CN102181394A - Method for preparing penicillin binding protein combined with beta-lactam antibiotic and special recombinant bacterium - Google Patents
Method for preparing penicillin binding protein combined with beta-lactam antibiotic and special recombinant bacterium Download PDFInfo
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Abstract
The invention discloses a method for preparing a penicillin binding protein combined with beta-lactam antibiotic and a special recombinant bacterium. The recombinant bacterium is prepared by the following steps of: importing the coding gene of the penicillin binding protein into initial escherichia coli to obtain recombinant escherichia coli. Experiments prove that the method for preparing the penicillin binding protein has high yield, and the problems of low yield and difficulty in preparation in the prior art are solved. Therefore, the method has significance.
Description
Technical field
The present invention relates to the method and the special recombinant bacterium of a kind of preparation and β-Nei Xiananleikangshengsu bonded penicillin-binding protein.
Background technology
Microbiotic residual in the food can destroy the gastrointestinal bacterial flora balance, and can cause secular diarrhoea, vitamin deficiency and influence the curative effect of other drug, thereby influences HUMAN HEALTH.Current residues of antibiotics problem has caused people's extensive concern, and as the requisite in people's life, the Determination of antibiotic in milk residue problem receives publicity especially day by day.How to guarantee milk qualities safety, in strictness control industrial chain the antibiotic use, set up that antibiotic method for quick then seems particularly important in the milk.
Determination of antibiotic in milk is residual to be mainly derived from feed of milk cow and the disease treatment of milk cow, and wherein the main disease of milk cow is a mazoitis, and the most frequently used medicine of treatment mazoitis is a β-Nei Xiananleikangshengsu.The detection method of β-Nei Xiananleikangshengsu mainly contains instrumental method (comprising high resolution gas chromatography, high performance liquid chromatography and application of gas chromatorgraphy/mass technology etc.), microbial method, immune analysis method and receptor assay method etc. in the milk at present.These methods respectively have characteristics: instrumental method is accurate, reliable, highly sensitive, is a kind of conclusive evidence method, but is not suitable for field quick detection; Microbial method can detect multiple microbiotic simultaneously, but sensitivity is not high, detection time is long, and can't be quantitative; Immune analysis method is based on the specific combination of antigen-antibody, but has advantage such as highly sensitive, high specificity detection by quantitative, but because antibody highly sensitive and that can discern same class medicine is difficult to obtain, restricted its application at the microbiotic detection range; Be subjected to body method based on the specific recognition between receptors ligand, early research arranged abroad, and have product to come out (comprising Belgian UniSensor, U.S. Charm etc.), but domesticly rarely have research.European Union has formulated the maximum residue limit(MRL) of β-Nei Xiananleikangshengsu in milk: penicillin, Ampicillin Trihydrate, amoxycilline Trihydrate bp are 4ppb; Oxazacillin, Cloxacillin, dicloxacillin, nafcillin are 30ppb; Cefquinome 20ppb; Ceftiofur, Cephalexin Monohydrate Micro/Compacted 100ppb; Kefzol, cefoperazone 50ppb.
(Penicillin-Binding Protein is a kind of membranin that extensively is present in bacterium surface PBP) to penicillin-binding protein, is β-antibiotic main target site of Nei phthalein amine.But the efficient for preparing penicillin-binding protein at present is not high.
Summary of the invention
An object of the present invention is to provide a kind of reorganization bacterium.
Reorganization bacterium provided by the present invention prepares according to the method that comprises the steps: import the encoding gene of penicillin-binding protein in the intestinal bacteria that set out, obtain recombination bacillus coli.
In the above-mentioned reorganization bacterium, the encoding gene of described penicillin-binding protein imports by recombinant expression vector; Described recombinant expression vector obtains for the encoding gene that inserts described penicillin-binding protein between the multiple clone site of the carrier pET28b that sets out.
