CN101003798A - Purified expression of recombined beta lactamase in superspectrum, and fermentation process in high density - Google Patents

Abstract

This invention discloses a method for expression, purification and high-density fermentation of recombinant extended-spectrum beta-lactamase. The method comprises: (1) inserting SHV-4 type ESBL gene into expression vector, and expressing in Escherichia coli; (2) performing high-density fermentation to obtain large quantities of bacteria, ultrasonically crushing the bacteria suspension, and centrifuging to collect the supernatant and the precipitate of inclusion body; (4) performing affinity chromatography to obtain high-purity recombinant SHV-4 type ESBL (7 mg/L fermentation solution, and specific activity is 476 IU/mg); (5) dissolving the inclusion body in urea, and purifying by molecular sieve chromatography to obtain high-purity recombinant SHV-4 type ESBL. The yield of recombinant SHV-4 type ESBL is 17 mg/L fermentation solution, and the specific activity is 438 IU/mg. This invention provides a method for mass production of ESBL.

Description

A kind of expression and purification of recombined beta lactamase in superspectrum and fermentation process in high density thereof

Technical field

The expression and purification of recombined beta lactamase in superspectrum of the present invention and fermentation process in high density thereof, belong to the medical bioengineering technical field, more specifically say so and be purified into ESBL at expression in escherichia coli by gene engineering method, utilize large scale fermentation and purification technique thereof to obtain a large amount of, inexpensive ESBL, for practical application lays the first stone.

Background technology

ESBL is the enzyme of amido linkage on class energy hydrolyzing penicillin class, cephalosporins and the monoamide class microbiotic acid amides ring, (TEM-1, SHV-1) compares with the wide spectrum β-Nei Xiananmei, the enzyme (Jacoby G A.Extended-Spectrum-Lactamases And Other Enzymes ProvidingResistance To Oxyimino-Lactams.Infect Dis Clin Noth Am 1997, the 11:875-887 that it is characterized in that energy hydrolysis following cynnematin β-Nei Xiananleikangshengsu of the 4th generation acid amides ring.) overwhelming majority these fermentoids be by plasmid-mediated pericentral siphon enzyme, can be secreted into that the shutoff signal peptide becomes maturing enzyme in the cell pericentral siphon, the hydrolysis β-Nei Xiananleikangshengsu, thereby avoided acting on penicillin-binding protein (penicillin bind proteins on the bacterium inner membrance because of microbiotic, and the synthetic bacterium death that causes of interference cell wall PBPs).

Because the widespread usage of different novel semi-synthetic β-Nei Xiananleikangshengsus, under antibiotic pressure, the ESBL of bacterium produces point mutation, has generated miscellaneous ESBL, so that it is wider to generate the substrate spectrum, and the β-Nei Xiananleikangshengsu that percent hydrolysis is higher.Wherein the most common and prevailing is SHV type and TEM type, they are respectively by the structural gene mutation that is positioned at encode on the plasmid SHV-1 and TEM-1, make one of enzyme active center or several amino acid generation replacement and derive a large amount of hypotypes (Medeiros AA.Evolution and dissemination of β-lactamases accelerated by generations of β-lactam antibioticsl Clin Infect Dis, 1997,24:19-45).As SHV-4 type ESBL: evolve by the SHV-1 β-Nei Xiananmei, be that with the difference of SHV-1 the 205th Leu is substituted Arg in the aminoacid sequence, the 238th Ser replaced by Glu by Gln replacement and 240 Lys, sudden change has caused SHV-4 type ESBL to enlarge the substrate spectrum, has higher degraded a new generation β-Nei Xiananleikangshengsu vigor.What resistant organism will contain different ESBL genes is delivered to R-plasmid of the same race or the xenogenesis bacterium, causes the propagation of ESBL gene.

Certainly, resistant organism produces how new resistance enzyme our clinical application is brought very big difficulty.But the resistant organism ESBL gene pool of expansion is composed extensively the high resistance enzyme of percent hydrolysis gene source for we provide an important substrate that obtains.After separating this kind gene, in intestinal bacteria, efficiently express the pure product that obtain behind the purifying and can be used to protect the normal microorganism of non-infection site to kill with exempting from beta-lactam, in the degraded environment and β-Nei Xiananleikangshengsu in the animal derived food.

