CN103864908A - Molecular modified penicillin-binding protein (PBP) capable of detecting beta-lactam antibiotic residues - Google Patents
Molecular modified penicillin-binding protein (PBP) capable of detecting beta-lactam antibiotic residues Download PDFInfo
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- CN103864908A CN103864908A CN201310332825.9A CN201310332825A CN103864908A CN 103864908 A CN103864908 A CN 103864908A CN 201310332825 A CN201310332825 A CN 201310332825A CN 103864908 A CN103864908 A CN 103864908A
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Abstract
The invention provides a molecular modified penicillin-binding protein (PBP) capable of detecting beta-lactam antibiotic residues. The molecular modified PBP is characterized by being obtained by respectively modifying leucine on the 147th position of the PBP of actinomadura yumaense R39 and histidine on the 351st position of the PBP of the actinomadura yumaense R39 into histidine and aspartic acid, so that the affinity of beta-lactam antibiotics is improved. The invention also provides a rapid detecting test strip based on production of the protein, and the rapid detecting test strip can be used for rapidly detecting the beta-lactam antibiotic residues of agricultural and sideline products.
Description
Technical field
The present invention relates to food safety detection field.More specifically, the present invention relates to high-affinity penicillin-binding protein and the application in belt-lactam antibiotics residues rapid detection in agricultural byproducts thereof that molecular modification obtains.
Background technology
Milk cow easily suffers from the diseases such as mastitis, vaginitis in breeding process; due to β-lactam antibitics good drug efficacy, cheap; dairy farmer uses in a large number in the time that milk cow is ill; cause in raw dairy belt-lactam antibiotics residues phenomenon general; strengthened to a great extent the difficulty of dairy enterprises milk source quality safety control, and the milk-product that contain belt-lactam antibiotics residues can have influence on human consumer's healthy and life security.Therefore, the Ministry of Agriculture has put into effect industry standard " pollution-free food Fresh Milk " (NY5045-2008), requires dairy enterprises must detect belt-lactam antibiotics residues in raw dairy.
Penicillin-binding protein is the synthetic important enzyme of bacteria cell wall, has the antibiotics of beta-lactam ring structure like its substrate, with it, irreversible fixation can occur.Therefore, penicillin-binding protein is the albumen of realizing the tool potentiality of belt-lactam antibiotics residues rapid detection.But at present known penicillin-binding protein and the avidity of β-lactam antibitics do not reach the examination criteria of national requirements, for realizing rapid detection β-lactam antibitics, in the urgent need to finding the penicillin-binding protein higher with β-lactam antibitics avidity.
At present, the β-lactam antibitics rapid detection product that dairy enterprises is used is mainly take imported product as main, as Snap company of the U.S. and Belgian UniSensor company.The detectability that the present invention is directed to China's β-lactam antibitics rapid detection product existence does not reach national standard problem, utilize the methods such as computer aided design (CAD), quantum chemistry, molecular biology, analytical chemistry, the 147th of Actinomycesa lmadurae R39 penicillin-binding protein (PBP) the and 351 amino acids are carried out to molecular modification, developed a kind of new high-affinity penicillin-binding protein.In the present invention, naming this high-affinity penicillin-binding protein is JN-Pro-β-lactam, and its corresponding gene is JN-Gen-β-lactam, and expression vector is JN-Bdzt-β-lactam.The present invention, using JN-Pro-β-lactam as molecular recognition original paper, has developed a kind of β-lactam antibitics Rapid detection test strip.
Summary of the invention
The object of this invention is to provide a kind of penicillin-binding protein (JN-Pro-β-lactam) higher with β-lactam antibitics avidity.The preservation strain the present invention relates to is colon bacillus Escherichia coli, be preserved in (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center, 100101, China), preservation date on July 19th, 2013, deposit number CGMCC7942.
Another object of the present invention is to provide a kind of belt-lactam antibiotics residues Rapid detection test strip that can meet or exceed national examination criteria of producing based on JN-Pro-β-lactam.
A first aspect of the present invention, provide JN-Pro-β-lactam: transform the leucine of the 147th of PBP and the 351st 's Histidine as Histidine and aspartic acid, prepare prokaryotic expression carrier conversion e. coli bl21 and make positive strain (called after JN-BL-β-lactam, CGMCC7942) express, and obtain JN-Pro-β-lactam by separation and purification, determination of activity and Identification of Fusion Protein.
A second aspect of the present invention, provides a kind of β-lactam antibitics Rapid detection test strip based on JN-Pro-β-lactam exploitation.
In embodiments of the present invention, provide the method based on JN-Pro-β-lactam processing test strip.
Accompanying drawing explanation
Fig. 1 is test strip schematic diagram.