In above-mentioned arbitrary described reorganization bacterium, the aminoacid sequence of described penicillin-binding protein is shown in SEQ ID NO:1.
In above-mentioned arbitrary described reorganization bacterium, the described intestinal bacteria that set out are e. coli bl21 (DE3), and the nucleotide sequence of the encoding gene of described penicillin-binding protein is shown in SEQ ID NO:2.
Above-mentioned arbitrary described reorganization bacterium specifically can be following reorganization bacterium: the reorganization bacterium, and its name is called colon bacillus (Escherichia coli) pET28b-PBP2x, and its deposit number is CGMCC No.4629.
Another object of the present invention provides proteic method shown in a kind of preparation SEQ ID NO:1.
Kind provided by the present invention prepares proteic method shown in the SEQ ID NO:1, and the above-mentioned arbitrary described reorganization bacterium that comprises the steps: to ferment obtains albumen shown in the SEQ ID NO:1.
In the aforesaid method, comprise in the described fermentation and followingly carry out the inductive step: at the A of the system of described fermentation with IPTG
600Be 0.6 o'clock, add IPTG in the system of described fermentation, making the concentration of IPTG in the system of described fermentation is ImM.
In above-mentioned arbitrary described method, described inductive method comprises the steps: that the temperature of described fermentation is 37 ℃, the substratum that uses in the described fermentation is prepared as follows and obtains: add kantlex in the LB liquid nutrient medium, making the concentration of kantlex in the LB liquid nutrient medium is 30ug/mL, and the mixing solutions that obtains is described substratum.
In above-mentioned arbitrary described method, after described fermentation, comprise following extraction step: get the reorganization bacterium thalline after the fermentation,, get supernatant liquor with its fragmentation.
In above-mentioned arbitrary described method, after described extraction, comprise following purification step: described supernatant liquor is carried out nickel post affinity chromatography, collect elutriant; In the described nickel post affinity chromatography, the elution buffer of use is prepared as follows and obtains: with 20mmol Tris, 0.5mol NaCl, 500mmol imidazoles water dissolution, transfer pH value to 8.0 with HCl, water is settled to 1L again, obtains 1 liter of elution buffer.
Experiment showed, that the present invention prepares the method yield height of penicillin-binding protein, solved the problem that yield is not high in the prior art, preparation is difficult.Therefore, the inventive method is significant.
Description of drawings
Fig. 1 is PBP 2x gene PCR agarose electrophoresis figure.
Fig. 2 cuts evaluation figure for PBP 2x gene is connected the back enzyme with the pET28b carrier.
Fig. 3 induces back different time points whole protein SDS-PAGE figure for IPTG.
Fig. 4 is PBP 2x protein purification SDS-PAGE figure.
Fig. 5 is PBP 2x albumen western blot figure.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The Ampicillin Trihydrate is available from U.S. Sigma-Aldrich company, and catalog number is A9518.
Expression vector pET28b is available from German Merk (Novagen) company, and catalog number is 69865; E. coli bl21 (DE3) is available from German Merk (Novagen) company, and catalog number is 69387; Protein purification His
Columns is available from German Merk (Novagen) company, and catalog number is 70971; T4DNA Ligase is available from U.S. Promega company, and catalog number is M1801S.
(American type culture collection ATCC), is numbered ATCC 49619 to streptococcus pneumoniae (Streptococcus pneumoniae) R6 available from the biological product of USS collecting center
TM(http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/ta bid/452/Default.aspx).Horseradish peroxidase (HRP) is available from U.S. Sigma company.BCA protein quantification test kit is available from U.S. Pierce company.
Carrier PMD19-T is available from TAKARA company, and catalog number is D102A.
The sheep anti-mouse antibody of His-tag monoclonal antibody and HRP mark is available from TIANGEN Biotech (Beijing) Co., Ltd., and catalog number is respectively AB102-02 and SA101-01.