Purposes about ESBL sees the CN1438315A patent the earliest; this patent disclosure a kind ofly have β-Nei Xiananmei and aminoglycoside modifying enzyme fusion rotein simultaneously; this albumen can be used to protect the normal microorganism of non-infection site to kill with exempting from beta-lactam and aminoglycoside antibiotics; in the degraded environment and beta-lactam and aminoglycoside antibiotics in the animal derived food; but this patent stresses the protection of this two fermentoids range of application, does not relate to merging any independent resistance expression of enzymes purification process patent application of enzyme.

WO 93/13795, A61K 37/54 patent disclosure obtain β-Nei Xiananmei with natural bacterial strain, the normal intestinal flora of protection during the antibiotic therapy; but this method is owing to be to extract enzyme from natural bacterium; so rate ratio is lower, purifying is difficulty comparatively, causes production cost higher thus.

At home relevant for the report of natural bacterium beta-lactam enzyme purification, but do not see the report of genetically engineered resistance expression of enzymes purifying, β-Nei Xiananmei as Zhao Xiuli purifying natural bacterium, successively used twice sieve chromatography, the primary ions displacement chromatography, loaded down with trivial details low, the high (Zhao Xiuli of cost of natural beta-lactam enzyme purification yield that causes of purge process, Li Jiatai. the characteristic research of plasmid-mediated extended spectrum (E S B L s). The Chinese Journal of Clinical Pharmacology, 1998,14 (1): 18-23.).

Natural all there is report with genetically engineered reorganization β-Nei Xiananmei resistance enzyme purification abroad, but also exists purifying loaded down with trivial details, or the shortcoming under yielding poorly.As Quinting B (Quinting B, Galleni M, Timm J et al.Purification and propertiesof the Mycobacterium smegmatis mc2155 β-lactamase.FEMS Microbiol Lett 1997,149 (1): the 11-15.) β-Nei Xiananmei among the purifying natural M. smegmatics mc2155, target protein output is about 1mg/L bacterium liquid, through QSepharose High Performance post (36/10), behind the NaCL gradient elution, last sample monoP HR 5/20 column, behind the pH wash-out, concentrating and going up the target protein yield that obtains behind sample Superdex 75 (10/30) posts only is 20%.Bouillenne F (Bouillenne F, Matagne A, Joris B et al.Technique for a rapid and efficient purification of theSHV-1 And PSE-2 β-lactamases.J Chromatogr B Biomed Sci 2000,737:261-265.) the SHV-1 gene is received expression plasmid p453 be transformed among the E.colik12 again, this methods and results is that expression amount is lower, purge process is more loaded down with trivial details, and purge process has been used a kind of ion exchange column and a kind of molecular sieve column.

Summary of the invention

Purpose of the present invention is exactly in order to address the above problem, and a kind of output height, purifying expression and purification method easy, recombined beta lactamase in superspectrum cheaply are provided, and the kind ESBL of various models can express and this zymoprotein of purifying according to this method.

Implementation method of the present invention:

The present invention designs the ESBL universal primer, and adds restriction enzyme site.Upstream primer is: 5 '-GGCCATGGGGATGCGTTATATTCGC-3 ' and downstream primer 5 '-TACTCGAGGCGTTGCCAGTGCTCGA-3 '.From the high resistance enzyme of screening, obtain total DNA,, carry out the PCR reaction as template.

PCR product glue is connected with pMD 18-T vector after reclaiming purifying, connects product transformed competence colibacillus JM109, after the screening of basket hickie, picking hickie, enlarged culturing again, extract plasmid pMD18T-SHV-4 then, through restriction enzyme digestion and electrophoresis, and the SHV-4 gene is confirmed as in order-checking.

