CN108642057A

CN108642057A - The method for detecting belt-lactam antibiotics residues using mutain

Info

Publication number: CN108642057A
Application number: CN201810290742.0A
Authority: CN
Inventors: 袁宗辉; 程古月; 宁佳囡; 王玉莲; 彭大鹏; 倪腾腾; 黄玲利; 戴梦红; 陶燕飞; 王旭; 谢书宇; 刘振利; 谢长清
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2018-03-30
Filing date: 2018-03-30
Publication date: 2018-10-12
Anticipated expiration: 2038-03-30
Also published as: CN108642057B

Abstract

The invention belongs to wild animal resources technical fields.The method for particularly relating to mutain detection beta-lactam antibiotics residue.The nucleotide sequence of the albumen such as SEQ ID NO：1, in 55,70 and 563 564 of sequence, there are allelic mutations.It discloses such as SEQ ID NO：The mutain of protein sequence shown in 2 simultaneously to the detection method of 40 kinds of beta-lactam antibiotics residues, including：The design in mutational site builds mutain, carries out induced expression and purifying to mutain, the detection method of beta-lactam antibiotics residue is established using mutain.35 kinds of beta-lactam class antibiotic assay methods of the present invention couple provide the technical parameters such as minimum detection limit, sample recovery rate and the coefficient of variation, accuracy has been done to the detection method of the present invention and repeatability verification is analyzed, the present invention can detect the residual quantity of beta-lactam class antibiotic in 35 kinds of animality food source products simultaneously.

Description

The method for detecting belt-lactam antibiotics residues using mutain

Technical field

The invention belongs to wild animal resources technical fields.Particularly relate to mutain detection beta-lactam antibiosis The remaining method of element.The present invention can detect simultaneously while to detect beta-lactam antibiotic in 35 kinds of animality food source products residual Allowance.

Background technology

Beta-lactam antibiotic refers to the major class antibiotic with beta-lactam nucleus in chemical constitution.In the past 50 years, Beta-lactam antibiotic be used as livestock and poultry prevent, treat and growth promotion.In modern agricultural production, animal food In such antibiotic addition and be considered essential to the prophylactic treatment of growth and development.However due to such The misuse or abuse of antibiotic causes residual in animal body, this jeopardizes Animal Food Security and human health.Generation The concern that remains height of the various countries of boundary to antibiotic formulates various correlation methods in animal feeding, production and sales process Laws ＆ Regulations carry out use and the residue detection of regulation antibiotic.

At present beta-lactam antibiotic there are many detection method exist, predominantly microbial method, physico-chemical analysis method, exempt from Epidemic disease analytic approach and receptor analysis method (Ahmed et al., 2017).Microbiological analysis is based on antibiotic to microbial physiology The inhibiting effect of function and metabolism selects bacillus subtilis, bacillus stearothermophilus and Sarcina lutea etc. quick mostly Bacterial strain etc. is felt as detection indicator bacteria, and this method is easy to operate, cheap, is suitable for the screening of a large amount of samples of base (Ferrini et al., 2008), but there are detection times it is long, stability is poor the shortcomings of, therefore Standardized quantitative difficult to realize Detection.Physico-chemical analysis method carries out quantitative or qualitative analysis to antibiotic, and accuracy is high, high sensitivity, but needs expensive instrument With the technology of profession, and pre-treatment program is complicated, be not suitable for base's sample screen on a large scale (Gaudin et al., 2001).Immunoassay is the analysis method that core reaction is combined into the specificity of antigen-antibody, invertibity, this method tool There is the features such as fast and convenient, high sensitivity, high specificity, big treating capacity, but wherein antibody production techniques are complex, and by It is high in the specificity of antibody, a kind of or a few antibiotic can only be detected simultaneously.Antibiotic receptor analysis method is to be based on antibiosis The analysis method of specific recognition reaction between element and receptor, can to contain active β-interior for specific recognition simultaneously The penicillins and cephalosporins of amide ring, to realize antibiotic multi-residue determination (journey Gu moon et al., 2014)。

At present receptor analysis method in the residue detection of antibiotic using more, as beta-lactam (Chen et al., 2015, Pazzola et al., 2015), tetracycline (Moeller et al., 2007), chloramphenicol, sulfamido (Gaudin Et al., 2012, Liang et al., 2013) etc. antibiotic.The residual that the technology is applied to beta-lactam antibiotic is examined Receptor protein main species in survey are more, wherein from streptococcus pneumonia PBP2x recombinant proteins (Zeng et al., 2013) can be used for detecting 15 kinds of beta-lactam antibiotics in milk sample, the PBP3 recombinant proteins from streptococcus pneumonia (quiet 2015) can be used for detecting 27 kinds of beta-lactam antibiotics in milk sample, minimum detection limit (Limit of Detection, LOD) it is 0.26-109.46 μ g/kg.

Hua Zhong Agriculture University's national basic veterinary drug where the applicant remains benchmarks room early-stage study beta-lactam The screening and ELISA kit development of antibiotic receptor construct prokaryotic expression carrier pET-28a (+)-BlaR-CTD, expression And purify and obtain the BlaR-CTD albumen of high-purity, it is interior to establish β-in milk, beef, Chicken Tissues using the albumen as receptor The LOD of the detection method of amide antibiotics residue, Cefquinome is 0.43 μ gL^-1, it is less than the MRL of European Union and China (Peng et al.,2013).However in the research albumen to Cloxacillin, cefalexin, cefadroxil affinity compared with Difference, and the stability of albumen is poor.

Invention content

It is an object of the invention to overcome the deficiencies of existing technologies, structure is carried out to BlaR-CTD by rite-directed mutagenesis and is changed It makes, improves the affinity and stability of the albumen, establish one kind and can be suitably used for beta-lactam in a variety of edibility animal products The how remaining detection method of antibiotic.

The albumen of the present invention can be applied to, can be with when the beta-lactam antibiotic type in tissue sample is known Semi-quantitative analysis is carried out to it.

In order to realize the task of the present invention, inventor is on the basis of prokaryotic expression carrier pET-28a (+)-BlaR-CTD Carrying out structure of modification to BlaR-CTD albumen, (mutain in the present invention, comes from whole outside the genosome of bacillus licheniformis It after closing pET-28a plasmids, then is transformed into Escherichia coli, that is to say, that the mutain has been integrated into Escherichia coli, profit It is exactly that mutain therein is utilized with the Escherichia coli KCC of preservation of the present invention), the prokaryotic expression for obtaining mutain carries Body pET-28a (+)-BlaR-CTD-I188K-S19C-G24C, applicant will contain the expression vector pET-28a (+)-BlaR- The Escherichia coli of CTD-I188K-S19C-G24C were named as Escherichia coli KCC, Escherichia coli KCC, in 2018 Delivered the China typical culture collection center preservation of the Chinese Wuhan Wuhan Universitys on March 16, deposit number is CCTCC NO: M2018132。

It is residual the present invention provides 40 kinds of beta-lactam antibiotics are detected while one kind being based on BlaR-CTD mutains The method stayed, it includes structure BlaR-CTD mutains, and beta-lactam is established in the induced expression for mutain of dashing forward and purifying Antibiotics residue detection method detects beta-lactam antibiotic, the specific steps packet of the method using mutain It includes：

(1) using prokaryotic expression carrier pET-28a (+)-BlaR-CTD as template, the mutant primer (sequence of mutant primer is utilized Row such as sequence table SEQ ID NO:Shown in 3-8) PCR amplification is carried out, obtain mutant plasmid pET-28a (+)-BlaR-CTD- I188K-S19C-G24C, after converting e. coli bl21 competent cell, it is CCTCC NO to obtain deposit number:M2018132 The Escherichia coli KCC of mutation；

(2) OD for the Escherichia coli being mutated when step (1)₆₀₀When being 0.6, the isopropyl-of final concentration of 1mM is added β-D- Thiogalactopyranosides (IPTG), 18 DEG C of constant-temperature shaking culture 12h carry out induced expression to the albumen, collect bacterium solution Ultrasonication afterwards takes supernatant to be purified, and obtains high-purity mutain I188K-S19C-G24C；

(3) the high-purity mutain I188K-S19C-G24C obtained by step (2) is coated on ELISA Plate, optimization is anti- It is detected after answering condition, investigates precision, the sensitivity and specificity of detection method；

(4) detection method for establishing step (3) is applied to the detection of various edibility animal samples, measures lowest detection Limit, sample recovery rate and the coefficient of variation, investigate accuracy and the repeatability of method.

