CN106188260A - Substrate protein, expression vector, construction method and the application of detection Sortase A activity - Google Patents

Abstract

The invention discloses the substrate protein of a kind of grouping system enzyme Sortase A activity, its nucleotide sequence is as shown in SEQ ID NO.1, its aminoacid sequence is as shown in SEQ ID NO.2, this substrate protein is to prepare with prokaryotic expression carrier expression, it is not necessary to chemosynthesis and modification, just can prepare under common experimental conditions, low cost, cycle are short, and do not limited by chemical synthesising technology, substrate protein to be prepared as power high, there is good application prospect.The invention also discloses substrate protein expression vector and the construction method thereof of a kind of grouping system enzyme Sortase A activity, a kind of method also disclosing sorting enzyme Sortase A activity utilizing above-mentioned substrate protein expression vector detection staphylococcus aureus.

Description

Substrate protein, expression vector, construction method and the application of detection Sortase A activity

Technical field

The invention belongs to technical field of biological, be specifically related to a kind of detect SortaseA activity substrate protein, table Reach carrier, construction method and application.

Background technology

Staphylococcus aureus sorting enzyme SortaseA has the function of protease and transpeptidase concurrently.Bacterial surface protein is becoming First synthesize in bacterial cytoplasm before ripe with N end signal peptide and the amyloid protein precursor of C end sorting signals, and divided by secretory pathway Secrete outside born of the same parents.Signal peptide is excised by signal peptidase in this course, and amyloid protein precursor is positioned at cell membrane by C end hydrophobic region On.Its signature motif is sorted enzyme SortaseA identification subsequently, and disconnects the T-G key in LPXTG, catalysis Thr and cell wall peptide The covalent bond of polysaccharide, thus surface protein is anchored on cell wall.Owing to bacterial surface protein is in the absorption sense of pathogen Dye, biofilm formation aspect has pivotal role, and therefore sorting enzyme SortaseA is the important of Gram-positive bacteria pathogenic Drug target, screening suppresses the inhibitor of its activity to have important practical significance at field of medicaments.

At present, the screening technique of sorting enzyme Sortase A inhibitor is broadly divided into four kinds at present: based on the little molecule of fragment Compound library high flux screening, FRET (fluorescence resonance energy transfer) method, HPLC detection method and fibronectin combined techniques.This several method In, suitable high-throughout screening technique be the substrate protein screening method quoting FRET, i.e. artificial synthesized sequence be the little of LPXTG Peptide, two ends are chemically modified respectively and make it contain Dabcyl-Edans or abz-dnp fluorescent quenching to modification, when sorting enzyme SortaseA catalytic substrate protein chain ruptures, and quenching group separates with fluorophor, and fluorescence intensity will change.Therefore may be used Inhibitor for screening impact sorting enzyme SortaseA activity.

Although the substrate protein screening method of FRET can carry out high throughput testing to the activity of sorting enzyme SortaseA, but Owing to its substrate protein is prepared by chemosynthesis, costly, the cycle is long, the most even synthesizes failure, needs repeatedly to optimize in synthesis Synthesis condition, limits its application to a certain extent.

Summary of the invention

It is an object of the invention to provide the substrate protein of a kind of grouping system enzyme Sortase A activity, expression vector, structure Method and application, solving substrate protein in existing sorting enzyme Sortase A activity test method needs chemosynthesis, expense Height, synthesis cycle is long, the most failed problem.

The invention provides the substrate protein of a kind of grouping system enzyme Sortase A activity, its nucleotide sequence such as SEQ Shown in ID NO.1, its aminoacid sequence is as shown in SEQ ID NO.2.

Present invention also offers the substrate protein expression vector of a kind of grouping system enzyme Sortase A activity, described substrate Protein expression vector is to insert such as SEQ ID NO.1 institute at NcoI, XhoI restriction enzyme site of coli expression carrier pET28a The nucleotide sequence shown is built-up.

