIRFYDNLQQYLNV fragment of the second polypeptide probe will combine with each other after forming a new chimeric polypeptide, thereby making two cysteine-cysteine doublets The molecules are spatially adjacent to form a specific conformation to bind the dye.

In a preferred embodiment, the amino acid sequence of the first polypeptide probe is: CCPSQPTYPGLPATGG (SEQ ID NO.1), the amino acid sequence of the second polypeptide probe is: GGVEDLIRFYDNLQQYLNVCC (SEQ ID NO.2), the first The amino acid sequence of the chimeric polypeptide formed by combining the polypeptide probe and the second polypeptide probe is CCPSQPTYPGLPATGGVEDLIRFYDNLQQYLNVCC (SEQ ID NO. 3).

In a preferred embodiment, the dye is a diarsenic dye.

The dual-arsenic dye-tetracysteine labeling system can specifically fluorescently label intracellular proteins. Among them, the tetracysteine tag is a hexapeptide or dodecapeptide containing four cysteine residues, which can be specifically labeled with arsenic dye. The cysteine-cysteine doublets in the tetracysteine tag can be directly linked by a proline-glycine dipeptide to form a linear tetracysteine tag. In addition, the cysteine-cysteine doublet can also be added to the end of the polypeptide or embedded in the protein, and through the folding and combination of the polypeptide and the protein, they are spatially adjacent to form a specific energy and diarsenic Dye bound conformation. In this labeling system, the spatial structure of the tetracysteine tag will affect the stability and quantum yield of the formed complex. In one embodiment of the present invention, the complementary region PSQPTYPG fragment of the first polypeptide probe and the complementary region VEDLIRFYDNLQQYLNV fragment of the second polypeptide probe form a new chimeric polypeptide after transpeptidation reaction mediated by transpeptidase A It will combine with each other, so that the two cysteine-cysteine doublets are spatially adjacent, and then combine with the diarsenic dye to produce extremely strong fluorescence. In contrast, in the absence of the action of transpeptidase A, the two cysteine-cysteine dyads cannot form a specific conformation to bind the diarsenic dye, and the diarsenic dye therefore does not emit light. Utilizing the characteristic that the fluorescence level of the dual arsenic dye-tetracysteine tag complex is extremely dependent on the space conformation of the tetracysteine tag, the activity and specificity of transpeptidase A can be quantitatively measured by measuring the change of the fluorescence level before and after the reaction. Strong, high sensitivity, and the determination results are accurate and reliable.

Preferably, the dye is FlAsH- _EDT2 , ReAsH- _EDT2 , F2FlAsH or F4FlAsH.

FlAsH-EDT ₂ , ReAsH-EDT2, F2FlAsH and F4FlAsH are diarsenic dyes with different excitation and emission wavelengths, so there are many ways to measure the activity of transpeptidase, and the detection method is flexible.

In the second aspect of the present invention, there is also provided a method for measuring transpeptidase A activity, comprising:

Mix and incubate the above-mentioned polypeptide probe pair for measuring the activity of transpeptidase A with the sample to be tested in the transpeptidation reaction buffer to obtain the reaction product, and the intensity of the fluorescent signal generated by the combination of the reaction product and the double arsenic dye Determine the activity of transpeptidase A.

Utilize the polypeptide probe pair for measuring transpeptidase A activity provided by the present invention to detect transpeptidase A activity, because the transpeptidation reaction mediated by transpeptidase A will make the first polypeptide probe of the polypeptide probe pair The needle and the second polypeptide probe form a new chimeric polypeptide, and the chimeric polypeptide folds back on itself through intramolecular complementarity, thereby forming a specific spatial conformation capable of binding the dye. Moreover, the fluorescence level of the dye-polypeptide complex depends on the number of specific spatial conformations formed by the chimeric polypeptide, that is, the more chimeric polypeptides are formed, the higher the fluorescence level of the complex will be. The formation of chimeric polypeptides depends on the transpeptidase A-mediated transpeptidation reaction. Therefore, the activity of transpeptidase A can be determined by the fluorescence level of the reaction product, and the stronger the fluorescence signal, the higher the activity of transpeptidase A. High, on the contrary, the weaker the fluorescence intensity, the lower the activity of transpeptidase A.

