CN102627693A - High-affinity penicillin binding protein and application thereof - Google Patents
High-affinity penicillin binding protein and application thereof Download PDFInfo
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- CN102627693A CN102627693A CN2012100891079A CN201210089107A CN102627693A CN 102627693 A CN102627693 A CN 102627693A CN 2012100891079 A CN2012100891079 A CN 2012100891079A CN 201210089107 A CN201210089107 A CN 201210089107A CN 102627693 A CN102627693 A CN 102627693A
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Abstract
The invention provides a high-affinity penicillin binding protein. The invention is characterized in that the high-affinity penicillin binding protein is obtained by respectively transforming isoleucine 526 and glutamine 547 in a streptococcus agalactiae penicillin binding protein (PBP 1b) into lysine and arginine, so that the affinity of beta-lactam antibiotics is improved. The invention further provides a quick detection test strip produced based on the protein, which can be used for quickly detecting beta-lactam antibiotic residues in agricultural and sideline products.
Description
Technical field
The present invention relates to the food safety detection field.More specifically, the present invention relates to high-affinity penicillin-binding protein and the application in the residual rapid detection of β-Nei Xiananleikangshengsu in agricultural byproducts thereof that molecular modification obtains.
Background technology
Milk cow is prone to suffer from diseases such as mastitis, vaginitis in breeding process; Because β-Nei Xiananleikangshengsu good drug efficacy, cheap; The dairy farmer uses when milk cow is ill in a large number; Cause in the raw dairy β-Nei Xiananleikangshengsu residual phenomena general, strengthened the difficulty of dairy enterprises milk source quality safety control to a great extent, and contained the healthy and life security that the residual milk-product of β-Nei Xiananleikangshengsu can have influence on the human consumer.Therefore, the Ministry of Agriculture has put into effect industry standard " the fresh cow's milk of pollution-free food " (NY5045-2008), and it is residual to require dairy enterprises must detect in the raw dairy β-Nei Xiananleikangshengsu.
Penicillin-binding protein is the important enzyme of bacteria cell wall synthetic, and the antibiotics with beta-lactam ring structure with it irreversible fixation can take place like its substrate.Therefore, penicillin-binding protein is an albumen of realizing the tool potentiality of the residual rapid detection of β-Nei Xiananleikangshengsu.But the avidity of present known penicillin-binding protein and β-Nei Xiananleikangshengsu does not reach the examination criteria of national requirements; For realizing the rapid detection β-Nei Xiananleikangshengsu, press for and find the penicillin-binding protein higher with β-Nei Xiananleikangshengsu avidity.
At present, the β-Nei Xiananleikangshengsu rapid detection product that dairy enterprises is used is main with imported product mainly, like U.S. Snap company and Belgian UniSensor company.The detectability that the present invention is directed to China's β-Nei Xiananleikangshengsu rapid detection product existence does not reach the national standard problem; Utilize methods such as CAD, quantum chemistry, molecular biology, analytical chemistry; The 526th of streptococcus agalactiae penicillin-binding protein (PBP 1b) and 547 amino acids are carried out molecular modification, developed a kind of new high-affinity penicillin-binding protein.Among the present invention, naming this high-affinity penicillin-binding protein is WFYZH-Pro-β-lactam, and its corresponding gene is WFYZH-Gen-β-lactam, and expression vector is WFYZH-Bdzt-β-lactam.The present invention as the molecular recognition original paper, has developed a kind of β-Nei Xiananleikangshengsu quick detection test paper bar with WFYZH-Pro-β-lactam.
Summary of the invention
The purpose of this invention is to provide the higher penicillin-binding protein of a kind of and β-Nei Xiananleikangshengsu avidity (WFYZH-Pro-β-lactam).The preservation strain that the present invention relates to is ETEC Escherichia coli; Be preserved in (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center; 100101; China), preservation date on October 24th, 2011, deposit number CGMCC No.5381.
Another object of the present invention provides a kind of residual quick test strip of the β-Nei Xiananleikangshengsu that can meet or exceed national examination criteria of producing based on WFYZH-Pro-β-lactam.