The present invention also provides for the structure side of the substrate protein expression vector of a kind of above-mentioned grouping system enzyme Sortase A activity Method, specifically includes following steps:

Step 1, the 5 ' ends at the nucleotide sequence shown in SEQ ID NO.1 add the restriction enzyme site of NcoI, and 3 ' ends add The restriction enzyme site of XhoI, designs and synthesizes the DNA fragmentation as shown in SEQ ID NO.3；

Step 2, the structure of substrate protein expression vector

With NcoI, XhoI double digestion DNA fragmentation as shown in SEQ ID NO.3, and reclaim the target DNA fragment of 1.6kb； With NcoI, XhoI double digestion coli expression carrier pET28a, and reclaim the vector DNA fragment of 5.2kb；By the mesh of 1.6kb DNA fragmentation be connected on the vector DNA fragment of 5.2kb, be built into substrate protein expression vector.

Present invention also offers a kind of above-mentioned substrate protein expression vector, at the sorting enzyme of detection staphylococcus aureus Application in Sortase A activity.

Present invention also offers a kind of sorting enzyme utilizing above-mentioned substrate protein expression vector detection staphylococcus aureus The method of Sortase A activity, comprises the following steps:

Step (1), prepares substrate protein expression vector；

Step (2), converts substrate protein expression vector to e. coli bl21 DE3 bacterium competence cell, induces table Reach substrate protein, obtain substrate protein expression product, then utilize Ni-NTA affinity column that substrate protein expression product is entered Row purification, obtains substrate protein solution；

Step (3), utilizes coli expression carrier pET28a to build and sorts containing in staphylococcus aureus gene group The expression vector pET28a-srtA of enzyme Sortase A gene, is then transformed into escherichia coli by expression vector pET28a-srtA In BL21DE3 bacterium competence cell, abduction delivering sorting enzyme Sortase A, obtain sorting enzyme SortaseA expression product, then Utilize Ni-NTA affinity column that sorting enzyme SortaseA expression product is purified, obtain sorting enzyme SortaseA solution；

Step (4), with substrate protein solution for blank group, utilizes microplate reader grouping system enzyme Sortase solution A The activity of middle sorting enzyme SortaseA.

Preferably, in the method for above-mentioned grouping system enzyme SortaseA activity, microplate reader grouping system enzyme is utilized SortaseA solution sorts the activity of enzyme Sortase A, specifically includes following steps:

Substrate protein solution and sorting enzyme Sortase solution A are mixed to join in ELISA Plate, as detection group, simultaneously With substrate protein solution for blank group；

ELISA Plate is placed in 37 DEG C, and lucifuge hatches 1h；

Microplate reader parameter is set as that 350nm excites, 495nm record, the fluorescence intensity of solution in mensuration ELISA Plate；

Relatively blank group and the fluorescence intensity size of detection group, it is judged that enzyme SortaseA is the most active in sorting.

The substrate protein of a kind of grouping system enzyme Sortase A activity that the present invention provides is to express with prokaryotic expression carrier Preparation, it is not necessary to chemosynthesis and modification, the substrate protein of preparation is the GFP albumen activated by sorting enzyme spcificity, is not having Before being sorted enzyme Sortase A cutting, do not produce the GFP fluorescin of maturation, fluorescence will not be produced, and be sorted enzyme After Sortase A cutting, the GFP fluorescin of maturation can be produced, produce fluorescence, sensitivity can substitute completely existing The substrate protein of chemosynthesis based on FRET principle.

The substrate protein of a kind of grouping system enzyme Sortase A activity that the present invention provides, just may be used under common experimental conditions Preparation, low cost, cycle are short, and are not limited by chemical synthesising technology, substrate protein to be prepared as power high, have well Application prospect.

Accompanying drawing explanation

Fig. 1 is the structural representation of substrate protein expression vector pET28a-GFP-QALPETGEE-QP；

Fig. 2 is the fluorescence intensity change figure of the sorting enzyme SortaseA reaction of substrate protein and staphylococcus aureus.