Main advantages of the present invention are as follows：

1, the present invention obtains to identify 40 kinds of beta-lactams simultaneously by carrying out rite-directed mutagenesis to BlaR-CTD albumen Antibiotic, and existing document and patent report cannot still identify it is so many to a variety of belt-lactam antibiotics residues Detection.

2, the detection method that the present invention establishes can detect the residual of 40 kinds of beta-lactam antibiotics, in addition to cefalexin The minimum detection limit of the other drugs MRL as defined in European Union is hereinafter, moreover, work as beta-lactam antibiotic type to be detected When being known, semi-quantitative analysis can be carried out to it.And existing micro-biological process does similar detection, detection is limited to height, i.e., The Antibiotics of existing method identification are limited, and without standard measure.

3, the applicable tissue of the present invention include milk, pig, chicken, ox musculature, it is applied widely.

4, sample treatment of the present invention is easy, and easy to operate, accuracy and precision are good.

Description of the drawings

Sequence table SEQ ID NO:1 is the nucleotide sequence of mutain I188K-S19C-G24C.In the sequence table, 55 Bit base replaces with T by A, and 70 bit bases replace with T by G, and 563,564 bit bases replace with AA by TT.

Sequence table SEQ ID NO:2 be the protein sequence of mutain I188K-S19C-G24C.In the sequence table, 19 Amino acids replace with C by S, and 24 amino acids replace with C by G, and 188 amino acids replace with K by I.

Sequence table SEQ ID NO:3 be the forward primer F1 for expanding mutant plasmid pET-28a (+)-BlaR-CTD-S19C Sequence.

Sequence table SEQ ID NO:4 be the reverse primer R1 for expanding mutant plasmid pET-28a (+)-BlaR-CTD-S19C Sequence.

Sequence table SEQ ID NO:5 be expand mutant plasmid pET-28a (+)-BlaR-CTD-S19C-G24C forward direction draw The sequence of object F2.

Sequence table SEQ ID NO:6 be to expand the reversed of mutant plasmid pET-28a (+)-BlaR-CTD-S19C-G24C to draw The sequence of object R2.

Sequence table SEQ ID NO:7 be amplification mutant plasmid pET-28a (+)-BlaR-CTD-I188K-S19C-G24C The sequence of forward primer F3.

Sequence table SEQ ID NO:8 be amplification mutant plasmid pET-28a (+)-BlaR-CTD-I188K-S19C-G24C The sequence of reverse primer R3.

Fig. 1：For the Technology Roadmap of the present invention.

Fig. 2：Plasmid construction for mutant plasmid pET-28a (+)-BlaR-CTD-I188K-S19C-G24C in the present invention shows It is intended to.

Fig. 3：For the plasmid map of mutant plasmid pET-28a (+)-BlaR-CTD-I188K-S19C-G24C in the present invention.

Fig. 4：For the BlaR-CTD albumen 3D structures for the bacillus licheniformis ATCC14580 that 1 step of embodiment (1) obtains Figure.

Fig. 5：For the BlaR-CTD eggs of Cefquinome and bacillus licheniformis ATCC14580 that 1 step of embodiment (1) obtains It is white to combine figure.

Fig. 6：For the sequencing result figure of mutant plasmid pET-28a (+)-BlaR-CTD-S19C in 1 step of embodiment (2).

Fig. 7：For the sequencing result of mutant plasmid pET-28a (+)-BlaR-CTD-S19C-G24C in 1 step of embodiment (2) Figure.

Fig. 8：For the survey of mutant plasmid pET-28a (+)-BlaR-CTD-I188K-S19C-G24C in 1 step of embodiment (2) Sequence result figure.

Fig. 9：For the canonical plotting that step (7) obtains in embodiment 3.

Specific implementation mode

Below by embodiment, the invention will be further described, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified Conventional biochemical reagent company is commercially available.Quantitative test in following embodiments is respectively provided with and repeats to test three times, as a result takes Average value.

Bacillus coli DH 5 alpha competent cell (lot number CD201) and e. coli bl21 competent cell (lot number CD601) It is purchased from Beijing Quan Shijin biotinylated biomolecules Technology Co., Ltd..Horseradish peroxidase (HRP) is purchased from U.S. Sigma- Aldrich.BCA determination of protein concentration kits are purchased from the green skies Bioisystech Co., Ltd (product identification in Shanghai P0010S)。

LB/KAN solid mediums are prepared：The routinely LB agar mediums after (121 DEG C, 15min) autoclave sterilization, About 60 DEG C are cooled to, is 2000 according to volume ratio：Kanamycins (100mg/mL) is added in 1 ratio, after mixing well, pours into In sterilized petri dishes, solidification to be cooled is placed on 4 DEG C of preservations.

Combination buffer：7.6g sodium phosphates, 29.22g sodium chloride are weighed, 0.68g imidazoles is dissolved in 900mL distilled waters, is mended It fills distilled water and is settled to 1L, adjust pH to 7.4, using 0.2 μm of water phase membrane filtration, 4 DEG C of preservations.

Elution buffer：7.6g sodium phosphates, 29.22g sodium chloride are weighed, 34.04g imidazoles is dissolved in 900mL distilled waters, Supplement distilled water is settled to 1L, adjusts pH to 7.4, using 0.2 μm of water phase membrane filtration, 4 DEG C of preservations.

Phosphate buffer (PBS)：Accurately weigh 8.0g sodium chloride, 0.2g sodium dihydrogen phosphates, 0.2g potassium chloride, 2.9g phosphorus Sour disodium hydrogen adds deionized water about 800mL, and pH to 7.4, supplement distilled water is adjusted to be settled to 1L.

The structure of 1 mutant plasmid of embodiment

(1) selection in mutational site

After the present invention is to the amino acid sequence and secondary structure analysis of the BlaR-CTD of bacillus licheniformis ATCC14580, It was found that it is without containing free cysteine and disulfide bond is not present, active pocket is formed in the interface of structural domain.BlaR-CTD Albumen can specifically identify beta-lactam antibiotic, and quickly can form conjugate with drug.From albumen flex region Start with, stablizes protein structure by introducing disulfide bond, it is made preferably to play bioactivity.Find ground by sequence alignment The homology of clothing bacillus ATCC14580 and 749/I are higher, therefore select 749/I for template, according to RCSB PDB data Crystal structure (the PDB ID of existing bacillus licheniformis 749/I bacterial strains in library：1nrf), pass through SYBYL X-2.0 computers Simulation softward carries out Blast search, constructs the 3D structures of the BlaR-CTD albumen of bacillus licheniformis ATCC14580, uses The Surflex-dock modules of SYBYL X-2.0 molecular simulation softwares are by beta-lactam antibiotic and receptor protein BlaR-CTD Molecular docking is carried out, binding pattern between the two is analyzed, and therefrom find out predominant intermolecular forces and key amino acid, in albumen The specific position of BlaR-CTD introduces disulfide bond, designs mutain, analyzes changing for improved protein affinity and stability Become.