The preparation of embodiment 1, reorganization bacterium
Streptococcus pneumoniae R6 genome is used following primer and is carried out pcr amplification as template, obtains the PCR product; Cut the PCR product with restriction enzyme NdeI and XhoI enzyme, the PCR product reclaims the target gene fragment (being the PBP2x goal gene) about 2250bp through 1% agarose gel electrophoresis; Electrophoresis result as shown in Figure 1, swimming lane 1-6 is a goal gene, swimming lane 7 is marker.
Upstream primer: 5 '-TT
CATATGATGAAGTGGACAAAAAGA-3 ' (5 ' end contains NdeI restriction enzyme site, protection base)
Downstream primer: 5 '-TT
CTCGAGTTAGTCTCCTAAAGTTAAT-3 ' (3 ' end contains XhoI restriction enzyme site, protection base)
Reclaim target gene fragment, it is connected with the PMD19-T carrier, connect product transformed into escherichia coli DH5 α,, positive monoclonal is carried out liquid culture, extract plasmid, check order by the resistance screening positive monoclonal.The result shown in SEQ ID NO:2, shows that the recombinant vectors of structure is correct at the nucleotide sequence of the gene that inserts among the PMD19-T, and note is made PMD 19-T-PBP2x.
Cut PMD19-T-PBP2x with restriction enzyme NdeI and XhoI enzyme, reclaim the target gene fragment about 2250bp; Cut pET28b with restriction enzyme NdeI and XhoI enzyme, reclaim the big fragment of carrier; The big fragment of target gene fragment and carrier is connected with T4 DNA Ligase, connect product transformed into escherichia coli DH5 α, by the resistance screening positive monoclonal, positive monoclonal is carried out liquid culture, extract plasmid, check order and make NdeI single endonuclease digestion, XhoI single endonuclease digestion and NdeI+XhoI double digestion respectively and identify.The nucleotide sequence of result's gene that (along the direction from NdeI to XhoI) inserted between the NdeI and XhoI restriction enzyme site of pET28b is shown in SEQ ID NO:2, and the aminoacid sequence of its encoded protein is (note is made penicillin-binding protein PBP2x) shown in SEQ ID NO:1.Show that gene direction of insertion and sequence are all correct in the recombinant vectors, positive recombinant vectors note is made recombinant expression vector pET28b-PBP2x.Enzyme is cut qualification result as shown in Figure 2, the single endonuclease digestion size about 7300bp, double digestion have about 2300bp and 5000bp about band, the illustration purpose gene has correctly connected into pET28b.Among Fig. 2, swimming lane 1:NdeI single endonuclease digestion; Swimming lane 2:XhoI single endonuclease digestion; Swimming lane 3:NdeI/XhoI double digestion; Swimming lane 4:Marker.
The recombinant expression vector pET28b-PBP2x transformed into escherichia coli BL21 (DE3) that builds, the kalamycin resistance screening obtains positive monoclonal; The positive monoclonal that obtains is inoculated in the LB liquid nutrient medium that contains kantlex 30ug/mL cultivates, extract plasmid, order-checking, the result shows that the plasmid of extraction is correct, shows that the reorganization bacterium of structure is correct, note is made reorganization bacterium BL21-pET28b-PBP2x.
Serve as the contrast bacterium with the e. coli bl21 (DE3) that changes pET28b over to simultaneously, note is made reorganization bacterium BL21-pET28b.