Detailed description of the invention

The present invention is described in detail with detailed description of the invention below in conjunction with the accompanying drawings, it is to be understood that the protection of the present invention Scope is not limited by detailed description of the invention.

The invention provides the substrate protein of a kind of grouping system enzyme Sortase A activity, its nucleotide sequence such as SEQ Shown in ID NO.1, its aminoacid sequence is as shown in SEQ ID NO.2, containing respectively in the nucleotide sequence of described substrate protein The coding sorting nucleotide sequence of enzyme Sortase A specific recognition, GFP fluorescent quenching sequence and the nucleotides sequence of GFP albumen Row.

Based on same inventive concept, present invention also offers the substrate protein of a kind of grouping system enzyme SortaseA activity Expression vector, described substrate protein expression vector is insertion such as SEQ ID NO.1 institute in coli expression carrier pET28a The nucleotide sequence shown is built-up, specifically builds according to following steps:

Step 1, synthetic DNA fragmentation as shown in SEQ ID NO.3.

According to the gene order of enhanced green fluorescence protein EGFP, coding staphylococcus aureus sorting enzyme Sortase The nucleotide sequence of the conserved motifs LPXTG contained by surface protein C end sorting signals that A can identify, and coding fluorescence cancellation The nucleotide sequence of albumen (quenching peptide, QP), the 5 ' ends at the nucleotide sequence shown in SEQ ID NO.1 add Adding the restriction enzyme site of NcoI, 3 ' ends add the restriction enzyme site of XhoI, design synthetic DNA as shown in SEQ ID NO.3 Fragment, the particular sequence fragment shown in SEQ ID NO.3 is as follows:

, Wherein 5 ' the dashed part held are the restriction enzyme site of Nco I, and the 3 ' dashed part held are the restriction enzyme site of XhoI, capitalization institute Containing the sequence of codified GFP in the nucleotide sequence shown, small letter darkens the nucleotide sequence shown in letter can quilt for coding Sorting enzyme SortaseA specific recognition the sequence of peptide fragment sheared, the nucleotides sequence shown in small letter italic is classified as coding fluorescence The nucleotide sequence of cancellation albumen.

It should be noted that the DNA fragmentation shown in SEQ ID NO.3 had both included the DNA sheet as shown in SEQ ID NO.1 Section, also includes the restriction enzyme site of Nco I, XhoI.

Step 2, the structure of substrate protein expression vector

By the DNA fragmentation as shown in SEQ ID NO.3 through NcoI and XhoI double digestion, and the product after double digestion is connected On coli expression carrier pET28a corresponding NcoI and XhoI double enzyme site, it is built into substrate protein expression vector, Specifically include:

Step 2.1, with NcoI, XhoI double digestion DNA fragmentation as shown in SEQ ID NO.3, and reclaims the purpose of 1.6kb DNA fragmentation；With NcoI, XhoI double digestion coli expression carrier pET28a, and reclaim the vector DNA fragment of 5.2kb；Will The target DNA fragment of 1.6kb is connected overnight (10-16h) with the vector DNA fragment of 5.2kb in 16 DEG C, is built into substrate protein table Reach carrier, by named for substrate protein expression vector, substrate protein expression vector pET28a-GFP-QALPETGEE-QP, this end The structure of thing protein expression vector pET28a-GFP-QALPETGEE-QP is as shown in Figure 1.

Step 2.2, is converted substrate protein expression vector to large intestine bar DH5 α bacterium competence cell, is obtained by resistance screening Obtaining positive strain, order-checking identifies whether substrate protein expression vector pET28a-GFP-QALPETGEE-QP successfully constructs.