Embodiment
The present invention transform the leucine of the 147th of PBP and the 351st 's Histidine as Histidine and aspartic acid, and by prokaryotic expression carrier preparation, expression and purification, determination of activity, Identification of Fusion Protein etc., obtained JN-Pro-β-lactam, the avidity of itself and β-lactam antibitics significantly improves.
1, site-directed point mutation: according to two mutant primers of DNA sequence dna design (Pt1 and Pt2) in wanted mutational site, coordinates with recombinant plasmid PGEX-6p-1-PBP primer (P1 and P2) and carry out 3 and take turns polymerase chain reaction (PCR) and react.The 1st takes turns PCR reaction: take recombinant plasmid PGEX-6p-1-PBP as template, with P1 and Pt2 collocation, amplification obtains PBP1b gene 5 ' end fragment, and with Pt1 and P2 collocation, amplification obtains 3 ' end fragment, reclaims respectively amplified production for subsequent use.The 2nd takes turns PCR reaction: with the 1st amplification of taking turns PCR and react template and 10 circulations of primer do each other of two fragments obtaining.The 3rd takes turns PCR reaction: take turns PCR take the 2nd and react the product obtaining as template, P1 and P2 collocation are increased for primer again, can obtain the goal gene with mutational site, be connected in carrier (pGEM-T), the positive colony transforming after identifying checks order, and can obtain cloning vector (called after pGEM-JN).
2, Prokaryotic expression vector construction: pGEM-JN and prokaryotic expression carrier (pGEX-6p-1) are carried out respectively to double digestion with restriction endonuclease (XhoI and EcoRI), reclaim respective segments after gel electrophoresis.Under the effect of ligase enzyme (T4DNA), connect, build the prokaryotic expression carrier containing goal gene, transform after bacillus coli DH 5 alpha constant temperature culture, extract recombinant plasmid.Recombinant plasmid is cut and is identified after correct and carry out gene sequencing through PCR and enzyme.Sequencing result shows (as accompanying drawing 1), and the 147th the base ctc that leucine is corresponding sported cac (Histidine); The base cac that the 351st hyte propylhomoserin is corresponding has sported gac (aspartic acid).
3, the conversion of albumen and expression: take out the 1.5mL centrifuge tube containing competence e. coli bl21 50 μ L in-70 ℃ of refrigerators, insert in wet ice and dissolve about 15min, the recombinant plasmid that adds wherein 5 μ L to build, mixes the rear 30min on ice that leaves standstill gently.42 ℃ of water-bath heat shock 90s, move into rapidly in ice bath and leave standstill 2min, add 800 μ L LB substratum (containing 15 μ g/mL tsiklomitsins), then in 37 ℃ of shaking culture (225rmp) 1h, get 50 μ L cultures and be coated with LB agar plate, cultivate 12h in 37 ℃; Select single bacterium colony, with LB substratum shaking culture 12h, upgrading grain, enzyme is cut evaluation, preserves positive colony bacterial classification.Positive colony bacterial classification is inoculated in 5mL LB substratum, cultivates 24h in 37 ℃, 220rmp, culture is forwarded to 100mL LB substratum, and 37 ℃ of shaking culture are to OD
600be 0.8, adding sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) to make its final concentration is lmmol/L, continues to cultivate 4h.Adopt 4 ℃, the centrifugal 15min results of 6000rmp thalline.Thalline is resuspended in phosphoric acid buffer (PBS, 0.01mol/L), and thalline is put ultrasonication in ice bath.Add Triton X-100 (TritonX-100) to final concentration 1%, stirring at room temperature 30min, 4 ℃, centrifugal 15 min of 12000 rmp, collect supernatant liquor.
4, protein purification: with the PBS of 10 times of volumes and the good gsh affinity column of PBS balance containing 1%Triton X-100 of 10 times of volumes, PBS with 20 times of column volumes washes away foreign protein, in affinity column, add zymoplasm, 25 ℃ of reaction 12h, collect endonuclease reaction liquid, be splined on through 10 mmol/LNH
4hCO
3the propylene dextrane gel S100 chromatography column of pre-equilibration, with 10mmol/LNH
4hCO
3wash-out, collects elutriant, concentrated.JN-Pro-β-lactam after purifying is stored in-20 ℃, 10% glycerine solution.