One, proteic preparation
BL21-pET28b-PBP2x is seeded in the LB liquid nutrient medium that contains kantlex 30ug/mL with the reorganization bacterium, 37 ℃ of joltings of 250rpm, and incubated overnight (12h) obtains seed liquor; Get the 1mL seed liquor and be inoculated in the LB liquid nutrient medium that 100mL contains kantlex 30ug/mL, 37 ℃ of joltings of 250rpm are to the A of culture system
600It is 0.6 o'clock, take out 1mL bacterium liquid earlier, as not inducing contrast, all the other bacterium liquid add IPTG to final concentration 1mM, 200pm shakes bacterium for 30 ℃, from inducing 3rd hour (note is done the 0th hour when adding IPTG), per hour take out 1mL bacterium liquid, induction time is respectively 3h, 4h, 5h, 6h, 7h, 8h and spend the night (12h); The bacterium liquid collected at 4 ℃ of centrifugal 5min of following 10000rpm, is collected thalline; With the resuspended thalline of Tris-HCL (10mM Tris pH7.0), the broken thalline of 200W power ice-bath ultrasonic (ultrasonic 10s stops 10s) becomes clarification to suspension, and 4 ℃ again, the centrifugal 20min of 12000 * g collect supernatant liquor, are the penicillin-binding protein extracting solution.All substances in culture vessel note is made bacterium liquid.
Contain the preparation of the LB liquid nutrient medium of kantlex 30ug/mL: add kantlex in the LB liquid nutrient medium, making the concentration of kantlex in the LB liquid nutrient medium is 30ug/mL, and the mixing solutions that obtains is the LB liquid nutrient medium that contains kantlex 30ug/mL.
The LB liquid nutrient medium is formed: be made up of yeast extract, peptone, NaCl and water, the concentration of each composition is in the solution: yeast extract 0.5% (quality percentage composition), peptone 1% (quality percentage composition), NaCl 1% (quality percentage composition).
Two, proteic purifying
Penicillin-binding protein purifying: utilize histidine-tagged (His-tag) mark that has on the expression vector conjugated protein by the nickel ion affinity chromatograph process to purify penicillin.First binding buffer balance His with 10 times of column volumes
Columns adds above-mentioned penicillin-binding protein extracting solution, uses the wash buffer wash-out foreign protein of 5 times of column volumes then, use the elution buffer wash-out target protein of 10 times of column volumes at last, collect elutriant, dialyse, obtain the penicillin-binding protein of purifying.
Expression product with reorganization bacterium BL21-pET28b is contrast simultaneously.
Binding buffer: with 20mmol Tris, 0.5mol Nacl and 10mmol imidazoles water dissolution, transfer pH value to 8.0 with HCL, water is settled to 1L again, obtains 1 liter of binding buffer.
Wash buffer: with 20mmol Tris, 0.5mol NaCl and 20mmol imidazoles water dissolution, transfer pH value to 8.0 with HCL, water is settled to 1L again, obtains 1 liter of wash buffer.
Elution buffer: with 20mmol Tris, 0.5mol Nacl and 500mmol imidazoles water dissolution, transfer pH value to 8.0 with HCL, water is settled to 1L again, obtains 1 liter of elution buffer.
Three, albumen checking
1, induce the front and back product to carry out the SDS-PAGE detection product before and after the purifying and IPTG, the result as shown in Figure 3 and Figure 4.
Fig. 3 is the result of reorganization bacterium BL21-pET28b-PBP2x, swimming lane 1: do not induce control group; Swimming lane 2～8: be respectively recombinant bacterial strain through 3,4,5,6,7,8 hours and spend the night induce after protein expression figure; Swimming lane 9:Marker.
Fig. 4 is the result of reorganization bacterium BL21-pET28b-PBP2x, swimming lane 1:Marker; Swimming lane 2: the unpurified recombinant bacterial strain whole protein of inductive not; Swimming lane 3: induce but unpurified recombinant bacterial strain whole protein; Swimming lane 4: induce and purifying after albumen.
The result shows, induces down at IPTG, and containing in IPTG inductive reorganization bacterium BL21-pET28b-PBP2x has tangible band about 80kD, consistent with the expection size of target protein, the success of PBP2x protein expression.And without not containing the purpose band among the IPTG inductive reorganization bacterium BL21-pET28b-PBP2x.
No matter whether induce, do not contain the purpose band in the expression product of reorganization bacterium BL21-pET28b.