Since the dual arsenic dye in the present invention will emit fluorescence only when combined with a chimeric polypeptide, and will not combine with the first polypeptide probe and the second polypeptide probe to emit fluorescence, the assay transpeptide provided by the present invention The method for enzyme A activity does not require separation or cleaning steps during the entire detection process, is simple and convenient to operate, and does not require expensive instruments or professional technicians to operate.

In a preferred embodiment, the transpeptide reaction buffer contains a reducing agent.

Reducing agent refers to the general term for substances such as free radicals and active oxygen scavengers, blocking agents and repairing agents. Adding a reducing agent in the transpeptidation reaction buffer can ensure that -SH in the cysteine residue in the polypeptide probe pair is in a reduced state and does not form a disulfide bond, so that it can be combined with the diarsenic dye.

It is preferably a disulfide bond reducing agent, more preferably DTT.

DTT is dithiothreitol, one of the disulfide bond reducing agents, which can keep -SH in the cysteine residue in a reduced state. Avoiding disulfide bond formation allows the chimeric peptide to bind to the diarsenic dye.

In a preferred embodiment, the transpeptide reaction buffer includes: Tris·HCl 40-60 mmol/L, NaCl 120-180 mmol/L, CaCl ₂ 1-10 mmol/L and DTT 0.5-1.5 mmol/L.

Wherein, Tris·HCl can be, for example, but not limited to 40mmol/L, 45mmol/L, 50mmol/L, 55mmol/L or 60mmol/L. NaCl can be, for example, but not limited to 120mmol/L, 130mmol/L, 140mmol/L, 150mmol/L, 160mmol/L, 170mmol/L or 180mmol/L. _CaCl can be, for example, but not limited to 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L or 10mmol/L. DTT can be, for example, but not limited to 0.5 mmol/L, 1 mmol/L or 1.5 mmol/L.

Preferably, the transpeptide reaction buffer is composed of the following components: Tris HCl 45-55mmol/L, NaCl 130-170mmol/L, CaCl ₂ 2-7mmol/L and DTT 0.7-1.3mmol/L;

Preferably, the transpeptide reaction buffer includes: Tris·HCl 50mmol/L, NaCl 150mmol/L, CaCl ₂ 5mmol/L and DTT 1mmol/L.

By selecting specific components and ratios, the prepared transpeptide reaction buffer can provide a suitable pH environment and salt ion concentration to ensure the catalytic activity of transpeptidase A, and at the same time ensure that the cysteine residues in the polypeptide probe Base-SH is in a reduced state.

In the third aspect of the present invention, the application of the above-mentioned polypeptide probe pair for measuring transpeptidase A activity or the method for measuring transpeptidase A activity in determining the potential pathogenicity of Gram-positive bacteria is provided.

Gram-positive bacteria are bacteria that can be stained dark blue or purple with Gram stain, and contain relatively large amounts of peptidoglycan in their cell walls. Among them, transpeptidase can anchor various virulence factor proteins to the cell wall of Gram-positive bacteria. The deletion of transpeptidase gene or the loss of transpeptidase activity will lead to the failure of localization of various surface proteins on the surface of bacteria, which in turn will reduce the ability of infection and adhesion of bacteria. Therefore, the detection of transpeptidase A activity can reflect the potential pathogenicity of Gram-positive bacteria, has high sensitivity, strong specificity, simple operation and cost saving.

Typical Gram-positive bacteria can be, for example, but not limited to, Streptococcus mutans, Streptococcus pyogenes, Streptococcus suis, Streptococcus pneumoniae, Streptococcus gasseri, Enterococcus or Listeria monocytogenes.

In order to further understand the design method, working principle and beneficial effect of the polypeptide probe pair for measuring transpeptidase A activity provided by the present invention, the present invention will be further described in detail below in conjunction with specific examples.

Embodiment 1 is used for measuring the design of the polypeptide probe pair of transpeptidase A activity