1) Blast search

The BlaR-CTD albumen of bacillus licheniformis ATCC14580 is constructed according to the following steps using Syble softwares 3D structures：

1. creating an ORCHESTRAR project：Bioploymer>Model Proteins>Input is arranged in FUGUE Sequence from buttons are FASTA File, put " ... " button with a line, select 14580.fasta in Selection, so After click OK, setting Proflies to Search buttons are All Proflies, and other parameters be default, click OK, beginning Run FUGUE.Open ORCHESTRAR, Bioploymer>Model Proteins>ORCHESTRAR pops up Protein Manager dialog boxes click New Project, confirm FUGUE Run in Slecte New Project Type dialog boxes Results is selected, clicks Import.

2. checking the sequence and structure of homolgous molecule：Build conserved region：Model is clicked in Protein Manager Conserved Regions, query sequence can be seen in Sequence Viewer with scrolling bar by two dialog boxes occur The case where being compared with homolgous molecule.Again it compares：Sequence homology is checked in Model Conserved Regions dialog boxes Property, Add Homolog are clicked, crystal structure (the PDB ID of bacillus licheniformis 749/I bacterial strains are added：1nrf), it removes simultaneously The low sequence of other homologys, clicking (Re) align Query to Homolog can find that Percent identity updates, Also some variations occur in Sequence Viewer.

3. merging ligand：Certain homologous molecules contain ligand, cofactor, metal in HOMSTRAD, water etc., Manager Ligand/Cofactor ... buttons, pop-up are clicked in Model Conserved Regions dialog boxes Manager Ligand/Cofactor dialog boxes.The small molecule being contained in homolgous molecule is identified, by Define List Type in the parts Ligand/Cofactor are set as All, it is contemplated that rear pattern plate in small molecule, selection is suitable Ligand.

4. establishing, analysis structural conserved regions：Build is clicked in Model Conserved Regions dialog boxes SCRs jumps out Model status therein in ORCHESTRAR-Analyze Conserved Region dialog boxes, , there are SCRs List in the homolgous molecule that structure model is listed in View Selected Homolog lists.It inspects Sequence Viewer dialog boxes：View>Text Style>Color by Secondary Structure, red are Helix, blue are sheet.Abnormal peptide chain length and C α-C α distances are checked, in Analyze Conserved Region Check Distances are clicked in dialog box, yellow is in the both sides gap and to identify, and red is peptide bond than ideal bond distanceIt is long 20% or more, blue is in normal range (NR).The undesirable dihedrals phi and psi are inspected, in Analyze Conserved Check Torsions are clicked in Region dialog boxes, red is, in the areas ramachandran map Ramachandran OUTSIDE, yellow is in ramachandran map Ramachandran The areas GENEROUS, blue is for perhaps can area in ramachandran map Ramachandran CORE.Steric clashes are inspected, in Analyze Conserved Check for Clashes are clicked in Region dialog boxes, red is that principle is collided, and yellow is that residue is at least one Collision, blue are that residue does not collide.

5. LOOP area searchings：Click Search Loops in Analyze Conserved Region dialog boxes, pop-up Search Loops dialog boxes.Inspect Gap options in the part View Gaps, the loop area searchings homolgous molecule of end and PRODAT.The Q or H of Loop Search list first rows indicate whether the search is submitted, or are not moved by hanging.It clicks Search Loops carry out Loop search.

6. opening Add/Analyze Loops windows, checks and add Loop：The structure for checking each gap, chooses Show All Marked Loops preview models click Jion All Marked Loops connection Loop, search for remaining lack Residue is lost, Remodel Loops is clicked in dialog box bottom, then click Search Loops, clicks Jion All Marked Loops, setting model title.

7. mould is built, analyzes side chain：Prepare addition side chain, Model is clicked in Add/Analyze Loops dialog boxes bottom Sidechains, Borrow Sidechain Conformations Form Homolog are selected, confirm Borrow Options options are Borrow Chi1+2+3/Restrict Chi1+2.Side chain is added, Add Sidechains are clicked, is occurred ORCHESTARA-Analyze Sidechains dialog boxes.The steric clashes between pendant atom are analyzed, Visualize is chosen Sidechain Clashes, red are that VDW is overlapped, and yellow is collision, and blue is other.

8. analysis model：Analyze Model are clicked in Analyze Sidechains dialog boxes bottom, are occurred Analyze Model dialog boxes.One simple inspection is carried out to model, clicks Check Backbone Distances, inspection Undesirable peptide bond bond distance and C α-C α distances are looked into, Check Backbone Torsions are clicked, red is deviation ideal zone. The dihedrals phi/psi are shown in ramachandran map Ramachandran with ProTable, click Analyze Model (ProTable), MSS：Protein >Ramachandran Plot.Click the Save Model of Analyze Model dialogue frame bottoms.

The BlaR-CTD protein structures of bacillus licheniformis ATCC14580 are obtained as shown in figure 4, main by Blast search It is made of two structural domains, the structural domain of α/β type is covered in one seven strands reversely by two α spirals (α 10 of α 1 and the ends C-) Parallel β plane (β 1- β 7), and covered by 3 spirals of α and 8 spirals of α in another side, second structural domain is mainly by four spiral shells It revolves (α 5, α 6, α 7 and α 9) and surrounds hydrophobic 4 spirals of α composition.The central area of the proteins carry pocket by S (55) TYK (58), The binding site of S (103) AT (105) and K (192) TG (194) compositions, beta-lactam antibiotic is Ser55.

2)Surflex-Dock

With established albumen 3D structures, the molecule pair of albumen and beta-lactam antibiotic is carried out according to operations described below Connect, according to molecular docking result obtain albumen active pocket and with pharmaceutically-active active site, in later stage mutational site These sites are avoided in selection course.

1. establishing activity conformation：It is big that protein is chosen from Protein structure databases (Protein Data Bank, PDB) Molecule and existing structure small molecule, the three-dimensional structure of compound and ligand is directed respectively into database.

2. setting joint mode, correcting albumen and extraction ligand：The interfaces Surflex-Dock are opened, are clicked Applications>Docking Suite>Dock Ligands confirm that " Docking mode " is " Surflex-Dock (SFXC) " Define, is clicked in the regions Docking Mode, opens Surflex-Dock (SFXC) dialog box. Receptor file formats are set as PDB in Surflex-Dock (SFXC) dialog box, " ... " button are clicked, from open text Part manager window selects the albumen file of a needs and clicks OK confirmations, clicks Prepare, opens Prepare Protein Structure dialog boxes, handle ligandin.Extract Ligand Substructures are clicked, The structure of ligand is selected in Other lists and clicks OK.Remove Substructures are clicked, in Water lists, point Select all buttons are hit, all hydrones are deleted, click OK.Click Analyze Selected Structure activation Albumen prepares button, clicks Add Hydrogens, selects " All " " Random ", clicks OK, adds Analyze after hydrogen Selected Structure buttons can be graying.Click the standard that the OK button below Receptor Preparation completes albumen Standby and ligand extraction.

3. setting docking pocket：In Surflex-Dock-Define SFXC File dialog boxes, Protomol is set Generation Mode are Residues, and Generate is clicked in setting key amino acid site, is clicked OK and is generated Surflex- Dock SFXC files.

4. preparing ligand file：It clicks Cancle buttons and temporarily exits the interfaces Surflex-Dock.Read in ligand file, point Hit Edit>Add Hydrogens are that ligand adds hydrogen, then click File>Export File preserve ligand file.

5. specified ligand to be docked simultaneously submits operation：The interfaces Surflex-Dock are reopened, are clicked Applications>Docking Suite>Dock Ligands confirm that " Docking mode " is " Surflex-Dock (SFXC) " " ... " button, is clicked in the regions Docking Mode, selects the ligand file set.In Ligand The regions Source, setting ligand file format are Mol2, click " ... ", select a ligand file to be docked. In the regions Options, Surflex-Dock is clicked, opens Surflex-Dock-Details windows；In Reference In the regions Molecule, combobox selection Mol2File and " ... " for clicking right side select a ligand reference paper, click OK retracts the interfaces Docking.Perform CScore Calculations are chosen in cancellation.Job title is set, is clicked OK submits operation.