Based on same inventive concept, present invention also offers a kind of above-mentioned substrate protein expression vector (substrate protein table Reach carrier pET28a-GFP-QALPETGEE-QP), in the sorting enzyme Sortase A activity of detection staphylococcus aureus Application, and the method for the sorting enzyme SortaseA activity of above-mentioned substrate protein expression vector detection staphylococcus aureus, specifically Comprise the following steps:

Step (1), prepares substrate protein expression vector pET28a-GFP-QALPETGEE-by the method for above-mentioned steps 1-2 QP。

Step (2), obtains substrate protein solution

Substrate protein expression vector pET28a-GFP-QALPETGEE-QP is converted to e. coli bl21 DE3 (large intestine bar Bacterium BL21 (DE3)) in, abduction delivering substrate protein, then utilize Ni-NTA affinity column to carry out pure to substrate protein product Change, obtain substrate protein solution, specifically include:

Step (2.1), is transformed into large intestine bar by substrate protein expression vector pET28a-GFP-QALPETGEE-QP In BL21DE3 bacterium competence cell, next day picking list bacterium colony, and overnight incubation in LB fluid medium, overnight incubation time Between be 10-16h, obtain culture fluid；

Step (2.2), takes 10mL culture fluid and is inoculated in the LB fluid medium containing kanamycin, and every milliliter of LB liquid Containing 25 μ g kanamycin in body culture medium, 37 DEG C of shaken cultivation to OD600=0.6-0.8.

Step (2.3), is down to 16 DEG C by the solution temperature of step (2.2), adds IPTG, and makes that IPTG's is final concentration of 1mmol/L, afterwards in 16 DEG C of overnight induction, the time of overnight induction is 10-16h, obtains induction broth.

Step (2.4), by induction broth in 4 DEG C, 14000rpm, centrifugal 30min, collects precipitation, and cracks with 50mL The resuspended precipitation of buffer, ultrasonication 30min on ice bath, wherein the frequency of ultrasonication is 400W, after in 4 DEG C, 14000rpm, Centrifugal 30min, collects supernatant, obtains the supernatant containing substrate protein expression product, finally by SDS-PAGE electrophoresis detection Substrate protein in supernatant.

Step (2.5), utilizes Ni-NTA affinity column (nickel post) to be purified substrate protein expression product, obtains on earth Thing protein solution.

After the imidazole wash buffer balance Ni-NTA affinity column that 5mL concentration is 10mmol/L, will be containing substrate The supernatant of protein expressioning product crosses post, because there being His label on the N end of substrate protein, therefore, it is possible to be attached to Ni-NTA parent With on chromatographic column, then it is 150mmol/L imidazole elution eluting by 5ml concentration, collects effluent, obtain substrate protein solution, Measure the concentration of substrate protein in substrate protein solution, and by named for substrate protein GFP-QALPETGEE-QP.

Wherein, the imidazole wash buffer of 10mmol/L is by NaH₂PO₄, NaCl, Tween-20, imidazoles and deionized water system For forming, and NaH in lysis buffer₂PO₄The concentration that concentration is 50mmol/L, NaCl be 300mmol/L, Tween-20 Concentration of volume percent is 0.05%, the concentration of imidazoles is 10mmol/L, is adjusted to pH with 3mol/L NaOH and is after preparing 7.0。

150mmol/L imidazole elution is by NaH₂PO₄, NaCl, Tween-20, imidazoles and deionized water be prepared from, and split Solve NaH in buffer₂PO₄The percent by volume that the concentration that concentration is 50mmol/L, NaCl is 300mmol/L, Tween-20 dense Degree is 0.05%, the concentration of imidazoles is that to be adjusted to pH with 3mol/L NaOH after 150mmol/L prepares be 7.0.

Step (3), obtains sorting enzyme SortaseA solution

Coli expression carrier pET28a is utilized to build containing staphylococcus aureus gene group sorts enzyme Sortase The expression vector pET28a-srtA of A gene, is then transformed into e. coli bl21 DE3 bacterium sense by expression vector pET28a-srtA By in state cell, abduction delivering sorts enzyme Sortase A, obtains sorting enzyme Sortase A expression product, then utilizes Ni-NTA Sorting enzyme Sortase A expression product is purified by affinity column, obtains sorting enzyme SortaseA solution, specifically includes:

Step (3.1), design of primers: according to the sorting enzyme SortaseA gene order of staphylococcus aureus, use Prime5.0 software design has synthesized a pair oligonucleotide primers, including forward primer: 5 '- CATGCCATGGGCCAAGCTAAACCT-3 ' (dashed part is NcoI restriction enzyme site)；Downstream primer: 5 '- CCGCTCGAGGCTGCTGCCTTTGACTTCTGTAGCTACAAAGA-3 ' (dashed part is XhoI restriction enzyme site).