With pMD18T-SHV-4 and pET26b with NcoI, XhoI double digestion, reclaim the test kit purifying through UNIQ-10 pillar DNA glue, reclaim the SHV-4 fragment and the big segment of plasmid thereof of double digestion, after the connection of T4 ligase enzyme, transformed competence colibacillus E.coliBL21 (DE3) coats transformed bacteria on the LB flat board that contains kantlex again, through 37 ℃ of overnight incubation, picking list bacterium colony enlarged culturing, through the nitrocefin qualitative test, conclusive evidence has active ESBL to produce.

The engineering bacteria that obtains forwards to and carries out high density fermentation in the fermentation culture agent, carries out under the condition of 35 ℃～40 ℃ and pH6.0～7.5, obtains highdensity fermentation culture bacterium liquid.

Nutrient chemical of the present invention is based on the LB substratum, and every liter is added compositions such as 40g glucose, 0.03% kantlex, 8ml inorganic salt solution.And inorganic salt solution is made up of 0.2%～0.5% SODIUM PHOSPHATE, MONOBASIC, 0.6%～0.8% dipotassium hydrogen phosphate, 0.01～0.05 zinc sulfate, 0.01%～0.05% sal epsom, 0.2%～0.8% ammonium phosphate.

Among the present invention, the expression of SHV-4 enzyme is controlled by the T7 promotor, and the T7 promotor can be activated by IPTG, when bacterial growth arrives the early stage of logarithmic phase, adds the final concentration of IPTG to 0.2mmol/L, and this moment, bacterial growth slowed down, the rising of target protein expression amount.

The purifying of solubility SHV-4 enzyme in the cell pericentral siphon among the present invention: get Ni ²⁺-NTA agarose dress post is with Binding buffer balance.Contain the ultrasonic supernatant liquor of SHV-4 enzyme bacterium behind 0.45 μ m membrane filtration, slowly cross post, then the Binding buffer of usefulness washes post, wash post with Wash buffer again, use Elute Buffer eluted protein at last, only obtain 95% above purity SHV-4 enzyme by the affinity chromatography in a step, obtain 7mg SHV-4 enzyme in average every liter of fermented liquid, enzyme is 476IU/mg than living.

The renaturation and the purifying of inclusion body SHV-4 enzyme among the present invention: zymocyte liquid is centrifugal, and collecting precipitation adds an amount of H ₂After O blew outstanding thalline, ice bath carries out ultrasonication, and was centrifugal again, and precipitation is the inclusion body crude extract.Add an amount of sex change liquid in the precipitation, blow outstanding precipitation with suction pipe and make it abundant dissolving, centrifuging and taking supernatant, supernatant are solubilization of inclusion bodies liquid.With an amount of inclusion body sex change liquid wash-out, collect the activated protein peak, the desalination of Sephadex G25 post, freeze-drying again obtain the pure product of albumen, and the checking of SDS-PAGE electrophoresis obtains proteic purity.

Enzyme activity determination method of the present invention adopts ultraviolet absorption method: measure the percent hydrolysis of enzyme to substrate, OD ₂₃₃The time measure the variation of penicillin G absorbancy, calculate enzyme activity unit.

The proteic assay of the present invention adopts the Bradford method: the bovine serum albumin of 1g/L is a reference liquid, OD ₅₉₅The time in adding sample 1h, survey absorbancy.

Beneficial effect of the present invention:

Adopt engineered method at expression in escherichia coli ESBL in this case, greatly improved target protein output, and the reorganization ESBL 3 ' end added 6 * His tail, made things convenient for the purifying in downstream with the fusion rotein of this His tail, by a step affinity chromatography, purity just reaches more than 95%.

Usually when genetically engineered improved target protein output, target protein can be assembled the inclusion body that forms non-activity, so just faced expensive and loaded down with trivial details annealing issues.In this case for the inclusion body that occurs, with urea-denatured dose and sieve chromatography combine method, in purifying, finished renaturation, and avoided the method elder generation renaturation of traditional oxygenant-reductive agent gsh, the price of repurity costliness and trivial step, thus greatly reduce production cost and the yield that has improved the SHV-4 enzyme.