5, determination of activity: under 4 ℃ of conditions, add a certain amount of JN-Pro-β-lactam solution in enzyme plate micropore, placement is spent the night, so that proteopexy is in micropore surface, use PBS to rinse after micropore, continue to add 2% casein solution sealing, for subsequent use after use PBS rinses three times.First should be the milk sample (please add) of preparation containing four kinds of beta-lactam antibioticss, in micropore, add the milk sample to be measured (doing three Duplicate Samples) after 100 μ L degreasings, cultivate 30min for 37 ℃, the β-lactam antibitics to be measured containing in milk sample is fully reacted with JN-Pro-β-lactam, generate albumen-antidusting element mixture to be measured.To the penbritin (B-AMPI) that adds 100 μ L dirt things element marks in micropore, cultivate 30min for 37 ℃, generate albumen-B-AMPI mixture.Clean after twice with cleaning solution (8.55g/LNaCl and 0.25mL/L Tween20) and PBS, to the avidin (1: 1500) that adds 100 μ L horseradish peroxidase-labeled in micropore, cultivate 30min for 37 ℃, so that albumen-B-AMPI mixture fully reacts with the avidin of horseradish peroxidase mark.Use cleaning solution and PBS to rinse after micropore, add 100 μ L horseradish peroxidase substrate tetramethyl biphenyl amine aqueous solutions, cultivate 10min for 37 ℃, in micropore, add 100 μ Llmol/L hydrochloric acid solns with enzymolysis reaction, and use microplate reader to measure the light absorption value of micropore at 450nm place.Utilize aforesaid method, measure respectively transformation front and back PBP and the JN-Pro-β-lactam limit of detection (the results are shown in following table) for four kinds of conventional β-lactam antibiticss.
The active comparing result of table PBP1b and JN-Pro-β-lactam
| Limit of detection | PBP1b | JN-Pro-β-lactam |
| Penicillin G (μ g/kg) | 4 | 2 |
| Penbritin (μ g/kg) | 4 | 2 |
| Cloxacillin Sodium (μ g/kg) | 30 | 20 |
| Ceftiofur (μ g/kg) | 100 | 50 |
But should understand, a variety of ordinary methods all can realize site-directed point mutation in JN-Pro-β-lactam preparation process, prokaryotic expression carrier preparation, expression and purification, determination of activity, Identification of Fusion Protein etc., aforesaid method is to be only explanation the present invention, rather than the restriction scope of the invention, if: transformed host cell can be prokaryotic cell prokaryocyte (as bacterial cell), eukaryotic cell (as corneal cell), the higher eucaryotic cells (as mammalian cell) etc. such as low; Substratum and culture condition can be selected according to the difference of host cell; Etc..
In addition, the present invention, using JN-Pro-β-lactam as molecular recognition original paper, adopts colloidal gold technique, has produced β-lactam antibitics Rapid detection test strip.The advantage of test strip of the present invention is: highly sensitive, simple and efficient, there is the ability that simultaneously detects multiple β-lactam antibitics, and be particularly suitable for dairy enterprises application in raw dairy and product detection.Further set forth below in conjunction with specific embodiment.Should be understood that embodiment is only for illustrating that JN-Pro-β-lactam can be applied in rapid detection field, rather than for limiting the scope of the invention.If test strip trace particle can be colloid gold particle, latex particle, electroselenium particle etc.In test strip making method, can be the JN-Pro-β-lactam that is fixed on β-lactam antibitics competition in penbritin coupled antigen and the testing sample on test strip surface and coats the colloid gold label in test strip, utilize penbritin coupled antigen and JN-Pro-β-lactam monoclonal antibody as detection line and nature controlling line.
The assembling of embodiment colloidal gold strip
A kind of β-lactam antibitics Rapid detection test strip comprises: base plate, sample pad, Radioactive colloidal gold pad, nitrocellulose filter, absorption pad, detection line, quality inspection line (seeing accompanying drawing 3).On Radioactive colloidal gold pad, have the JN-Pro-β-lactam of colloid gold label, nitrocellulose filter is provided with detection line and quality inspection line, has penbritin coupled antigen on detection line, has the monoclonal antibody of JN-Pro-β-lactam on nature controlling line.
A kind of making method of β-lactam antibitics Rapid detection test strip comprises:
(1) JN-Pro-β-lactam preparation
Preparation process refers to " embodiment ".
(2) bovine serum albumin and the preparation of penbritin coupled antigen
25mg penbritin is dissolved in pH9.6Na
2cO
3in buffered soln, after fully mixing, add 5mg BSA, regulate pH value to 11,37 ℃ are stirred 12h, fully dialyse and no longer detected bacteriostatic activity in 0.01mol/LPBS to dialyzate.By dialysate packing and-20 ℃ of preservations.
(3) JN-Pro-β-lactam monoclonal antibody preparation
Take JN-Pro-β-lactam as immunogen immune mouse, get mouse spleen cell and myeloma cell and merge, obtain the cell of stably excreting JN-Pro-β-lactam antibody through screening and cloning, apply this cell and prepare monoclonal antibody.