6. checking docking result：The interfaces Surflex-Dock are opened, Applications is clicked>Docking Suite> Analyze Results.At the interfaces Results Browser, " ... " on the right side of jobname is clicked, opens job catalog.With The top score docking conformation of docking marking and ligand is checked in body list box.Click the File of SYBYL main interfaces>Import File, reads in ligand file, the front and back ligand conformational of observation docking, position difference.Click top in ligand list box Then Table clicks ligand file, can check the whole conformations exported after ligand docking and marking details.

The albumen obtained after mould is built carries out molecular docking with beta-lactam antibiotic.Cefquinome and BlaR-CTD eggs White docking result as shown in Fig. 5, between protein target and Cefquinome ligand formed 9 hydrogen bonds, in addition to existing document report Form hydrogen bond between key amino acid Ser55, Ser103, Thr105, Thr193 for leading, wherein with Ser55, Ser103, Be respectively formed between Thr105 1 between hydrogen bond, with Thr193 formed 3 hydrogen bonds, in addition, Cefquinome ligand also with Tyr87 it Between formed 2 between hydrogen bond and Thr195 formation 1 hydrogen bond.Hydrogen bond is as the strongest non-binding effect of intermolecular interaction, hydrogen bond Quantity it is more, show that the binding ability of the ligand molecular and albumen is stronger.

It is as shown in table 1 to enumerate the amino acid residue that participation hydrogen bond is formed in docking operation, in addition to active site and closely It outside amino acid Ser55, Ser101, Ser102, Ser103, Thr105, Thr193, Thr195 of active site, also space Closer Tyr87, Glu89, Arg229, Ser233 amino acid in positional distance activated centre also assists in the formation of hydrogen bond, therefore These sites are avoided in the selection course in follow-up mutational site, in order to avoid reduce the binding ability of albumen and drug.

The amino acid sites and marking result of 1 BlaR-CTD albumen of table and beta-lactam antibiotic specific binding

3) determination in simple point mutation site

In addition to disulfide bond, salt bridge can also increase the stability of albumen, and I188 is located at 5 segments of β, is sported Lys, can Salt bridge is formed with the E212 in 6 segments of β.In addition other blueness can be utilized by comparing the similar penicillin binding protein of structure The corresponding site of BlaR-CTD albumen is sported the amino acid, to carry by the active site amino in mycin binding protein The affinity of high protein, the PBP3 albumen from streptococcus pneumonia R6 is to the affinity of cefadroxil relative to BlaR-CTD It is higher (quiet 2013), analyze the molecular docking of PBP3 and cefadroxil as a result, in KTGTTD sequences D244 and cephalo hydroxyl Ammonia benzyl formed hydrogen bond, by sequence alignment in BlaR-CTD the sequence be KTGTSV, therefore design mutant V197D to Improve affinity of the BlaR-CTD albumen to cefadroxil.Mutational site is predicted using SIFT and PloyPhen2 softwares, After selecting mutational site, whether which is caused by shadow to the function of albumen after other amino acid by software prediction It rings, the marking of SIFT is higher, and the marking of PloyPhen2 softwares is lower, illustrates that mutation is more reasonable, is designed according to marking result Protein mutant.In conjunction with these factors, it is as shown in table 2 that suitable simple point mutation site can be selected.

The prediction result in 2 amino acid mutation site of table

4) disulfide bond is introduced

The 3D structures of the BlaR-CTD of the bacillus licheniformis ATCC14580 obtained according to Blast search utilize 2.0 softwares of Disulfide by Design obtain the amino acid sites for being likely to form disulfide bond, as shown in table 3.Exclude activity Site, key amino acid and introducing easily cause the amino acid sites of steric hindrance, according to two cysteines for forming disulfide bond In the position of BlaR-CTD secondary structures, four classes are classified as, focus primarily upon the areas loop, β segments and the adjacent α of Ω samples Between spiral, α spirals between β segments.The data simulated according to software, according to bonding energy smaller, temperature factor compared with The molecular docking of high principle, binding protein and drug filters out conjunction as a result, exclude the amino acid sites near activated centre Suitable mutational site S76C-L96C, S135C-S145C, R50C-Q147C, S19C-G24C, E183C-I188C.

The amino acid sites for the disulfide bond being likely to form in the BlaR-CTD of 3 bacillus licheniformis ATCC14580 of table

In order to improve the affinity and stability of albumen simultaneously, pass through receptor analysis method (the Peng et of Peng Juan foundation Al., activity identification 2013) is carried out to mutain, compares the activity (as shown in table 4) of albumen, shows mutain S76C- L96C, S135C-S145C are in the detection without activity, then respectively with disulfide bond mutain S19C-G24C, S135C- S145C, E183C-I188C are template, carry out simple point mutation.Compare mutain inhibiting rate (as shown in table 5), filters out prominent It is best to the affinity of various beta-lactam antibiotics to become albumen I188K-S19C-G24C, as purpose of the present invention egg In vain.

The activity identification of 4 mutain of table

Inhibiting rate of 5 drug of table to mutain

(2) acquisition of mutant plasmid

1) a small amount of pET-28a (+)-BlaR-CTD expression bacterium of picking (detects from patent belt-lactam antibiotics residues Receptor analysis method and kit, application number：CN201310196144.4, publication number：CN103897046A, publication date： Freeze-dried powder 2014-07-02) is seeded in the LB broth bouillons of 2mL, 37 DEG C, and culture is shaked in 220rpm constant-temperature tables Overnight.A small amount of bacterium solution streak inoculation is chosen in LB/Kan agar plates, 37 DEG C overnight.Picking single bacterium falls within the LB/Kan meat of 10mL In soup culture medium, 37 DEG C, plasmid pET-28a (+)-BlaR-CTD is extracted after overnight incubation is shaked in 220rpm constant-temperature tables ,- 20 DEG C freeze.

2) using pET-28a (+)-BlaR-CTD plasmids as template, PCR amplification is carried out with primers F 1, R1, by the production after amplification Object (sequencing result is as shown in Figure 6) is transformed into bacillus coli DH 5 alpha competent cell and is coated on LB/Kan agar plates.

Primers F 1, R1 sequences are as follows：

F1:GATGACTGCACCTTTTTTGATGGCT, (SEQ ID NO:3)

R1:GGTGCAGTCATCTTCGTATTCTACA；(SEQ ID NO:4)

3) single bacterium is fallen in the LB/Kan broth bouillons of 10mL on picking step 2) tablet, 37 DEG C, and 220rpm constant temperature shakes Mutant plasmid S19C is extracted after shaking overnight incubation in bed, using mutant plasmid pET-28a (+)-BlaR-CTD-S19C as template, PCR amplification is carried out with primers F 2, R2, the product (sequencing result is as shown in Figure 7) after amplification is transformed into bacillus coli DH 5 alpha impression State cell is simultaneously coated on LB/Kan agar plates.

Primers F 2, R2 sequences are as follows：

F2:TTTGATTGCTTCTCAGGAGGTTTTG, (SEQ ID NO:5)

R2:AGAAGCAATCAAAAAAGGTGCAGTC；(SEQ ID NO:6)

4) single bacterium is fallen in the LB/Kan broth bouillons of 10mL on picking step 3) tablet, 37 DEG C, and 220rpm constant temperature shakes Mutant plasmid S19C/G24C is extracted after shaking overnight incubation in bed, with mutant plasmid pET-28a (+)-BlaR-CTD-S19C- G24C is template, and PCR amplification is carried out with primers F 3, R3, and the product (sequencing result is as shown in Figure 8) after amplification is transformed into large intestine Bacillus DH5 α competent cells are simultaneously coated on LB/Kan agar plates.