Because all types of ESBL sequence similarities, the expression and purification of SHV-4 enzyme and the method for fermentation thereof also are applicable to the preparation of the ESBL of other type.

Description of drawings:

Fig. 1: the design of graphics of expression vector pET26-SHV-4;

Fig. 2: 1:DL15000 molecule object of reference; 2:pET26-SHV-4 plasmid single endonuclease digestion (NcoI); 3:pET26-SHV-4 plasmid double digestion (NcoI+XhoI);

Fig. 3: the SDS-PAGE electrophorogram of reorganization bacterium (pET26-SHV-4/BL21) supernatant purified product; 1: total supernatant; 2: cross post supernatant (being mainly) to not being attached to the albumen on the affinity column; 3:Bind elutriant (mainly cleaning foreign protein unconjugated on the affine resin); 4:Wash elutriant (the flush away foreign protein on affine resin of combining closely.5:Elution elutriant (the SHV-4 enzyme of purity more than 95%); 6: molecular weight standard;

Fig. 4: the SDS-PAGE electrophorogram of reorganization bacterium (pET26-SHV-4/BL21) inclusion body purification product;

1:Sephadex G75 elutriant (the SHV-4 enzyme of purity more than 95%); 2: inclusion body sex change liquid (the denseest band is the SHV-4 enzyme); 3: molecular weight standard;

Material of the present invention

1. restriction enzyme NcoI, XhoI are available from the precious bioengineering in Dalian Co., Ltd;

2.T4 ligase, Tag DNA plus polymerase, Taq archaeal dna polymerase, UNIQ-10 pillar DNA glue reclaim examination The agent box is available from Shanghai bioengineering Co., Ltd;

3. plasmid extracts kit available from Shanghai China Shun bioengineering Co., Ltd;

4.HisTag affine resin is available from U.S. Novagen, Inc. (Madison, WI);

5.Nitrocefin available from Britain OXOID company;

6.LB culture medium oneself configuration (NaCL 1% for peptone 1%, dusty yeast 0.5%g)

Embodiment:

The clone of embodiment 1:SHV-4 gene

(1) the segmental pcr amplification of SHV-4

With SHV-1 type ESBL gene is template, design SHV type ESBL universal primer, and add restriction enzyme site.Upstream primer is: 5 '-GG CCATGGGGATGCGTTATATTCGC-3 ' contains NcoI restriction enzyme site and downstream primer 5 '-TA CTCGAGGCGTTGCCAGTGCTCGA-3 ' contains the XhoI restriction enzyme site.Extract the plasmid of above-mentioned product enzyme E.coli,, carry out the PCR reaction as template.Reaction conditions is: 94 ℃ of pre-sex change 5min, again by following parameter circulation 25 times: 94 ℃ of sex change 55s, 58 ℃ of annealing 50s, 72 ℃ are extended 1min, and last 1 circulation is extended 10min for 72 ℃.

(2) purifying of PCR product

(Low melting point agarose, LMPA) method reclaims to adopt low melting point agarose.The PCR product is behind 1% low melting point agarose gel electrophoresis, cut out and contain the segmental blob of viscose of purpose, put into 1.5ml Eppendorf pipe, add the about 200 μ l of TE, add the saturated phenol of isopyknic Tris again in 70 ℃ of thawings, the centrifugal 10min of 10000rpm/min, isopyknic phenol: chloroform (1: 1), each extracting of chloroform once, the dehydrated alcohol and 1/10 volume 3mol/L NaAc (pH5.2) the deposit D NA that add 2 times of volumes, be dissolved in the 6-10 μ l water according to reclaiming segmental amount, get 1 μ l electrophoresis detection, preserve standby or be directly used in ligation for-4 ℃.

(3) ligation

Purified PCR product fragment is connected with the pMD18T fragment.

Ligation system: pMD18T carrier segments 2 μ l

PCR fragment 2 μ l

10×T4?Buffer 2μl

T4 ligase enzyme 2.5U 1 μ l

Add distilled water to 20 μ l

Mixing adds the ligation liquid that finishes, place 16 ℃ connect 5h after, place 4 ℃ standby or be directly used in conversion reaction.