2, albumen checking Western blot:
Collect each stage product in the above-mentioned preparation process, carry out the 10%SDS-PAGE electrophoresis; The electrophoretic band trace to the NC film, is hybridized the DAB colour developing more successively with the sheep anti-mouse antibody of His-tag monoclonal antibody and HRP mark.
The result as shown in Figure 5.Among Fig. 5, swimming lane 1:Marker; Swimming lane 2: the unpurified reorganization of inductive bacterium BL21-pET28b-PBP2x whole protein not; Swimming lane 3: induce but unpurified reorganization bacterium BL21-pET28b-PBP2x whole protein; Swimming lane 4: reorganization bacterium BL21-pET28b-PBP2x induce and purifying after albumen.
The result shows: it is about 80kD that reorganization bacterium BL21-pET28b-PBP2x expresses the molecular weight of albumen that obtains, consistent with the molecular weight of albumen of expection.Do not contain target protein in the expression product of reorganization bacterium BL21-pET28b.
Four, protein yield:
The bacterium colony (being labeled as BL21-pET28b-PBP2x-1～-8 respectively) of 8 reorganization of picking bacterium BL21-pET28b-PBP2x on the solid screening culture medium, it is inoculated in 2mL respectively contains in the LB liquid nutrient medium of kantlex 30ug/mL, 37 ℃ of joltings of 250rpm, after cultivating 8h, obtain seed culture fluid; The 1mL seed culture fluid is inoculated in the LB liquid nutrient medium that 200mL contains kantlex 30ug/mL, and 37 ℃ of joltings of 250rpm are to the A of culture system
600Be 0.6 o'clock, take out 1mL bacterium liquid earlier, as not inducing contrast, all the other bacterium liquid add IPTG to final concentration 1mM, and 200rpm30 ℃ is shaken bacterium, collects all bacterium liquid behind the 6h; The bacterium liquid collected at 4 ℃ of centrifugal 5min of following 10000rpm, is collected thalline; With the resuspended thalline of Tris-HCL (10mM Tris pH7.0), the broken thalline of 200W power ultrasonic (ultrasonic 10s stops 10s) becomes clarification to suspension, and 4 ℃ again, the centrifugal 20min of 12000 * g collect supernatant liquor, are the penicillin-binding protein extracting solution.All substances in culture vessel note is made bacterium liquid.
The penicillin-binding protein extracting solution that 8 strain bacterium obtain is respectively used the nickel ion affinity chromatograph column purification respectively, purification process is with consistent described in the experiment two, the albumen that purifying is obtained is remembered respectively and is done PBP2x-1～8, with BCA protein quantification kit measurement protein content, and then the amount of the target protein of calculating purifying.PBP2x-1～-8 gained target protein total amounts are respectively 1.27mg, 1.36mg, 1.11mg, 2.02mg, 2.54mg, 2.33mg, 1.87mg and 2.08mg, this shows that the PBP2x-5 output that is obtained by reorganization bacterium BL21-pET28b-PBP2x-5 abduction delivering is the highest.
This reorganization bacterium BL21-pET28b-PBP2x-5 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 3rd, 2011 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.4629, the classification called after colon bacillus (Escherichia coli) of this bacterial strain, strain name is pET28b-PBP2x.
Utilize reorganization bacterium BL21-pET28b-PBP2x-5 to prepare target protein according to the method described above, experiment repeats 3 times, 3 average out to 2.5mg, 2.54mg, 2.6mg as a result, the good stability of the generation target protein that shows.
1, bag is cushioned liquid with bag and is configured to 1ug/mL by the penicillin-binding protein of preparation among the enzyme plate of penicillin-binding protein: the embodiment 2; Every hole 100uL.
2, enzyme mark thing: the Ampicillin Trihydrate of HRP mark, its working concentration 1ug/mL, enzyme mark thing working fluid obtains with sample diluting liquid dilution enzyme mark thing.