This embodiment provides a pair of polypeptide probes for measuring the activity of transpeptidase A, including a first polypeptide probe and a second polypeptide probe, and the two designed polypeptide probes are named aPP- N and aPP-C, wherein the amino acid sequence of aPP-N is: CCPSQPTYPGLPATGG (SEQ ID NO.1), and the amino acid sequence of aPP-C is: GGVEDLIRFYDNLQQYLNVCC (SEQ ID NO.2). Each probe contains three different functional regions: among them, LPATGG and GG will participate in the transpeptidase A-mediated transpeptidation reaction; CC is the site combined with arsenic dye to read the activity of transpeptidase A ; PSQPTYPG and VEDLIRFYDNLQQYLNV will bind to each other so that the two cysteine-cysteine doublets are spatially adjacent. The transpeptidation reaction mediated by transpeptidase A will form a new chimeric polypeptide, its amino acid sequence is: CCPSQPTYPGLPATGGVEDLIRFYDNLQQYLNVCC (SEQ ID NO.3), named CC-aPP-CC. For the formed chimeric polypeptide, the mutual combination between PSQPTYPG and VEDLIRFYDNLQQYLNV will make the two cysteine-cysteine doublets close to each other in space, and then combine with the double arsenic dye to produce extremely strong fluorescence; In contrast, in the absence of the action of transpeptidase A, the two cysteine-cysteine dyads cannot form a specific conformation to bind the diarsenic dye, and the diarsenic dye therefore does not emit light. Utilizing the characteristic that the quantum yield and fluorescence level of the dual arsenic dye-tetracysteine label complex are extremely dependent on the space conformation of the tetracysteine label, the activity of transpeptidase A can be quantitatively determined by measuring the change of the fluorescence level before and after the reaction. Activity, and then determine the potential pathogenicity of Gram-positive bacteria.

Embodiment 2 is used for measuring the performance measurement of the polypeptide probe pair of transpeptidase A activity

In order to determine whether transpeptidase A can catalyze aPP-N and aPP-C provided in Example 1 of the present invention to form CC-aPP-CC, this example adds 10 micromoles per liter of aPP-N, 40 micromoles per liter of a Add PP-C and 0.1 μg of transpeptidase A to 20 μl of transpeptidation reaction buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl ₂ and 1 mM DTT), and incubate two Hour. Subsequently, the ten-fold diluted reaction product was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results are shown in Figure 1A. The theoretical molecular weight of CC (3920.51 Daltons) was consistent, indicating that transpeptidase A could recognize the response element in aPP-N/aPP-C and connect aPP-N and aPP-C correctly. In order to test whether the formed chimeric polypeptide can form a specific conformation capable of binding to arsenic dye, this example also provides a polypeptide that has lost its intramolecular complementarity, named CC-aPP ^P -CC, and its sequence is CCPSQPTYPGLPATGGVEDLIRPYDNLQQPL For NVCC (SEQ ID NO.4), the N-terminal and C-terminal of the polypeptide are modified by acetylation and amidation respectively. In this example, the binding conditions between the double arsenic dye FlAsH-EDT ₂ and the mixture of aPP-N and aPP-C, CC-aPP-CC and CC-aPP ^P -CC were determined, and the results are shown in Figure 1B. Dual arsenic dye can combine with CC-aPP-CC to produce a strong fluorescent signal; on the contrary, only some background signal and comparison between dual arsenic dye and aPP-N/aPP-C mixture and CC-aPP ^P -CC can be produced. Weak fluorescent signal indicated that CC-aPP-CC could self-assemble into a specific conformation to bind diarsenic dye.

Embodiment 3 is used for measuring the activity of the polypeptide probe of transpeptidase A activity in vitro quantitative determination of transpeptidase A

This example further verifies that aPP-N and aPP-C can specifically measure the activity of transpeptidase A through experiments. In this example, the linking effects of transpeptidase A, T4 DNA ligase and ubiquitin ligase on aPP-N and aPP-C were determined in the transpeptidation reaction buffer. The specific experimental operation is as follows: 10 micromoles per liter of aPP-N and 10 micromoles per liter of aPP-C were mixed with 0.1 micrograms of transpeptidase A, 0.1 micrograms of T4 DNA ligase and 0.1 micrograms of ubiquitin ligase in Incubate _at 37 degrees for two hours, then add the reaction mixture to 80 microliters of double arsenic dye labeling buffer (100 mM Tris Cl, pH 7.4, 75 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.05 mM BAL) and incubate at room temperature for 30 minutes in the dark. After the reaction, the reaction product was analyzed by fluorescence imaging, and the results are shown in Figure 2A. It can be seen from Figure 2A that T4 DNA ligase and ubiquitin ligase can hardly catalyze the generation of fluorescent signals, thus indicating that the polypeptide probes aPP-N and aPP-C can specifically measure the activity of transpeptidase A. Further, the aPP-N and aPP-C probes were mixed with concentrations of 0 μg/mL, 0.02 μg/mL, 0.04 μg/mL, 0.06 μg/mL, 0.08 μg/mL, 0.1 μg/mL, 0.2 μg/mL, 0.4μg/mL, 0.6μg/mL, 0.8μg/mL and 1μg/mL of transpeptidase A were incubated, and each concentration of transpeptidase A could lead to an increase in fluorescence signal ΔF, ΔF and transpeptidase The relationship between A concentrations is shown in Figure 2B. As the concentration of transpeptidase A increases, ΔF increases, and the calculated minimum detection limit is 8.1 ng/mL, which is better than the commonly used fluorescence quenching activity. detection method with high sensitivity.