(4) preparation of competent cell and conversion thereof

Be equipped with competent cell with Lime Chloride, each step of operation is all strict aseptic.Scrape with the aseptic inoculation ring earlier and get frozen E.coli JM109 bacterial classification in-80 ℃ of refrigerators, streak inoculation is cultivated about 16h for 37 ℃ in not containing antibiotic LB agar plate.Picking list bacterium colony is inoculated in the 5ml LB substratum, and 37 ℃, the 250rpm/min jolting is cultivated, and gets the 0.5ml culture and is inoculated in the 50ml LB substratum 37 ℃ of shaking culture 2-3h to OD ₆₀₀Be 0.4-0.6.Again bacterial cultures is transferred in the aseptic polypropylene centrifuge tube of 0 ℃ of precooling, ice bath 10min, 4 ℃, the centrifugal 10min of 5000rpm/min abandon supernatant, with the 100mmol/L CaCl of 10ml ice precooling ₂, 10mmol/LTrisCl (pH8.0) the resuspended precipitation of solution.Last ice bath 20min, 4 ℃, the centrifugal 10min of 5000rpm/min abandon supernatant, with the 100mmol/L CaCl of 2ml ice bath precooling ₂The resuspended sedimentation cell of-glycerine solution is competent cell.Competent cell is sub-packed in the aseptic Eppendorf tube, and every pipe 200 μ l indicate bacterial strain, date, and it is frozen standby to put-80 ℃ of refrigerators.

(5) transform and identify

10 μ l ligation liquid are added in the 200 μ l E.coli JM109 competent cells, mixing gently, ice bath 45min, 42 ℃ of water-bath 2min are reentered into ice bath 2min, add the LB substratum to 1ml, 37 ℃, 1h is cultivated in the 150rpm/min jolting, get the above-mentioned culture of 200 μ l, be distributed in penbritin, the LB flat board of X-gal and IPTG is through 37 ℃ of overnight incubation, the picking hickie, enlarged culturing is extracted plasmid DNA, then through restriction enzyme digestion and electrophoresis again, nucleotide sequencing is finished by precious biotech firm, and check nucleotide sequence, confirm that the SHV-4 type nucleotide sequence of announcing with NCBI is consistent, so with this cloned plasmids called after pMD18T-SHV-4.

Embodiment 2:pET26-SHV-4 construction of recombinant plasmid, evaluation

(1) pET26b-SHV-4 construction of recombinant plasmid and evaluation

PMD18T-SHV-4 and pET26b with NcoI, XhoI double digestion, are reclaimed the test kit purifying through UNIQ-10 pillar DNA glue, reclaim the SHV-4 fragment and the big segment of plasmid thereof of double digestion, by 5: 1 molecule number, with 16 ℃ of connections of T4 ligase enzyme (see figure 1) of spending the night.Then, transformed competence colibacillus E.coli BL21 (DE3).Again transformed bacteria is coated on the LB flat board that contains kantlex, through 37 ℃ of overnight incubation, picking list bacterium colony enlarged culturing, through the nitrocefin qualitative test, conclusive evidence has active ESBL to produce.Extract plasmid, the restriction enzyme digestion and electrophoresis (see figure 2).

The fermentation expression of embodiment 3:SHV-4 enzyme

(1) preparation of fermentation culture agent

10L LB fermention medium, the 100g peptone, the 50g yeast powder, 100gNaCL, 40g glucose, 0.03% kantlex, 8ml inorganic salt solution, inorganic salt solution is made up of 0.2%～0.5% SODIUM PHOSPHATE, MONOBASIC, 0.6%～0.8% dipotassium hydrogen phosphate, 0.01%～0.05% zinc sulfate, 0.01%～0.05% sal epsom, 0.2%～0.8% ammonium phosphate.Behind 120 ℃ of fermented liqs, 30min autoclaving, add kantlex to 30 μ g/ml, fermentation culture agent preparation finishes, and defoamer is high-quality soya-bean oil (sterilization in advance); Strong aqua and concentrated hydrochloric acid (regulating fermented liquid pH value);