3, β-Nei Xiananleikangshengsu standard substance: standard substance are the penicillin G lyophilized powder, and penicillin G is available from U.S. Sigma-Aldrich company; Catalog number is 46616; Penicillin G is dissolved with sample diluting liquid, obtain the standard solution of following different concns: 8.1 μ g/L, 2.7 μ g/L, 0.9 μ g/L, 0.3 μ g/L, 0.1 μ g/L.
4, substrate colour developing liquid: be made up of A liquid and B liquid, A liquid is the aqueous solution of 2% urea peroxide, and B liquid is the aqueous solution of 1% tetramethyl benzidine;
5, stop buffer: 0.2M aqueous sulfuric acid;
6, washings: per 1 liter of described washings is prepared as follows and is obtained: 0.5ml polysorbas20,5g sodiumazide and 990ml phosphate buffered saline buffer are mixed, obtain described washings; The concentration of described phosphate buffered saline buffer is 0.01M, and the pH value is 7.4;
7, the phosphate buffered saline buffer of sample diluting liquid: 0.1mol/L, pH value are 7.2.
8, bag is cushioned the aqueous solution, the pH9.6 of the yellow soda ash of liquid: 0.05mol/L.
9, confining liquid: per 1 liter of confining liquid is prepared as follows: 50mgBSA, 1g sodiumazide, 30g casein are mixed, with the phosphate buffered saline buffer dissolving and be settled to 1000ml, obtain confining liquid; Wherein, the concentration of phosphate buffered saline buffer is 0.02M, and the pH value is 7.2.
The preparation of the Ampicillin Trihydrate of HRP mark
A. get the 50mg Ampicillin Trihydrate, (0.05mol/L is pH7.0) in the solution to be dissolved in 5mL PBS;
B. add 40mg EDC, 4 ℃ of stirring reaction 1h;
C. get the HRP of 100mg and 10mg EDC be dissolved in 10mLPBS (0.05mol/L, pH7.0) in the solution,
D. the dialysis tubing of packing into, PBS (0.05mol/L, pH7.0) dialysis obtain the conjugate of Ampicillin Trihydrate and HRP,
Be enzyme mark thing.
0.05M, the pH value is the preparation of 7.0 PBS damping fluid: dispose earlier 0.05mol/L Na respectively
2HPO
4Solution (takes by weighing Na
2HPO
4.12H
2O 17.9g adds deionized water to 1000m L) and 0.05mol/L KH
2PO
4Solution (takes by weighing KH
2PO
4.2H
2O 8.6g adds deionized water to 1000m L); Get 0.05mol/L Na
2HPO
4Solution 60m L and 0.05mol/LKH
2PO
4Solution 40m L adds 8.5g NaCL, mixes.
0.01M, the pH value is the preparation of 7.4 PBS damping fluid: per 1 liter of phosphate buffered saline buffer (PBS) is prepared as follows: with 8g NaCl, 0.2g KCl, 3.53g Na
2HPO
4.12H
2O, 0.24g KH
2PO
4Mix with water, water is settled to 1 liter.The concentration of the phosphate buffered saline buffer that so obtains is 0.01M, and the pH value is 7.4.
0.1M, the pH value is the preparation of 7.2 PBS damping fluid: dispose earlier 0.1mol/L Na respectively
2HPO
4Solution (takes by weighing Na
2HPO
4.12H
2O 35.8g adds deionized water to 1000mL) and 0.1mol/L NaH
2PO
4Solution (takes by weighing NaH
2PO
4.2H
2O 15.6g adds deionized water to 1000m L); Get 0.1mol/L Na
2HPO
4Solution 72m L and 0.1mol/LNaH
2PO
4Solution 28m L adds 8.5g NaCL, mixes.
0.02M, the pH value is the preparation of 7.2 PBS damping fluid: get 0.1M, pH value and be 7.2 PBS damping fluid 200mL, add deionized water 800mL, mixing is promptly.
0.05mol/L, the pH value is the preparation of the aqueous solution of 9.6 yellow soda ash: Na
2CO
31.59g, NaHCO
32.93g, add water and be settled to 1 liter.