First aspect of the present invention; WFYZH-Pro-β-lactam is provided: transform the 526th the Isoleucine of PBP 1b and the 547th Stimulina as Methionin and l-arginine; Preparation prokaryotic expression carrier transformed into escherichia coli BL21 makes positive strain (called after WFYZH-Gchj-β-lactam; CGMCC No.5381) expresses, and obtain WFYZH-Pro-β-lactam through separation and purification, determination of activity and Identification of Fusion Protein.
Second aspect of the present invention provides a kind of β-Nei Xiananleikangshengsu quick detection test paper bar based on WFYZH-Pro-β-lactam exploitation.
In embodiments of the present invention, the method for processing test strip based on WFYZH-Pro-β-lactam is provided.
Description of drawings
Fig. 1 is the corresponding gene sequencing result of WFYZH-Pro-β-lactam.
Fig. 2 is the corresponding amino acid sequencing result of WFYZH-Pro-β-lactam.
Fig. 3 is the test strip synoptic diagram.
Embodiment
The present invention transform the 526th the Isoleucine of PBP 1b and the 547th Stimulina as Methionin and l-arginine; And through prokaryotic expression carrier preparation, expression and purification, determination of activity, Identification of Fusion Protein etc.; Obtained WFYZH-Pro-β-lactam, the avidity of itself and β-Nei Xiananleikangshengsu significantly improves.
1, site-directed point mutation:, cooperates with recombinant plasmid PGEX-6p-1-PBP 1b primer (P1 and P2) and to carry out 3 and take turns polymerase chain reaction (PCR) and react according to the dna sequence dna in want mutational site design two mutant primers (Pt1 and Pt2).The 1st takes turns the PCR reaction: with recombinant plasmid PGEX-6p-1-PBP 1b is template, and with P1 and Pt2 collocation, amplification obtains PBP 1b gene 5 ' end fragment, and with Pt1 and P2 collocation, amplification obtains 3 ' end fragment, and it is subsequent use to reclaim amplified production respectively.The 2nd takes turns the PCR reaction: take turns two fragments template and 10 round-robin amplification of primer do each other that the PCR reaction obtains with the 1st.The 3rd takes turns PCR reaction: taking turns the product that the PCR reaction obtains with the 2nd is template; P1 and P2 collocation are increased for primer again; Can obtain having the goal gene in mutational site; Be connected in the carrier (pGEM-T), the positive colony that transforms after identifying checks order, and can obtain cloning vector (called after pGEM-WFYZH).
2, prokaryotic expression carrier makes up: with restriction endonuclease (XhoI and EcoRI) pGEM-WFYZH and prokaryotic expression carrier (pGEX-6p-1) are carried out double digestion respectively, reclaim respective segments after the gel electrophoresis.Under the effect of ligase enzyme (T4DNA), connect, make up the prokaryotic expression carrier that contains goal gene, after the transformed into escherichia coli DH5 α constant temperature culture, extract recombinant plasmid.Recombinant plasmid is cut through PCR and enzyme and is identified and carry out gene sequencing after correct.Sequencing result shows (like accompanying drawing 1), and the 526th the corresponding base ata of Isoleucine sported aaa (Methionin); The 547th the corresponding base caa of Stimulina sported cga (l-arginine).