Step (3.2), extracts staphylococcus aureus gene group DNA, and with staphylococcus aureus gene group DNA as mould Plate DNA, carries out sorting the PCR amplification of enzyme Sortase A gene, after reclaim PCR primer through 1.0% sepharose electrophoresis.

PCR cumulative volume 25 μ L, including: template DNA 1 μ L (template DNA concentration is 50ng/ μ L), 2.5 μ L 10 × Buffer, forward primer 1 μ L (forward primer concentration is 10 μm ol/L), (downstream primer concentration is 10 μm ol/ to downstream primer 1 μ L L), the 2.5mmol/L dNTP of 2.0 μ L, 0.2 μ L ExTaq polymerase, 17.3 μ L ddH₂O。

The reaction condition of PCR amplification: 94 DEG C of denaturations 4min, 94 DEG C of degeneration 45s, 60.2 DEG C of annealing 1min, 72 DEG C of extensions 1min, totally 28 circulations, then 72 DEG C extend 7min, and last 4 DEG C terminate reaction.

Step (3.3), PCR primer and the coli expression carrier pMD18T of step (3.2) connect overnight in 4 DEG C, even Taking over the time at night is 10-16h, will connect product and convert to bacillus coli DH 5 alpha competent cell, and carries out order-checking and confirm, obtains The positive cell of the pMD18T carrier containing sorting enzyme SortaseA gene.

Step (3.4), in the positive cell of extraction step (3.3), the pMD18T containing sorting enzyme Sortase A gene carries Body, then through NcoI, XhoI double digestion, reclaims the target DNA fragment of 0.46kb；Simultaneously with NcoI, XhoI double digestion large intestine bar Bacterium expression vector pET28a, reclaims the vector DNA fragment of 5.2kb；The target DNA fragment of 0.46kb and the carrier DNA sheet of 5.2kb Section connects overnight in 16 DEG C, and this connection overnight time is 10-16h, is built into sorting enzyme SortaseA expression vector.

Step (3.5), is transformed into large intestine bar BL21DE3 bacterium competence by sorting enzyme Sortase A expression vector through thermal shock In cell, it is thus achieved that carry the prokaryotic expression bacterium of sorting enzyme SortaseA expression vector.

Step (3.6), single bacterium colony of picking prokaryotic expression bacterium, it is seeded in the LB culture medium containing kanamycin, and often Containing 50ug kanamycin in milliliter LB fluid medium, 37 DEG C of overnight incubation, this overnight incubation time is 10-16h, obtains former Nuclear expression bacterium culture.

Step (3.7), by prokaryotic expression bacterium culture: the volume ratio of the LB culture medium=1:100 containing kanamycin, Prokaryotic expression bacterium culture is seeded in the LB culture medium containing kanamycin of 20mL, and in every milliliter of LB fluid medium Containing 50ug kanamycin, 37 DEG C of 220r/min, cultivate to OD600=0.6.

Step (3.8), is down to 28 DEG C by the solution temperature of step (3.7), adds IPTG, and makes that IPTG's is final concentration of 1mmol/L, afterwards in 28 DEG C of 180r/min, abduction delivering 6h, obtains prokaryotic expression bacterium induction broth.

Step (3.9), takes prokaryotic expression bacterium induction broth 400 μ L, 4 DEG C, 10000r/min, centrifugal 30s, collects precipitation Thing, and precipitate is resuspended in 5mL lysis buffer, add 50 μ L 100mg/mL lysozyme, place 30min on ice, then 400W ultrasonic disruption 5min, sequentially adds 0.5 μ L 5U/mL DnaseI, 5 μ L 10 μ g/mL RnaseA, 125 μ L 1mol/L MgCl₂, 250 μ L 20%TrionX-100, place 30min, 14 000g for 4 DEG C and be centrifuged 10min, collect supernatant, obtain Supernatant containing sorting enzyme Sortase A expression product.