Primers F 3, the sequence of R3 are as follows：

F3:GTCCCGGTTTTACCGGATAGTTTTCTGCCATTTGATTCTTCT, (SEQ ID NO:7)

R3:AGAAGAATCAAATGGCAGAAAACTATCCGGTAAAACCGGGAC；(SEQ ID NO:8)

5) single bacterium is fallen in the LB/Kan broth bouillons of 10mL on picking step 4) tablet, 37 DEG C, and 220rpm constant temperature shakes Mutant plasmid pET-28a (+)-BlaR-CTD-I188K-S19C-G24C is extracted after shaking overnight incubation in bed.

The acquisition of 2.2 mutain I188K-S19C-G24C

Mutant plasmid pET-28a (+)-BlaR-CTD-I188K-S19C-G24C is transformed into e. coli bl21 competence Cell is simultaneously coated on LB/Kan agar plates, obtains the Escherichia coli KCC for including BlaR-CTD mutains.

The induced expression of 2 mutain of embodiment and purifying

(1) induced expression of mutain

Mutant plasmid pET-28a (+)-BlaR-CTD-I188K-S19C-G24C is transformed into e. coli bl21 competence Cell is simultaneously coated on LB/Kan agar plates, obtains the Escherichia coli KCC for including mutain I188K-S19C-G24C.Using E. coli bl21 expression system carries out vivoexpression (Peng et al., 2013) to mutain, obtains mutain Albumen supernatant after I188K-S19C-G24C total proteins, ultrasound and precipitation.

(2) SDS-PAGE is detected

By after mutain I188K-S19C-G24C total proteins that step 2.1 obtains, ultrasound albumen supernatant and precipitate into Row SDS-PAGE detection (Peng et al., 2013).The result shows that the total eggs of mutain I188K-S19C-G24C of the present invention In vain, the albumen supernatant after ultrasound and the precipitation band containing 26kD or so, but protein content is much larger than egg in precipitation in supernatant Bai Hanliang, mutain illustrate that mutain exists with soluble protein with this condition nearly all in supernatant.

(3) purifying of mutain

1) with the combination buffer of 10mL come balance nickel column.

2) the albumen supernatant that step (1) is collected is added, makes sample and the Ni Sepharose 6Fast Flow in purification column Suspension fully reacts 90min at 4 DEG C, and a pillar is vibrated per 10min or so, His labels is made fully to be combined with Ni.When After all samples are by pillar, the combination buffer washing of about 10 column volumes is added.

3) elution buffer and combination buffer are mixed by different proportion in advance, elution buffer liquid proportional is respectively 5%, 10%, 20%, 40%, 60%, 80%, 100%, corresponding imidazole concentration is about 35,60,100,200,300,400, 500mmol/L。

4) by the buffer solution mixing of different proportion, the sequence from low concentration to high concentration is added in pillar to elute egg In vain, 6 column volumes are about added in each concentration, and collect imidazole concentration be 60,100,200mmol/L when eluent.

6. the eluent of collection is dialysed with PBS, mutain is obtained.

(4) verification of mutain

1) albumen carries out SDS-PAGE verifications after the dialysis for obtaining step (3), the results showed that has obtained purity after purification Higher mutain.

2) after the mutain for obtaining step (3) carries out SDS-PAGE, (institute is verified with Western blot methods With antibody, primary antibody is His-tag monoclonal antibodies, and secondary antibody is the sheep anti-mouse antibody of HRP labels), it is used in combination DAB to develop the color.As a result it shows Show that mutain is the positive.

(5) detection of mutain yield

The mutain that step (3) is obtained detects albumen concentration with BCA determination of protein concentration kits.The result shows that prominent Become a concentration of 1.3mg/mL of albumen.

The foundation of 3 beta-lactam antibiotic detection method of embodiment

(1) ampicillin of HRP labels is prepared

Using carbodiimide/n-hydroxysuccinimide (EDC/NHS) method (Peng et al., 2013) synzyme mark medicine Object, i.e. the ampicillin HRP-AMP of horseradish peroxidase-labeled, respectively measure AMP, HRP and HRP-AMP be put into it is ultraviolet All-wave length figure, obtains the maximum absorption wavelength of each substance, and whether identification is coupled success.

(2) determination of the best peridium concentration of mutain

Using the concentration of the tentatively selected best coating mutain of square formation titration, selection criteria is with OD₄₅₀Value is located at Near 2.0, and it is optimal combination that adjacent holes OD values, which have the peridium concentration of large change and corresponding enzyme marker dilution,. After mutain is diluted with PBS buffer solution, 100 μ L of protein solution are added per hole into ELISA Plate, 4 DEG C are coated with overnight, outwell hole 250 μ L of PBST are added per hole for interior liquid, stand 2min, discard liquid in hole and pat dry, and wash repeatedly 3 times；Envelope is added per hole It closes buffer solution 250 μ L, 4 DEG C of closing 12h and discards liquid in hole, 250 μ L of PBST are added per hole, standing 2min discards liquid in hole Body simultaneously pats dry, and washes repeatedly 3 times；Every hole is added 50 μ L of PBS buffer solution and is 1 according to volume ratio:150、1:300、1:600、1: 1200、1:2400 are diluted the 50 μ L of HRP-AMP solution that step (1) obtains, and are incubated 45min for 37 DEG C after mixing, discard in hole 250 μ L of PBST are added per hole for liquid, stand 2min, discard liquid in hole and pat dry, and wash repeatedly 3 times；It is added per hole and now matches 100 μ L of Substrate cocktail, 37 DEG C are protected from light colour developing 15min；50 μ L of terminate liquid are added per hole；Each hole OD is measured with microplate reader₄₅₀ Value.

The result shows that when albumen extension rate is 1300 times (i.e. a concentration of 1 μ g/mL of albumen), enzyme marker extension rate is 1200 times of (i.e. a concentration of 0.36 μ gL of enzyme marker^-1) when, it is its best peridium concentration.

(3) determination of coating buffer

CBS buffer solutions that the PBS buffer solution and pH value for being respectively 7.4 with pH value are 9.6 dilution mutain is to most preferably dilute Degree of releasing after mixing well, 100 μ L is added per hole, 4 DEG C are coated with overnight, are subsequently carried out according to ELISA operation sequences, measure coating The OD of liquid₄₅₀Value.

The result shows that the OD values using PBS buffer solution as coating buffer are apparently higher than CBS buffer solutions, therefore the application selects PBS buffer solution is as coating buffer.

(4) determination of sealing condition

The optimization of confining liquid：Mutain is diluted using identified buffer solution after optimization, 100 μ L, 4 DEG C of mistakes are added per hole Night is coated with, and the OVA (ovalbumin) and BSA (bovine serum albumin(BSA)) confining liquid for being then respectively adding 1% are closed, and are set Negative control hole is set, per 250 μ L of hole, in 4 DEG C of wet box overnight, is subsequently carried out according to ELISA programs, measures the OD of confining liquid₄₅₀ Value.

The result shows that it is with obvious effects better than OVA buffer solutions using BSA buffer solutions as confining liquid, therefore select BSA bufferings Liquid is as confining liquid.

The optimization of closing mode：Mutain is diluted using PBS buffer solution, 100 μ L are added per hole, 4 DEG C are coated with overnight, use BSA confining liquids are respectively put into 2h and 4 DEG C of closing 12h of 37 DEG C of closings, are subsequently carried out according to ELISA programs, measure confining liquid OD₄₅₀Value.

The result shows that 4 DEG C, the effect for closing 12h is better than 37 DEG C, closes the effect of 2h, therefore the application selects sealing condition It is 4 DEG C, closes 12h.