3, proteic conversion and expression: in-70 ℃ of refrigerators, take out the 1.5mL centrifuge tube that contains competence e. coli bl21 50 μ L, insert the about 15min of dissolving in the wet ice,, leave standstill 30min on ice gently behind the mixing to wherein adding the recombinant plasmid that 5 μ L build.42 ℃ of water-bath heat shock 90s move into rapidly and leave standstill 2min in the ice bath, 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 are coated with the LB agar plate, cultivate 12h in 37 ℃; Select single bacterium colony, with LB substratum shaking culture 12h, the upgrading grain, enzyme is cut evaluation, preserves the positive colony bacterial classification.The positive colony bacterial classification inoculation in 5mL LB substratum, is cultivated 24h in 37 ℃, 220rmp, and culture is forwarded to 100mL LB substratum, and 37 ℃ of shaking culture are to OD
600Be 0.8, it is 1mmol/L that adding sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) makes its final concentration, continues to cultivate 4h.Adopt 4 ℃, the centrifugal 15min results of 6000rmp thalline.Thalline is resuspended in phosphoric acid buffer, and (PBS, 0.01mol/L), thalline is put ultrasonication in the ice bath.Add polyoxyethylene octyl phenyl ether (TritonX-100) to final concentration 1%, stirring at room 30min, 4 ℃, the centrifugal 15min of 12000rmp collect supernatant.
4, protein purification: with the PBS of 10 times of volumes and the good gsh affinity column of the PBS balance that contains 1%Triton X-100 of 10 times of volumes; PBS flush away foreign protein with 20 times of column volumes; Add zymoplasm in the affinity column; 25 ℃ of reaction 12h collect endonuclease reaction liquid, are splined on the NH through 10mmol/L
4HCO
3The propylene polydextran gel S100 chromatography column of pre-equilibration is with 10mmol/L NH
4HCO
3Wash-out is collected elutriant, concentrates.WFYZH-Pro-β-lactam behind the purifying is stored in-20 ℃, 10% glycerine solution.
5, determination of activity: under 4 ℃ of conditions, in the enzyme plate micropore, add a certain amount of WFYZH-Pro-β-lactam solution, placement is spent the night; So that proteopexy is in micropore surface; After use PBS washes micropore, continue to add the sealing of 2% casein solution, subsequent use after use PBS washes three times.It at first should be the milk sample (please add) that preparation contains four kinds of beta-lactam antibioticss; Milk sample to be measured in micropore after the adding 100 μ L degreasings (doing three parallel appearance); Cultivate 30min for 37 ℃; The β-Nei Xiananleikangshengsu to be measured and the WFYZH-Pro-β-lactam that contain in the milk sample are fully reacted, generate albumen-antibiotic complex to be measured.In micropore, add the biotin labeled penbritins of 100 μ L (B-AMPI), cultivate 30min for 37 ℃, generate albumen-B-AMPI mixture.After cleaning twice with cleaning solution (8.55g/L NaCl and 0.25mL/L Tween20) and PBS; The avidin (1: 1500) that in micropore, adds 100 μ L horseradish peroxidase-labeled; Cultivate 30min for 37 ℃, so that the avidin of albumen-B-AMPI mixture and horseradish hydrogen peroxide enzyme labelling fully reacts.After using cleaning solution and PBS flushing micropore; Add 100 μ L horseradish hydrogen peroxide enzyme substrates TMB solution; Cultivate 10min for 37 ℃, in micropore, add 100 μ L 1mol/L hydrochloric acid solns, and use ELIASA to measure the light absorption value of micropore at the 450nm place with enzymolysis reaction.Utilize aforesaid method, measure transformation front and back PBP 1b and WFYZH-Pro-β-lactam limit of detection (result sees the following form) respectively for four kinds of β-Nei Xiananleikangshengsus commonly used.
The active comparing result of table PBP 1b and WFYZH-Pro-β-lactam
| Limit of detection | PBP?1b | WFYZH-Pro-β-lactam |
| Penicillin G (μ g/kg) | 4 | 2 |
| Penbritin (μ g/kg) | 4 | 2 |
| Cloxacillin Sodium (μ g/kg) | 30 | 18 |
| Ceftiofur (μ g/kg) | 100 | 55 |
But should understand; A variety of ordinary methods can realize that all WFYZH-Pro-β-lactam prepares site-directed point mutation in the process, prokaryotic expression carrier preparation, expression and purification, determination of activity, Identification of Fusion Protein etc.; Aforesaid method only is to be explanation the present invention; Rather than the restriction scope of the invention, can be prokaryotic cell prokaryocyte (like bacterial cell), eukaryotic cell (like corneal cell), higher eucaryotic cells (like mammalian cell) etc. such as low like: transformed host cell; Substratum can be selected according to the different of host cell with culture condition; Or the like.