Wherein, the lysis buffer of step (2.4) and step (3.9) is by NaH₂PO₄, NaCl, Tween-20 and deionized water It is prepared from, and NaH in lysis buffer₂PO₄The concentration that concentration is 50mmol/L, NaCl be 300mmol/L, Tween-20 Concentration of volume percent be 0.05%, with 3mol/L NaOH regulation to pH=8.0 after preparing.

Step (3.10), utilizes the Ni-NTA affinity column (nickel post) supernatant to sorting enzyme SortaseA expression product It is purified, obtains sorting enzyme SortaseA solution, including:

It is after balance Ni-NTA affinity column washed by 20mmol/L imidazole wash buffer by 5mL concentration, will be containing sorting The supernatant of enzyme Sortase A expression product crosses post, then is 250mmol/L imidazole elution eluting by 5mL concentration, collects and flows out Liquid, obtains sorting enzyme SortaseA solution.

The imidazole wash buffer of 20mmol/L is by NaH₂PO₄, NaCl, Tween-20, imidazoles and deionized water preparation and Become, and NaH in lysis buffer₂PO₄The volume that the concentration that concentration is 50mmol/L, NaCl is 300mmol/L, Tween-20 Percent concentration is 0.05%, the concentration of imidazoles is 20mmol/L, and being adjusted to pH with 3mol/L NaOH after preparing is 7.0.

250mmol/L imidazole elution is by NaH₂PO₄, NaCl, Tween-20, imidazoles and deionized water be prepared from, and split Solve NaH in buffer₂PO₄The percent by volume that the concentration that concentration is 50mmol/L, NaCl is 300mmol/L, Tween-20 dense Degree is 0.05%, the concentration of imidazoles is that to be adjusted to pH with 3mol/L NaOH after 250mmol/L prepares be 7.0.

Step (4), with substrate protein solution for blank group, utilizes microplate reader in sorting enzyme Sortase solution A The activity of sorting enzyme SortaseA detects

Step (4.1), is mixed to join substrate protein solution and sorting enzyme SortaseA solution in ELISA Plate, as inspection Survey group, simultaneously with substrate protein solution for blank group；

Step (4.2), is placed in 37 DEG C by ELISA Plate, and lucifuge hatches 1h；

Step (4.3), microplate reader parameter is set as that 350nm excites, and 495nm record measures the fluorescence intensity in every hole；

Step (4.4), compares the fluorescence intensity size of blank group and detection group, it is judged that sorting enzyme Sortase A is No active.

Fig. 2 is the fluorescence intensity change figure of the sorting enzyme Sortase A reaction of substrate protein and staphylococcus aureus, altogether Count three groups of parallel laboratory tests, as in figure 2 it is shown, when after substrate protein and sorting enzyme Sortase A reaction, can detect under microplate reader The phenomenon raised to fluorescent value；And substrate protein group fluorescent value is the lowest, illustrate that the substrate protein that the present invention provides can be effective The activity of grouping system enzyme Sortase A.

It should be noted that above-mentioned e. coli bl21 DE3 competent cell and bacillus coli DH 5 alpha competent cell are equal Purchased from New England Biolabs, Inc. (US) Massachusetts, United States of America, Ni-NTA affinity column is by the Ni-purchased from Merck KGaA Biological Science Co., Ltd NTAHis Bind resin (article No. 70666) and empty chromatographic column (article No. 69673) are prepared from according to a conventional method.

It should be noted that in above-mentioned test, utilize Ni-NTA affinity column (nickel post) that substrate protein expression product is entered The step of row purification, and utilize Ni-NTA affinity column (nickel post) that sorting enzyme Sortase A expression product is purified Step, is all to be purified according to conventional Ni-NTA affinity column (nickel post) using method.