(5) determination of reaction condition

Mutain is diluted using PBS buffer solution, 100 μ L are added per hole, 4 DEG C are coated with overnight, with 4 DEG C of envelopes of BSA confining liquids 12h is closed, after HRP-AMP (ampicillin of horseradish peroxidase-labeled) is added, is placed in 25,30,33,35,37 DEG C respectively Reaction 15,30,45,60min are subsequently carried out according to ELISA programs, measure confining liquid OD₄₅₀Value.

The result shows that the OD values of confining liquid are maximum when 30 DEG C of reaction 30min, therefore reaction condition selects 30 DEG C of reactions 30min。

(6) preparation of related reagent

1) it is coated with the ELISA Plate of mutain

The mutain that the Escherichia coli KCC being mutated in embodiment 2 is prepared with step (3) is dilute with PBS buffer solution It releases to 1 μ g/mL, the 100 μ L of mutain solution is added per hole into polystyrene ELISA Plate, in 4 DEG C of wet box overnight Coating, outwells liquid in hole, and PBST washing buffers are added per hole and (see below described) 250 μ L, stands 2min, discards liquid in hole Body simultaneously pats dry, and washes repeatedly 3 times；250 μ L confining liquids are added per hole, closes 12h in 4 DEG C of wet box, discards liquid in hole, do It is preserved with aluminium film vacuum sealing after dry.

2) preparation of other reagents

It is coated with buffer solution (preferably PBS buffer solution, that is, phosphate buffer)：Accurately weigh 8.0g sodium chloride, 0.2g phosphoric acid Sodium dihydrogen, 0.2g potassium chloride, 2.9g disodium hydrogen phosphates add distilled water about 800mL, pH to be adjusted to 7.4, distilled water is added to be settled to 1L.

Washing buffer (PBST)：0.05% Tween-20 is added in PBS buffer solution, it is spare after mixing.

Block buffer (preferably BSA)：Bovine serum albumin(BSA) 10g accurately is weighed, the PBS buffer solution of 1L is added, stirring is extremely Albumen is completely dissolved.

Substrate A liquid：The accurate tetramethyl benzidine (TMB) for weighing 160mg, is added the dimethylacetylamide of 10mL, stirs To being completely dissolved.

Substrate B liquid：13.70g citric acids, 10.14g trisodium citrates and 282.00mg carbamide peroxides accurately are weighed, is added Enter distilled water dissolving, constant volume stops 1L.

Substrate cocktail：It is accurate to weigh 10mL substrate B liquid, 100 μ L substrate A liquid are added, mixing is now with the current.

Terminate liquid：It is accurate to measure concentrated sulfuric acid 100mL, it is slowly added dropwise into the distilled water of 800mL.

3) preparation of Cefquinome standard solution

Cefquinome standard items storing solution：It is accurate to weigh Cefquinome standard items 4.815mg (Germany Dr.Ehrenstorfer companies, purity 96.3%), a small amount of methanol-water (methanol：Water volume ratio=1：1) it dissolves, uses distilled water It is settled to 5mL, as 1mgmL^-1Mother liquor, dispense simultaneously stored at -20 DEG C.

The preparation method of Cefquinome series concentration standard solution：

10μg·mL^-1Titer：Accurate 10 μ L standard items storing solutions of drawing are mixed well in the PBS of 990 μ L, vortex 20s, Obtain 10 μ gmL^-1Titer.

100μg·L^-1Titer：It is accurate to draw 10 μ gmL^-1For 10 μ L of titer in the PBS of 990 μ L, vortex 20s is abundant Mixing obtains 100 μ gL^-1Titer.

8μg·L^-1Titer：It is accurate to draw 100 μ gL^-1For 10 μ L of titer in the PBS of 1840 μ L, vortex 20s is fully mixed It is even, obtain 8 μ gL^-1Titer.

4μg·L^-1Titer：It is accurate to draw 8 μ gL^-1Titer 1mL is mixed well, is obtained in 1mL PBS, vortex 20s 4ng/mL titers.

2 μ gL-1 titers：It is accurate to draw 4 μ gL^-1Titer 1mL is mixed well, is obtained in 1mL PBS, vortex 20s 2μg·L^-1Titer.

1μg·L^-1Titer：It is accurate to draw 2 μ gL^-1Titer 1mL is mixed well, is obtained in 1mL PBS, vortex 20s 1μg·L^-1Titer.

0.5μg·L^-1Titer：It is accurate to draw 1 μ gL^-1Titer 1mL is mixed well, is obtained in 1mL PBS, vortex 20s To 0.5 μ gL^-1Titer.

(7) standard curve of Cefquinome is detected with mutain

It is as follows：

1) 250 μ L of PBST are added per hole into the ELISA Plate for be coated with mutain, stands 2min, discards liquid in hole And pat dry, it washes repeatedly 3 times.

2) 50 μ L Cefquinomes standard solutions are added per hole and 50 μ L enzyme markers is put into after plank in cover plate lid 30min is reacted in 30 DEG C of water-baths, dries liquid in hole.

3) 250 μ L of PBST are added per hole, stands 2min, discards liquid in hole and pat dry, wash repeatedly 3 times.

4) the 100 μ L of substrate developing solution now matched are added per hole, colour developing 15min is protected from light in 37 DEG C of insulating boxs.

5) 50 μ L of terminate liquid are added per hole, each hole OD is measured with microplate reader₄₅₀Value.

The OD values in zero hole are B₀, the OD values in each drug hole are B, using the logarithm of drug concentration as abscissa, inhibiting rate (B/ B0) it is ordinate, draws standard curve as schemed, it is respectively y=-0.4547x+0.7687 to obtain regression equation and related coefficient, R=0.9998 calculates IC₅₀Value is 3.90 μ gL^-1, the range of linearity is 0.5-8 μ gL^-1。

(8) precision of Cefquinome is detected with mutain

According to step (7) step, by 0.5,1,2,4,8 μ gL of Cefquinome standard concentration^-1Corresponding OD values point It is not updated in the calibration curve equation of above-mentioned foundation, calculates corresponding drug concentration, calculated with standard concentration measured value The coefficient of variation in the plate of ELISA method standard curve and between plate.The results are shown in Table 6, variation lines between the coefficient of variation and plate in plate Number is no more than 10%, shows there is preferable precision.

The coefficient of variation of 6 standard curve of table measures

(9) sensitivity of Cefquinome is detected with mutain

According to step (7) step, to 20 0 μ gL^-1Standard solution is detected, and detected value is substituted into standard Corresponding standard concentration is calculated in curvilinear equation, and finds out average value (C) and standard deviation (SD).According to formula Z=C+3 × SD (formula 1) is calculated, and obtained data are the sensitivity of the method.The results are shown in Table 7, shows that this receptor analytic approach is correct The sensitivity of spore quinoline oxime is 0.73 μ gL^-1。

The sensitivity determination of 7 ELISA method of table

(10) specificity of beta-lactam antibiotic is detected with mutain

Other beta-lactam antibiotic standard items are subjected to gradient dilution with PBS buffer solution, according to step (7) step Suddenly each standard dilutions are detected.The OD values in zero hole are B₀, the OD values in each drug hole are B, with pair of drug concentration Numerical value is abscissa, inhibiting rate (B/B₀) it is ordinate, standard curve is drawn, obtains regression equation and related coefficient, and calculate IC₅₀Value.

Various beta-lactam antibiotics the results show that absorbance value and per hole be added each drug concentration be inversely proportional, demonstrate,prove Bright mutain has the characteristic for identifying a variety of beta-lactam antibiotics, and linear relationship is presented, and illustrates that mutain can be with Detection for beta-lactam antibiotic.

Using Cefquinome as standard drug, cross reacting rate (Cross reactivity, CR) is calculated according to following formula： CR (%)=IC50 (Cefquinome)/IC50 (other drugs) × 100 (formula 2), the results are shown in Table 8.