The β-Nei Xiananleikangshengsu that adds the 50uL gradient dilution in the enzyme plate micropore that is coated with penicillin-binding protein (is 0～100ppb between the penicillins gradient zones; Be 0～500ppb) between the cephalosporins gradient zones, add enzyme mark thing working fluid 50 μ L again, with cover plate film shrouding, react 30min in 37 ℃ of thermostat containers, dry liquid in the hole, every hole adds 350 μ L washingss, pour out liquid in the hole behind the 30s, so repetitive operation is washed plate 4 times altogether, pats dry with thieving paper.Every hole adds substrate colour developing liquid, the mixing that vibrates gently, and 37 ℃ of thermostat container lucifuge colour developing 10min, every hole adds stop buffer 50 μ L, and the mixing that vibrates is gently used microplate reader, measures every hole absorbance.
With the β-Nei Xiananleikangshengsu solution absorbency mean value (B) of each concentration divided by first 0 concentration solution absorbency value (B
0) multiply by 100% again, obtain the percentage absorbance.Semilog value with β-Nei Xiananleikangshengsu concentration (μ g/L) is an X-axis, and the percentage absorbance is a Y-axis, and curve plotting obtains 50% inhibition concentration (IC50) of every kind of β-Nei Xiananleikangshengsu.
β-Nei Xiananleikangshengsu is as follows: penicillin G, Ampicillin Trihydrate, amoxycilline Trihydrate bp, Oxazacillin, Cloxacillin, dicloxacillin, nafcillin, Gepcillin, X-1497, Cephalexin Monohydrate Micro/Compacted, S 578, Kefzol, cefoperazone, ceftriaxone, cefotaxime, cefoxitin, ceftiofur, cefquinome.
3 repetitions are established in experiment.
Ppb promptly is ng/ml in the literary composition.
The result is as follows:
1) each β-Nei Xiananleikangshengsu concentration of being added of absorbance and every hole is inversely proportional to, prove that the penicillin-binding protein that expressed purifying obtains has the characteristic of the multiple β-Nei Xiananleikangshengsu of identification, and present linear relationship, illustrate that this penicillin-binding protein can be used for the detection of multiple β-Nei Xiananleikangshengsu.
2) cross reacting rate: the IC50 that calculates various medicines earlier: the absorbance of positive control hole (promptly not adding the hole of standard solution) is B
0, the absorbance of each experimental port is B, works as B/B
0The concentration that is 50% o'clock pairing standard solution is half inhibiting rate (IC50); With the Ampicillin Trihydrate is standard drug, calculates cross reacting rate, and promptly the IC50 of Ampicillin Trihydrate multiply by 100% promptly divided by the IC50 of each medicine.The results are shown in Table 1.
The result is as shown in table 1, and PBP 2x albumen can be discerned at least 16 kinds of Beta-lactam medicines that comprise penicillin, amoxycilline Trihydrate bp, Ampicillin Trihydrate, Cephalexin Monohydrate Micro/Compacted etc.
The cross reacting rate of many kinds of beta-lactam medicines of table 1
The preservation of embodiment 4, recombinant bacterial strain
The BL21-pET28b-PBP2x-5 bacterial strain that protein yield is higher is frozen.
Picking BL21-pET28b-PBP2x-5 mono-clonal bacterium colony from transform flat board adds the LB liquid nutrient medium that 2mL contains kantlex 30ug/mL, cultivates slightly to be muddiness to bacterium liquid in 4 hours; On the flat board that contains kantlex 30ug/mL, rule, cultivate 20h for 37 ℃; Get the mono-clonal bacterium colony, inoculation is gone into 2ml and is contained in the LB substratum of kantlex 30ug/mL, is cultured to A
600Reach 0.5; Bacterium liquid and 0.1ml 80% glycerine of 0.9ml is mixed, be stored in-70 ℃ of refrigerators.