In addition, the present invention as the molecular recognition original paper, adopts colloidal gold technique with WFYZH-Pro-β-lactam, has produced β-Nei Xiananleikangshengsu quick detection test paper bar.The advantage of test strip of the present invention is: highly sensitive, simple and efficient, have the ability that detects multiple β-Nei Xiananleikangshengsu simultaneously, and be particularly suitable for dairy enterprises and in raw dairy and product detection, use.Further set forth below in conjunction with specific embodiment.Should be understood that embodiment only is used to explain that WFYZH-Pro-β-lactam can be applied in the rapid detection field, rather than be used to limit scope of the present invention.Like the test strip trace particle can be colloid gold particle, latex particle, electroselenium particle etc.On the test strip making method; Can be to be fixed in the penbritin coupled antigen on test strip surface and WFYZH-Pro-β-lactam that the colloid gold label on the test strip is coated in the competition of the β-Nei Xiananleikangshengsu in the testing sample, utilize penbritin coupled antigen and WFYZH-Pro-β-lactam monoclonal antibody as detection line and nature controlling line.
The assembling of embodiment colloidal gold strip
A kind of β-Nei Xiananleikangshengsu quick detection test paper bar comprises: base plate, sample pad, Radioactive colloidal gold pad, nitrocellulose filter, absorption pad, detection line, quality inspection line (seeing accompanying drawing 3).WFYZH-Pro-β-lactam that colloid gold label is arranged on the Radioactive colloidal gold pad, nitrocellulose filter are provided with detection line and quality inspection line, and the penbritin coupled antigen is arranged on the detection line, and the monoclonal antibody of WFYZH-Pro-β-lactam is arranged on the nature controlling line.
A kind of making method of β-Nei Xiananleikangshengsu quick detection test paper bar comprises:
(1) WFYZH-Pro-β-lactam preparation
The preparation process sees " embodiment " for details.
(2) bovine serum albumin and penbritin coupled antigen preparation
The 25mg penbritin is dissolved in pH9.6 Na
2CO
3In the buffered soln, fully add 5mg BSA behind the mixing, regulate pH value to 11,37 ℃ are stirred 12h, and in 0.01mol/L PBS, fully dialysing has no longer detected bacteriostatic activity to dialyzate.With dialysate packing and-20 ℃ of preservations.
(3) WFYZH-Pro-β-lactam Monoclonal Antibody
With WFYZH-Pro-β-lactam is the immunogen immune mouse, gets mouse spleen cell and myeloma cell and merges, and the process screening and cloning obtains the cell of stably excreting WFYZH-Pro-β-lactam antibody, uses this cell preparation monoclonal antibody.
(4) Radioactive colloidal gold pad preparation
The preparation of colloidal gold solution: get 0.01% tetrachloro gold solution 100mL, be heated to boiling.The sodium citrate aqueous solution that adds 10-50mL 1% then keeps boiling to solution again and appears orange red.
The preparation of the WFYZH-Pro-β-lactam of colloid gold label: get the 50mL Radioactive colloidal gold, stir adding WFYZH-Pro-β-lactam down.Get two 1.5mL test tubes, add 1.2mL 10nM Radioactive colloidal gold respectively, add an amount of 25mM K
2CO
3Be adjusted into 7.5 to pH; Adding 10 μ L concentration respectively is 1mg/mL WFYZH-Pro-β-lactam, mixing, and room temperature is placed 10min; Add 6 μ L 2%PEG20000 respectively, room temperature is placed 5min; The centrifugal 20min of 10000rpm absorbs supernatant gently; With the loose Radioactive colloidal gold deposition of 40 μ L PBS solution weight suspension, and focus in the new pipe; Add 40 μ L glycerine respectively, abundant mixing ,-20 ℃ of preservations are subsequent use.