As shown in table 8,35 kinds of beta-lactam antibiotics of mutain pair of the invention have higher cross reacting rate, It can be used for the foundation of β in animal tissue-belt-lactam antibiotics residues detection method；But mutation of the invention in the present embodiment Albumen and the pungent furan of cephalo, Cefradine, cefalexin, cefadroxil, the cross reacting rate of Imipenem are relatively low, and fail to see Other aztreonam.It is shown in Table 8.

8 mutain I188K-S19C-G24C of table measures the cross reacting rate of beta-lactam antibiotic

Embodiment 4 detects the application test of beta-lactam antibiotic with mutain

(1) processing of sample

It accurately weighs fresh 1.00 ± 0.02g of samples of whole milk to be placed in the centrifuge tube of 1.5mL, adds beforehand dilution Good various Beta-lactam medicines, after abundant vortex mixed is uniform, extracting solution PBS buffer solution, which is added, makes sample be diluted to conjunction Suitable concentration takes mixed solution for analyzing after mixing.

Accurate weigh is placed in 50mL's by the fresh pork of homogenized, chicken, 2.00 ± 0.02g of beef tissue sample In centrifuge tube, extracting solution PBS buffer solution is added in the good various Beta-lactam medicines of addition beforehand dilution, after mixing well in 4000rpm centrifuges 10min, and the abundant vortex mixed of supernatant is taken uniformly to be used to analyze afterwards.

(2) minimum detection limit

According to 3 step of embodiment (7) step, measures 20 parts of milk blank samples or organize the OD values of blank sample. It carries it into standard curve and calculates its corresponding sample concentration, and calculate the average value (C) and standard deviation of 20 parts of samples (SD).It is calculated according to formula Z=C+3 × SD (formula 1), obtained data are the minimum detection limit (LOD) of method for organizing, root It is calculated according to formula Q=C+10 × SD (formula 3), obtained data are the minimum quantitative limit (LOQ) of method for organizing.

Beta-lactam antibiotic minimum detection limit is as shown in table 9 in milk, pork, chicken, beef tissue, such drug Minimum detection limit be below maximum residue limit as defined in European Union (MRL).

(3) recovery test

Blank sample is taken, beta-lactam antibiotic is added to blank sample according to 1 × LOQ, 2 × LOQ, 4 × LOQ In, each concentration repeats 3 times, and each sample does 5 parallel samples, handles sample.The side established according to embodiment 3 Method is detected, and detected value is brought into the standard curve of corresponding β-lactam antibiotics and is calculated the measured value of sample concentration, The rate of recovery and the coefficient of variation are calculated according to following formula：

The rate of recovery (%)=measured concentration/addition concentration × 100 (formula 4)

Coefficient of variation CV (%)=sample standard deviation/sample mean × 100 (formula 5)

The rate of recovery, the accuracy of wire examination method are calculated according to formula 4；Within-run and between-run analysis coefficient is calculated according to formula 5, The repeatability of wire examination method.TIANZHU XINGNAO Capsul of the beta-lactam antibiotic in milk, pork, chicken, beef tissue and batch It is interior to be shown in Table 10,11 with interassay coefficient of variation measurement result.The result shows that average recovery rate in sample 51.77%~ Between 119.77%, most of drug substantially conforms to the Ministry of Agriculture to the rate of recovery in 60%~120% requirement, illustrates accuracy Well；Within-run and between-run analysis coefficient illustrates repeated good within 20%.

The detection limit and quantitative limit of beta-lactam antibiotic in 9 sample of table

The TIANZHU XINGNAO Capsul of beta-lactam antibiotic in 10 milk of table, pork sample

The TIANZHU XINGNAO Capsul of beta-lactam antibiotic in 11 chicken of table, beef sample

The preservation of 5 Escherichia coli KCC of embodiment

Monoclonal colonies from picking I188K-S19C-G24C on the agar plate of conversion are Escherichia coli KCC, in In the LB/Kan broth bouillons of 2mL, 37 DEG C, culture is shaked in 220rpm constant-temperature tables to OD₆₀₀Reach 0.5 or so, takes 500 40% glycerine of 500 μ L is added in μ L Escherichia coli KCC bacterium solutions, is placed in -70 DEG C of refrigerators and freezes.

The preparation of 40% glycerine：4mL glycerine is taken to be added in the deionized water of 6mL, after mixing in 121 DEG C of sterilizings 15min。

Bibliography：

Cheng Guyue etc., the progress of belt-lactam antibiotics residues receptor analysis method, journal of animal science and veterinary medicine, 2014, 45:354-362,

The preliminary of Beta-lactam medicine residue detection and protein structure of the quiet based on penicillin binding protein PBP3 is opened to grind Study carefully [D] China Agricultural University, 2015.

Ahmed S,Ning J,et al.Receptor-based screening assays for the detection of antibiotics residues-a review. Talanta,2017,166:176-186；

Chen Y,et al.A gold immunochromatographic assay for the rapid and simultaneous detection of fifteen beta-lactams.Nanoscale,2015,7:16381-16388；

Ferrini AM,et al.Detection and identification of beta-lactam residues in milk using a hybrid biosensor.Journal of Agricultural&Food Chemistry,2008, 56:784-788；

Gaudin,V,et al.Validation of a commercial receptor kit sulfasensor(r) honey for the screening of sulfonamides in honey according to commission decision 2002/657/ec.Food Additives And Contaminants Part a-Chemistry Analysis Control Exposure&Risk Assessment,2012,29:942-950；

Gaudin V,et al.Screening of penicillin residues in milk by a surface plasmon resonance-based biosensor assay: Comparison of chemical and enzymatic sample pre-treatment.Analytica Chimica Acta,2001,436:191-198；

Liang X,et al.A proof-of-concept receptor-based assay for sulfonamides.Analytical Biochemistry,2013,438: 110-116；

Moeller N,et al.A new strategy for the analysis of tetracycline residues in foodstuffs by a surface plasmon resonance biosensor.European Food Research&Technology,2007,224:285-292；

Pazzola M,et al.Evaluation of the rapid assay betastar combo 3.0 for the detection of penicillin,amoxicillin, cefazolin and oxytetracycline in individual sheep milk.Small Ruminant Research,2015,124:127-131

Peng J,et al.Development of a direct elisa based on carboxy-terminal of penicillin-binding protein blar for the detection of beta-lactam antibiotics in foods.Analytical and bioanalytical chemistry,2013,405:8925- 8933；

Zeng K,et al.Development of a rapid multi-residue assay for detecting β-lactams using penicillin binding protein 2x.Biomedical&Environmental Sciences Bes,2013,26:100-109。

Sequence table

<110>Hua Zhong Agriculture University

<120>The method for detecting beta-lactam antibiotics residue using mutain

<141> 2018-03-24

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213>Escherichia coli (Escherichia coli)

<220>

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<222> (1)..(765)

<220>

<221> mutation

<222> (563)..(564)

<220>

<221> mutation

<222> (70)..(70)

<220>

<221> mutation

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<220>

<221> CDS

<222> (1)..(765)

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atg caa aga gat acg cac ttt tta tct ggt gca aat gta gaa tac gaa 48