Although preferred embodiments of the present invention have been described, but those skilled in the art once know basic creation Property concept, then can make other change and amendment to these embodiments.So, claims are intended to be construed to include excellent Select embodiment and fall into all changes and the amendment of the scope of the invention.

Obviously, those skilled in the art can carry out various change and the modification essence without deviating from the present invention to the present invention God and scope.So, if these amendments of the present invention and modification belong to the scope of the claims in the present invention and equivalent technologies thereof Within, then the present invention is also intended to comprise these change and modification.

Claims (6)

1. the substrate protein of a grouping system enzyme Sortase A activity, it is characterised in that its nucleotide sequence such as SEQ ID Shown in NO.1, its aminoacid sequence is as shown in SEQ ID NO.2.

2. the substrate protein expression vector of a grouping system enzyme Sortase A activity, it is characterised in that described substrate protein table Reaching carrier is to insert core as shown in SEQ ID NO.1 at NcoI, XhoI restriction enzyme site of coli expression carrier pET28a Nucleotide sequence is built-up.

The construction method of the substrate protein expression vector of grouping system enzyme Sortase A activity the most according to claim 2, its It is characterised by, specifically includes following steps:

Step 1, the 5 ' ends at the nucleotide sequence shown in SEQ ID NO.1 add the restriction enzyme site of NcoI, and 3 ' ends add XhoI Restriction enzyme site, design and synthesize the DNA fragmentation as shown in SEQ ID NO.3；

Step 2, the structure of substrate protein expression vector

With NcoI, XhoI double digestion DNA fragmentation as shown in SEQ ID NO.3, and reclaim the target DNA fragment of 1.6kb；With NcoI, XhoI double digestion coli expression carrier pET28a, and reclaim the vector DNA fragment of 5.2kb；By the purpose of 1.6kb DNA fragmentation is connected on the vector DNA fragment of 5.2kb, is built into substrate protein expression vector.

4. utilize the substrate protein expression vector described in claim 2, at the sorting enzyme of detection staphylococcus aureus Application in Sortase A activity.

5. the sorting enzyme of the substrate protein expression vector detection staphylococcus aureus that a kind utilizes described in claim 2 The method of Sortase A activity, it is characterised in that comprise the following steps:

Step (1), prepares substrate protein expression vector；

Step (2), converts substrate protein expression vector to e. coli bl21 DE3 bacterium competence cell, at the bottom of abduction delivering Thing albumen, obtains substrate protein expression product, then utilizes Ni-NTA affinity column to carry out pure to substrate protein expression product Change, obtain substrate protein solution；

Step (3), utilizes coli expression carrier pET28a to build containing sorting enzyme in staphylococcus aureus gene group The expression vector pET28a-srtA of Sortase A gene, is then transformed into escherichia coli by expression vector pET28a-srtA In BL21 DE3 bacterium competence cell, abduction delivering sorting enzyme Sortase A, obtain sorting enzyme Sortase A expression product, so After utilize Ni-NTA affinity column to sorting enzyme Sortase A expression product be purified, obtain sort enzyme Sortase A molten Liquid；

Step (4), with substrate protein solution for blank group, utilizes in microplate reader grouping system enzyme Sortase solution A and divides Select the activity of enzyme Sortase A.

The method of substrate protein expression vector grouping system enzyme Sortase A the most according to claim 5 activity, its feature It is, utilizes the activity sorting enzyme Sortase A in microplate reader grouping system enzyme Sortase solution A, specifically include following step Rapid:

Substrate protein solution and sorting enzyme Sortase solution A are mixed to join in ELISA Plate, as detection group, simultaneously the end of with Thing protein solution is blank group；

ELISA Plate is placed in 37 DEG C, and lucifuge hatches 1h；

Microplate reader parameter is set as that 350nm excites, 495nm record, the fluorescence intensity of solution in mensuration ELISA Plate；

Relatively blank group and detection group fluorescence intensity size, it is judged that enzyme Sortase A is the most active in sorting.