(4) Radioactive colloidal gold pad preparation
The preparation of colloidal gold solution: get 0.01% four chlorogold solution 100mL, be heated to boiling.Then add the sodium citrate aqueous solution of 10-50mL1%, then keep boiling to solution and presenting orange red.
The preparation of the JN-Pro-β-lactam of colloid gold label: get 50mL Radioactive colloidal gold, add JN-Pro-β-lactam under stirring.Get two 1.5mL test tubes, add respectively 1.2mL10nM Radioactive colloidal gold, add appropriate 25mM K
2cO
3pH is adjusted into 7.5; Adding respectively 10 μ L concentration is 1mg/mL JN-Pro-β-lactam, mixes, and room temperature is placed 10min; Add respectively 6 μ L2%PEG20000, room temperature is placed 5min; The centrifugal 20min of 10000rpm, absorbs supernatant gently; The Radioactive colloidal gold precipitation that suspends loose by 40 μ LPBS solution weight, and focus in new pipe; Add respectively 40 μ L glycerine, fully mix ,-20 ℃ save backup.
JN-Pro-β-the lactam of colloid gold label is dispersed in and on glass fibre, makes Radioactive colloidal gold pad.
(5) detection line of nitrocellulose filter preparation
Rule as detection line with the conjugate of avidin and carrier proteins.
(6) the quality inspection line of nitrocellulose filter preparation
Rule as quality inspection line with the antibody of JN-Pro-β-lactam.
(7) test strip assembling
By reference to the accompanying drawings 3, the assembling process of test strip is described.
The first step adds the nitrocellulose filter of detection line and quality inspection line on base plate.
Second step, adds Radioactive colloidal gold pad in one end of nitrocellulose filter (detection line end).
The 3rd step adds sample pad above Radioactive colloidal gold pad.
The 4th step, adds absorption pad at the other end (quality inspection line end) of nitrocellulose filter.
The 5th step, cuts into the test strip that width is 4mm with cutting machine.
The test strip that the present embodiment is assembled is respectively penicillin G for four kinds of conventional microbiotic limit of detection: 4 μ g/kg, penbritin: 4 μ g/kg, Cloxacillin Sodium: 30 μ g/kg, ceftiofur: 100 μ g/kg.
Sequence table
Claims (9)
1. the penicillin-binding protein after molecular modification, is characterized in that, this albumen obtains based on Actinomycesa lmadurae R39 penicillin-binding protein (PBP) molecular modification, and this protein gene comes from the gene of PBP.
2. the penicillin-binding protein after a kind of molecular modification according to claim 1, is characterized in that, the gene of this albumen be the 147th of PBP and 351 amino acids the combination mutant of corresponding base.
3. the penicillin-binding protein after a kind of molecular modification according to claim 1, is characterized in that, transform the 147th leucine of PBP as Histidine.
4. the penicillin-binding protein after a kind of molecular modification according to claim 1, is characterized in that, transform the 351st hyte propylhomoserin of PBP as aspartic acid.
5. the penicillin-binding protein after a kind of molecular modification according to claim 1, it is characterized in that, wherein said β-lactam antibitics is a large class microbiotic with beta-lactam nucleus, comprising: Penicillin antibiotics and cephalosporins.
6. a β-lactam antibitics Rapid detection test strip, is characterized in that, this test strip is to produce and obtain based on penicillin-binding protein mutant claimed in claim 1.
7. according to described in claim 1 and claim 6, the penicillin-binding protein after this molecular modification and the Rapid detection test strip of production thereof can be used for the residual quantity of rapid detection β-lactam antibitics.
8. β-lactam antibitics Rapid detection test strip claimed in claim 6 application in β-lactam antibitics in detection agricultural byproducts.
9. express a bacterial strain for molecular modification penicillin-binding protein as claimed in claim 1, its preserving number is: CGMCC7942.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106085982A (en) * | 2016-08-18 | 2016-11-09 | 武汉职业技术学院 | Penicillin-binding protein Bt pbp2X and application thereof |
| CN106591265A (en) * | 2016-08-18 | 2017-04-26 | 武汉职业技术学院 | Penicillin-binding protein and application thereof |
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2013
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106085982A (en) * | 2016-08-18 | 2016-11-09 | 武汉职业技术学院 | Penicillin-binding protein Bt pbp2X and application thereof |
| CN106591265A (en) * | 2016-08-18 | 2017-04-26 | 武汉职业技术学院 | Penicillin-binding protein and application thereof |
| CN106591265B (en) * | 2016-08-18 | 2019-08-13 | 武汉职业技术学院 | A kind of penicillin binding protein and its application |
| CN106085982B (en) * | 2016-08-18 | 2019-08-13 | 武汉职业技术学院 | Penicillin binding protein Bt-pbp2X and its application |
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