The preparation of 80% glycerine: get 8mL glycerine and the 2mL deionized water mixes, be sub-packed in the frozen pipe, every pipe 0.1mL, 121 ℃ of sterilization 20min.
Claims (10)
1. a reorganization bacterium prepares according to the method that comprises the steps: import the encoding gene of penicillin-binding protein in the intestinal bacteria that set out, obtain recombination bacillus coli.
2. reorganization bacterium according to claim 1, it is characterized in that: the encoding gene of described penicillin-binding protein imports by recombinant expression vector; Described recombinant expression vector obtains for the encoding gene that inserts described penicillin-binding protein between the multiple clone site of the carrier pET28b that sets out.
3. reorganization bacterium according to claim 1 and 2 is characterized in that: the aminoacid sequence of described penicillin-binding protein is shown in SEQ ID NO:1.
4. according to arbitrary described reorganization bacterium among the claim 1-3, it is characterized in that: the described intestinal bacteria that set out are e. coli bl21 (DE3), and the nucleotide sequence of the encoding gene of described penicillin-binding protein is shown in SEQ ID NO:2.
5. strain reorganization bacterium, its name is called colon bacillus (Escherichia coli) pET28b-PBP2x, and its deposit number is CGMCC No.4629.
6. one kind prepares proteic method shown in the SEQ ID NO:1, and arbitrary described reorganization bacterium obtains albumen shown in the SEQ ID NO:1 among the claim 1-5 that comprises the steps: to ferment.
7. method according to claim 6 is characterized in that: comprise in the described fermentation and followingly carry out the inductive step with IPTG: at the A of the system of described fermentation
600Be 0.6 o'clock, add IPTG in the system of described fermentation, making the concentration of IPTG in the system of described fermentation is 1mM.
8. according to claim 6 or 7 described methods, it is characterized in that: described inductive method comprises the steps: that the temperature of described fermentation is 37 ℃, the substratum that uses in the described fermentation is prepared as follows and obtains: add kantlex in the LB liquid nutrient medium, making the concentration of kantlex in the LB liquid nutrient medium is 30ug/mL, and the mixing solutions that obtains is described substratum.
9. according to arbitrary described method among the claim 6-8, it is characterized in that: in the described method, after described fermentation, comprise following extraction step: get the reorganization bacterium thalline after the fermentation,, get supernatant liquor its fragmentation.
10. according to arbitrary described method among the claim 6-9, it is characterized in that: in the described method, after described extraction, comprise following purification step: described supernatant liquor is carried out nickel post affinity chromatography, collect elutriant; In the described nickel post affinity chromatography, the elution buffer of use is prepared as follows and obtains: with 20mmol Tris, 0.5mol NaCl, 500mmol imidazoles water dissolution, transfer pH value to 8.0 with HCl, water is settled to 1L again, obtains 1 liter of elution buffer.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102627693A (en) * | 2011-11-22 | 2012-08-08 | 平原县伟峰永驻科技有限公司 | High-affinity penicillin binding protein and application thereof |
| CN104560903A (en) * | 2014-12-25 | 2015-04-29 | 中山大学 | Application of glutathione reductase to binding of penicillin antibiotics |
| 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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| CN101813698A (en) * | 2010-04-02 | 2010-08-25 | 上海优你生物科技股份有限公司 | Kit for detecting beta-lactam antibiotic ligand in milk by receptor method and detection method thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102627693A (en) * | 2011-11-22 | 2012-08-08 | 平原县伟峰永驻科技有限公司 | High-affinity penicillin binding protein and application thereof |
| CN102627693B (en) * | 2011-11-22 | 2013-05-22 | 平原县伟峰永驻科技有限公司 | High-affinity penicillin binding protein and application thereof |
| CN104560903A (en) * | 2014-12-25 | 2015-04-29 | 中山大学 | Application of glutathione reductase to binding of penicillin antibiotics |
| 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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