(2) shake flask fermentation

The reorganization bacterium is inoculated in fermention medium (containing 30 μ g/ml kantlex), behind 37 ℃, 150r/min shaking culture 7h, activatory kind daughter bacteria is inoculated in the LB substratum (containing 30 μ g/ml kantlex) of 1L with 1: 100 ratio, in 37 ℃, 150rpm/min shaking culture.When bacterial growth reaches OD ₆₀₀Be 1.5 o'clock, add IPTG, after continuing to cultivate 4h, get bacterium liquid,, collect thalline through the centrifugal 5min of 12000rpm/min to final concentration 0.2mmol/L.

(3) high density fermentation

Fermentation parameter is set: feature is that described fermentation culture is carried out under the condition of 35～40 ℃ of temperature and pH6.0～7.5, and keeps water-soluble oxygen＞15%, and stirring velocity 400rpm/min when dissolved oxygen＜15%, improves rotating speed automatically.

7L LB after the sterilization, adding 2.1ml kantlex (100mg/ml), to make LB substratum final concentration be 30ug/ml, again seed is inserted in the fermentor tank in 5% ratio, when thalli growth to OD _600nmBegin during desired concn 1.5 to induce, adding inductor IPTG is 0.2mmol/L to final concentration, and other fermentation condition remains unchanged.Treat that thalline OD value no longer raises and begin to remove jar when keeping for some time to begin to descend, fermentation stops.

Embodiment 4: the preparation of original enzyme liquid before the reorganization SHV-4 enzyme bacteria purification

3L zymocyte liquid 8000rpm/min, 4 ℃, 20min are centrifugal, and collecting precipitation adds 30ml H ₂After O blew outstanding thalline, ice bath carried out ultrasonication: output rating control shelves are 4, continue 7s, interval 7s, time 35min.Homogenate after ultrasonic, 20000rpm/min, 4 ℃, centrifugal 30min, ultrasonic supernatant liquor is mainly solubility reorganization SHV-4 enzyme, and ultrasound precipitation is mainly insoluble reorganization SHV-4 enzyme.

Embodiment 5: the purifying of solubility SHV-4 enzyme

(1) purifying of solubility SHV-4 enzyme

Get 1ml Ni ²⁺-NTA agarose (the adsorbable 5-10mg 6 * His of every ml resin fusion rotein), the dress post is with Binding buffer balance.100ml contains the ultrasonic supernatant liquor of SHV-4 enzyme behind 0.45 μ m membrane filtration, slowly cross post, then the Bindingbuffer with 1000ml washes post, uses Wash buffer (0.5M NaCl, the 60mM imidazole of 600ml again, 20mM Tris-HCl, pH7.9) wash post, use 600ml Elute Buffer (1M imidazole, 0.5M NaCl at last, 20mM Tris-HCl, pH7.9) eluted protein.

(2) desalination of activated protein peak, freeze-drying

Lead balance with aseptic double-distilled water balance Sephadex G25 XK 50/100 to pH and electricity.With sample Sephadex G25XK 50/100 post (applied sample amount is at every turn less than 350ml) on the active peak, carry out wash-out with aseptic double-distilled water, collect the desalination peak.Through the 0.22um membrane filtration degerming of the beta-lactam enzymic activity peak of desalination, above-mentioned sterile liquid is propped up branch by 1-1.5ml/ and is installed in the aseptic peace bottle,-65 ℃ freeze after, pack into and carry out freeze-drying in the freeze-drying pin, obtain the pure product of freeze-drying, obtain 7mg SHV-4 enzyme in average every liter of fermented liquid, enzyme is 476IU/mg than living.Pure product of albumen and different purification step component S DS-PAGE electrophoresis (see figure 3) thereof.

Embodiment 6: the purifying of SHV-4 enzyme in the inclusion body

(1) inclusion body sex change

Ultrasound precipitation adds 5ml sex change liquid (10mol/L urea, 10mmol/L DTT) by every liter of bacterium liquid among the embodiment 4.Blow outstanding precipitation with suction pipe and make it abundant dissolving, room temperature is placed more than the 1h.Lysate 20000rpm/min, 20 ℃, 30min, centrifuging and taking supernatant.