Embodiment 4 is used for measuring the polypeptide probe of transpeptidase A activity to measuring curcumin to the inhibition of transpeptidase A activity

In this example, a pair of polypeptide probes was used to determine the inhibitory effect of curcumin on the activity of transpeptidase A. Curcumin is a polyphenolic compound present in turmeric, and its molecular structure is shown in Figure 3A. The transpeptidase A was incubated with different concentrations of curcumin at room temperature for 10 minutes, and then incubated with the mixture of aPP-N and aPP-C to measure the fluorescence signal of the formed product, the results are shown in Figure 3B. As can be seen from the result figure, curcumin can inhibit the activity of transpeptidase A, and the activity of transpeptidase A will gradually decrease with the increase of curcumin concentration, and the calculated half-inhibitory concentration is 13.4 micrograms per milliliter, which is the same as The 13.8 ± 0.7 micrograms reported in the literature are consistent with each milliliter, explaining that the polypeptide probe for measuring transpeptidase A activity provided by the present invention can reliably and accurately measure the activity of transpeptidase A, and then can measure small molecule Inhibition of transpeptidase A by inhibitors.

Embodiment 5 is used for measuring the activity of the polypeptide probe of transpeptidase A activity to measuring Staphylococcus aureus internal transpeptidase A

In order to confirm whether the polypeptide probe pair used for determining the activity of transpeptidase A provided by the present invention can determine the activity of transpeptidase A in Gram-positive bacteria, this embodiment takes Staphylococcus aureus as an example, and the present invention provides The peptide probe pair used to measure transpeptidase A activity was incubated with the lysate of Staphylococcus aureus, and the fluorescence levels before and after incubation were measured. As shown in FIG. 4A , the lysate of Staphylococcus aureus can lead to an increase in fluorescence, indicating that transpeptidase A in bacteria can promote the transpeptidation reaction between aPP-N and aPP-C. Further, the present embodiment uses curcumin with a concentration of 0 μg/mL, 1 μg/mL, 3 μg/mL and 5 μg/mL to treat Staphylococcus aureus, and measures the endotranspeptidase activity of Staphylococcus aureus treated with curcumin active. As shown in Figure 4B, with the gradual increase of curcumin concentration, the activity of transpeptidase A in Staphylococcus aureus gradually decreases, so the polypeptide probe pair for measuring transpeptidase A activity provided by the present invention can be effectively measured Staphylococcus aureus endotranspeptidase A activity.

In summary, the polypeptide probe pair used for measuring the activity of transpeptidase A provided by the present invention can specifically participate in the transpeptidation reaction mediated by transpeptidase A, which ensures that the polypeptide probe pair has strong specificity and accuracy. The advantages of good accuracy and high sensitivity. The new chimeric polypeptide formed by the transpeptidation reaction mediated by the first polypeptide probe and the second polypeptide probe through transpeptidase A, its complementary region can undergo self-folding through intramolecular interaction to form a specific spatial conformation To bind the double arsenic dye and emit fluorescence, while the unconnected first polypeptide probe and the second polypeptide probe will not combine with the double arsenic dye, so that the polypeptide probe pair provided by the invention can measure transpeptidase A High activity, simple operation, saving manpower and material resources. Moreover, using the polypeptide molecule as the detection probe has good biocompatibility with the assay target transpeptidase A.

In the method for measuring the activity of transpeptidase A provided by the present invention, the dual arsenic dye only combines with the chimeric polypeptide formed after the connection of the polypeptide probe pair to generate fluorescence, and does not generate fluorescence with the unconnected polypeptide probe pair, so The method for measuring the activity of transpeptidase A provided by the present invention does not need separation or cleaning steps in the whole detection process, is simple and convenient to operate, and does not require expensive instruments or professional technicians to operate.