Met Gln Arg Asp Thr His Phe Leu Ser Gly Ala Asn Val Glu Tyr Glu

1 5 10 15

gat gac tgc acc ttt ttt gat tgc ttc tca gga ggt ttt gtc ctg ttt 96

Asp Asp Cys Thr Phe Phe Asp Cys Phe Ser Gly Gly Phe Val Leu Phe

20 25 30

gat tct aac agg aaa aag tat aca ata tac aat agg aaa gaa agc acc 144

Asp Ser Asn Arg Lys Lys Tyr Thr Ile Tyr Asn Arg Lys Glu Ser Thr

35 40 45

gcc aga ttc gca cct gct tct aca tac aag gtg ttc agc gca ttg ctc 192

Ala Arg Phe Ala Pro Ala Ser Thr Tyr Lys Val Phe Ser Ala Leu Leu

50 55 60

gca ctg gaa tcc ggg atc atc acg aag aac aat tct caa atg acg tgg 240

Ala Leu Glu Ser Gly Ile Ile Thr Lys Asn Asn Ser Gln Met Thr Trp

65 70 75 80

gac gga act caa tat ccg tat aaa gaa tgg aat caa gac cag gat tta 288

Asp Gly Thr Gln Tyr Pro Tyr Lys Glu Trp Asn Gln Asp Gln Asp Leu

85 90 95

ttc tct gcg atg agc agc tcc gca aca tgg tat ttt caa aaa ctg gac 336

Phe Ser Ala Met Ser Ser Ser Ala Thr Trp Tyr Phe Gln Lys Leu Asp

100 105 110

cgg caa att gga gag gat cat ttg cgt cgc tat ctc aaa tct atc cat 384

Arg Gln Ile Gly Glu Asp His Leu Arg Arg Tyr Leu Lys Ser Ile His

115 120 125

tat gga aat gag gat ttc tca ggt ccg gcg aat tat tgg ctg gat ggc 432

Tyr Gly Asn Glu Asp Phe Ser Gly Pro Ala Asn Tyr Trp Leu Asp Gly

130 135 140

tct ctt caa att tcc cct ctt gaa cag gtt aat atg tta aaa aag ttt 480

Ser Leu Gln Ile Ser Pro Leu Glu Gln Val Asn Met Leu Lys Lys Phe

145 150 155 160

tat gat aac gaa ttt gat ttt aaa cag tct aat att caa act gtg aaa 528

Tyr Asp Asn Glu Phe Asp Phe Lys Gln Ser Asn Ile Gln Thr Val Lys

165 170 175

gat tcg ata cgt tta gaa gaa tca aat ggc aga aaa cta tcc ggt aaa 576

Asp Ser Ile Arg Leu Glu Glu Ser Asn Gly Arg Lys Leu Ser Gly Lys

180 185 190

acc ggg act tca gtt atc aac gga gaa ctt cat gcc ggc tgg ttt gtc 624

Thr Gly Thr Ser Val Ile Asn Gly Glu Leu His Ala Gly Trp Phe Val

195 200 205

gga tat gta gaa act gcc gaa aat act ttt ttc ttt gct gtt cat att 672

Gly Tyr Val Glu Thr Ala Glu Asn Thr Phe Phe Phe Ala Val His Ile

210 215 220

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Gln Gly Glu Lys Arg Ala Ala Gly Ser Thr Ala Ala Glu Ile Ala Leu

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Ser Ile Leu Asp Lys Lys Gly Ile Tyr Pro Ser Ala Ser Arg

245 250

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Met Gln Arg Asp Thr His Phe Leu Ser Gly Ala Asn Val Glu Tyr Glu

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Asp Asp Cys Thr Phe Phe Asp Cys Phe Ser Gly Gly Phe Val Leu Phe

20 25 30

Asp Ser Asn Arg Lys Lys Tyr Thr Ile Tyr Asn Arg Lys Glu Ser Thr

35 40 45

Ala Arg Phe Ala Pro Ala Ser Thr Tyr Lys Val Phe Ser Ala Leu Leu

50 55 60

Ala Leu Glu Ser Gly Ile Ile Thr Lys Asn Asn Ser Gln Met Thr Trp

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Asp Gly Thr Gln Tyr Pro Tyr Lys Glu Trp Asn Gln Asp Gln Asp Leu

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Phe Ser Ala Met Ser Ser Ser Ala Thr Trp Tyr Phe Gln Lys Leu Asp

100 105 110

Arg Gln Ile Gly Glu Asp His Leu Arg Arg Tyr Leu Lys Ser Ile His

115 120 125

Tyr Gly Asn Glu Asp Phe Ser Gly Pro Ala Asn Tyr Trp Leu Asp Gly

130 135 140

Ser Leu Gln Ile Ser Pro Leu Glu Gln Val Asn Met Leu Lys Lys Phe

145 150 155 160

Tyr Asp Asn Glu Phe Asp Phe Lys Gln Ser Asn Ile Gln Thr Val Lys

165 170 175

Asp Ser Ile Arg Leu Glu Glu Ser Asn Gly Arg Lys Leu Ser Gly Lys

180 185 190

Thr Gly Thr Ser Val Ile Asn Gly Glu Leu His Ala Gly Trp Phe Val

195 200 205

Gly Tyr Val Glu Thr Ala Glu Asn Thr Phe Phe Phe Ala Val His Ile

210 215 220

Gln Gly Glu Lys Arg Ala Ala Gly Ser Thr Ala Ala Glu Ile Ala Leu

225 230 235 240

Ser Ile Leu Asp Lys Lys Gly Ile Tyr Pro Ser Ala Ser Arg

245 250

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<213>Escherichia coli (Escherichia coli)

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<222> (1)..(25)

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<213>Escherichia coli (Escherichia coli)

<220>

<221> primer_bind

<222> (1)..(25)

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<210> 5

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<213>Escherichia coli (Escherichia coli)

<220>

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<213>Escherichia coli (Escherichia coli)

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gtcccggttt taccggatag ttttctgcca tttgattctt ct 42

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<213>Escherichia coli (Escherichia coli)

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Claims

1. a kind of mutain of identification belt-lactam antibiotics residues, it is characterised in that：The nucleotides sequence of the mutain Row such as SEQ ID NO:Shown in 1.

2. a kind of mutain of identification belt-lactam antibiotics residues, it is characterised in that：The protein sequence of the mutain Row such as SEQ ID NO:Shown in 2.

3. a kind of Escherichia coli of the mutain BlaR-CTD of expression beta-lactam antibiotic, during which is deposited in State's Type Tissue Collection, deposit number are CCTCC NO:M2018132.

4. the Escherichia coli described in claim 3, it is turned by plasmid pET-28a (+)-BlaR-CTD-I188K-S19C-G24C Change obtained by e. coli bl21 competent cell.

5. it is a kind of using mutain while to the detection method of 40 kinds of beta-lactam antibiotics residues, including：Structure is prominent Become albumen, induced expression and purifying are carried out to mutain, establish beta-lactam antibiotic detection method and utilizes mutation egg White detection beta-lactam antibiotic, feature further includes following steps：

(1) using prokaryotic expression carrier pET-28a (+)-BlaR-CTD as template, with mutant primer (primer sequence such as SEQ ID NO:Shown in 3-8) PCR amplification obtains mutant plasmid, e. coli bl21 competent cell is converted, obtaining preserving number is CCTCCNO:The E. coli mutant bacterium KCC of M2018132；

(2) as the OD of E. coli mutant bacterium KCC₆₀₀When being 0.6, the IPTG of final concentration of 1mM, 18 DEG C of constant temperature oscillation trainings are added 12h is supported, ultrasonication after bacterium solution is collected, supernatant is taken to be purified to obtain the mutain I188K-S19C-G24C of high-purity, Protein sequence such as SEQ ID NO：Shown in 2；

(3) high-purity mutain I188K-S19C-G24C is coated in ELISA Plate, is detected, examines after optimizing reaction condition Examine precision, sensitivity and the specificity of method；

(4) detection method for establishing step (3) is applied to the detection of beta-lactam class antibiotic in sample, measures minimum inspection Limit, sample recovery rate and the coefficient of variation are surveyed, accuracy and the repeatability of method are investigated.

6. applications of the mutain I188K-S19C-G24C described in claim 1 in the detection of beta-lactam class antibiotic.

7. the answering in the detection of beta-lactam antibiotics residue in edibility animal product of the method described in claim 5 With.