WFYZH-Pro-β-the lactam of colloid gold label is dispersed in making Radioactive colloidal gold pad on the spun glass.
(5) detection line of nitrocellulose filter preparation
Conjugate with avidin and carrier proteins is rule as detection line.
(6) the quality inspection line of nitrocellulose filter preparation
Antibody with WFYZH-Pro-β-lactam is rule as the quality inspection line.
(7) test strip assembling
In conjunction with accompanying drawing 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.
In second step, add the Radioactive colloidal gold pad at the end (detection line end) of nitrocellulose filter.
In the 3rd step, above the Radioactive colloidal gold pad, add sample pad.
In the 4th step, add absorption pad at the other end (quality inspection line end) of nitrocellulose filter.
In the 5th step, use cutting machine to cut into the test strip of width as 4mm.
The test strip that present embodiment is assembled is respectively penicillin G for four kinds of microbiotic limit of detection commonly used: 4 μ g/kg, penbritin: 4 μ g/kg, Cloxacillin Sodium: 20 μ g/kg, ceftiofur: 80 μ g/kg.
Claims (5)
1. a high-affinity penicillin-binding protein is characterized in that, said albumen obtains based on streptococcus agalactiae penicillin-binding protein (PBP 1b) molecular modification, and its aminoacid sequence is SEQ 2.
2. express the bacterial strain of high-affinity penicillin-binding protein according to claim 1 for one kind, its preserving number is: CGMCC No.5381.
3. a β-Nei Xiananleikangshengsu quick detection test paper bar is characterized in that, this test strip is based on the production of the described high-affinity penicillin-binding protein of claim 1 and obtains.
4. β-Nei Xiananleikangshengsu quick detection test paper bar according to claim 3 is in the application of the residual quantity that is used for the rapid detection β-Nei Xiananleikangshengsu; Wherein said β-Nei Xiananleikangshengsu is one big type of microbiotic with beta-lactam nucleus, comprising: PCs, cephalosporins, cephamycin-type microbiotic, sulfomycin class microbiotic and monocycle beta-lactam antibiotics etc.
5. β-Nei Xiananleikangshengsu quick detection test paper bar according to claim 3 application in the β-Nei Xiananleikangshengsu in the rapid detection agricultural byproducts; Wherein said β-Nei Xiananleikangshengsu is one big type of microbiotic with beta-lactam nucleus, comprising: PCs, cephalosporins, cephamycin-type microbiotic, sulfomycin class microbiotic and monocycle beta-lactam antibiotics etc.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103864908A (en) * | 2013-08-02 | 2014-06-18 | 吉林省农业科学院 | Molecular modified penicillin-binding protein (PBP) capable of detecting beta-lactam antibiotic residues |
| CN104560903A (en) * | 2014-12-25 | 2015-04-29 | 中山大学 | Application of glutathione reductase to binding of penicillin antibiotics |
| CN106591265A (en) * | 2016-08-18 | 2017-04-26 | 武汉职业技术学院 | Penicillin-binding protein and application thereof |
| CN110187117A (en) * | 2019-06-18 | 2019-08-30 | 清华大学深圳研究生院 | The detection kit and its application of beta-Lactam antibiotic |
| CN110579606A (en) * | 2019-09-04 | 2019-12-17 | 武玉香 | detection card for rapidly and quantitatively detecting penicillin and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103864908A (en) * | 2013-08-02 | 2014-06-18 | 吉林省农业科学院 | Molecular modified penicillin-binding protein (PBP) capable of detecting beta-lactam antibiotic residues |
| CN104560903A (en) * | 2014-12-25 | 2015-04-29 | 中山大学 | Application of glutathione reductase to binding of penicillin antibiotics |
| 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 |
| CN110187117A (en) * | 2019-06-18 | 2019-08-30 | 清华大学深圳研究生院 | The detection kit and its application of beta-Lactam antibiotic |
| CN110579606A (en) * | 2019-09-04 | 2019-12-17 | 武玉香 | detection card for rapidly and quantitatively detecting penicillin and application thereof |
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