Applying the polypeptide probe pair for measuring the activity of transpeptidase A provided by the present invention or the method for measuring the activity of transpeptidase A to determine the potential pathogenicity of Gram-positive bacteria, the sensitivity is high, the specificity is strong, and the detection result is accurate , and the operation is simple.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.

SEQUENCE LISTING

<110> Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences

<120> Polypeptide probe pair for measuring transpeptidase A activity, method for measuring transpeptidase A activity and application of both

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 16

<212> PRT

<213> Artificial sequence

<400> 1

Cys Cys Pro Ser Gln Pro Thr Tyr Pro Gly Leu Pro Ala Thr Gly Gly

1 5 10 15

<210> 2

<211> 21

<212> PRT

<213> Artificial sequence

<400> 2

Gly Gly Val Glu Asp Leu Ile Arg Phe Tyr Asp Asn Leu Gln Gln Tyr

1 5 10 15

Leu Asn Val Cys Cys

20

Claims (10)

1. A polypeptide probe pair for measuring transpeptidase A activity is characterized in that, the polypeptide probe pair comprises a first polypeptide probe and a second polypeptide probe; The first polypeptide probe and the second polypeptide probe respectively include a reaction zone, a complementary zone and a dye binding zone; The reaction region is used to participate in the transpeptidation reaction mediated by transpeptidase A, so that the first polypeptide probe and the second polypeptide probe are combined into a chimeric polypeptide; the complementary region is used to make the chimeric polypeptide The complex polypeptide self-assembles into a hairpin structure; the dye-binding region is used to bind the dye. 2. The polypeptide probe pair according to claim 1, wherein the complementary region is a polypeptide fragment capable of forming a hairpin structure. 3. The polypeptide probe according to claim 1 is characterized in that, the amino acid sequence of the reaction zone of the first polypeptide probe is LPXTGX, and the amino acid sequence of the complementary zone of the first polypeptide probe is PSQPTYPG, the amino acid sequence of the dye-binding region of the first polypeptide probe is CC; Wherein, X in the amino acid sequence LPXTGX of the reaction region of the first polypeptide probe is any amino acid except cysteine; Preferably, the amino acid sequence of the reaction region of the first polypeptide probe is LPATGG. 4. polypeptide probe according to claim 1 is paired, it is characterized in that, the aminoacid sequence of the reaction zone of described second polypeptide probe is GG, and the aminoacid sequence of the complementary zone of described second polypeptide probe is VEDLIRFYDNLQQYLNV, the amino acid sequence of the dye-binding region of the second polypeptide probe is CC. 5. The polypeptide probe pair according to claim 1, wherein the first polypeptide probe has an amino acid sequence as shown in SEQ ID NO.1, and the second polypeptide probe has an amino acid sequence as shown in SEQ ID NO.1. The amino acid sequence shown in NO.2. 6. The polypeptide probe pair according to any one of claims 1-5, wherein the dye is a double arsenic dye; Preferably, the dye is FlAsH-EDT ₂ , ReAsH-EDT ₂ , F2FlAsH or F4FlAsH. 7. A method for measuring transpeptidase A activity, characterized in that, comprising: The polypeptide probe pair for measuring the activity of transpeptidase A described in any one of claims 1-6 is mixed with the sample to be tested in the transpeptide reaction buffer and incubated to obtain a reaction product, through which the reaction product is combined with The activity of transpeptidase A was determined by the fluorescence level of the complex formed by dual arsenic dye binding. 8. the method for measuring transpeptidase A activity according to claim 7, is characterized in that, comprises reducing agent in described transpeptidation reaction buffer, and described reducing agent is preferably disulfide bond reducing agent, more preferably DTT . 9. The method for measuring transpeptidase A activity according to claim 7 or 8, characterized in that, the transpeptidation reaction buffer comprises: Tris HCl 40-60mmol/L, NaCl 120-180mmol/L, CaCl ₂ 1-10mmol/L and DTT 0.5-1.5mmol/L; Preferably, the transpeptide reaction buffer mainly consists of the following components: Tris·HCl 45-55mmol/L, NaCl 130-170mmol/L, CaCl ₂ 2-7mmol/L and DTT 0.7-1.3mmol/L. 10. as described in any one of claim 1-6, be used for measuring the active polypeptide probe of transpeptidase A pair or the method for measuring transpeptidase A activity described in any one of claim 7-9 is in assay target Application of potential pathogenicity of Lambert-positive bacteria.