(2) renaturing inclusion bodies and enzyme purification

(2mol/L urea+0.05mol/L Sodium phosphate dibasic-citric acid, pH6.6) balance Sephadex G75 (XK50/100) post is led balance to pH and electricity with separating Buffer.With the last sample of inclusion body sex change liquid (Guanidinium hydrochloride of 6mol/L) 15ml, flow velocity 5ml/min carries out wash-out, and 280nm detects, and collects the activated protein peak, and is qualitative with nitrocefin, remains with the protein peak of high vigor.

(3) desalination of activated protein peak, freeze-drying

Lead balance with aseptic double-distilled water balance Sephadex G25 XK 50/100 to pH and electricity.With sample Sephadex G25XK 50/100 post (applied sample amount is at every turn less than 350ml) on the active peak, carry out wash-out with aseptic double-distilled water, collect the desalination peak.With the degerming of 0.22um membrane filtration, above-mentioned sterile liquid is propped up branchs by 1-1.5ml/ and is installed in the aseptic peace bottle through the beta-lactam enzymic activity peak of desalination ,-65 ℃ freeze after, carry out freeze-drying in the freeze-drying pin of packing into.Freeze Drying Equipment: vacuum tightness 0.05bar, water vessel-55 ℃.Ambient temperature overnight obtains the pure product of freeze-drying, on average obtains obtaining in every liter of fermented liquid 17mg SHV-4 enzyme, and enzyme is than living for 438IU/mg, pure product of albumen and different purification step component S DS-PAGE electrophoresis (see figure 4) thereof.

Embodiment 7: the mensuration of protein content

The Bradford method is surveyed proteic content, and the bovine serum albumin of 1g/L is a reference liquid.OD ₅₉₅The time in adding sample 1h, survey absorbancy.

(1) reagent: CBG250 reagent: Coomassie brilliant blue G250 (Coomassie Blue G250) 100mg is dissolved in 95% alcohol 50ml, and adding distil water 800ml adds 85% phosphatase 11 00ml, supplies distilled water to 1000, adds 12mol/L hydrochloric acid 10ml behind the filter paper filtering.Protein standard liquid: 1mg/ml bovine serum albumin

(2) measuring method

Blank pipe: CBG250 5ml+100ul distilled water

Standard pipe: CBG250 5ml+100ul protein standard liquid

Measure pipe: CBG250 5ml+100ul sample (the sample protein concentration dilution is extremely near protein standard liquid concentration)

Calculation formula: M sample=A sample * V mark/A mark * V sample, M is total=and M sample * V is total/the V sample

A sample, A mark are respectively measures pipe, standard pipe OD ₅₉₅The time absorbancy, V sample, V mark, be respectively and measure the protein liquid volume that pipe, standard pipe add, V is always long-pending for surveying total protein liquid, M sample, M always are respectively the quality that adds albumen, total protein.

With blank pipe is contrast, measures OD ₅₉₅The time A sample, A target absorbancy, calculate the quality of total protein by top calculation formula.

Embodiment 8: the mensuration of enzyme activity

(1) ultraviolet absorption method is measured the percent hydrolysis of enzyme to substrate, OD ₂₃₃The time measure the variation of penicillin G absorbancy ^[6], calculate enzyme activity unit.Enzyme activity unit is defined as pH7.0,37 ℃, and per minute hydrolysis 1 μ mol substrate is an international unit.

(2) reagent

Substrate: penicillin G, molecular weight 356.4, concentration 480 μ g/mL.

Damping fluid: 0.1mol/L PBS (0.1mol/LNa ₂HPO4,0.1mol/LNaH ₂PO4, pH7.0)

Stop buffer: 1mol/L hydrochloric acid

(3) measuring method

Blank pipe: 4ml distilled water+PBS 1ml+ adds 1ml stop buffer termination reaction, mixing.

The full pipe that decomposes: substrate 1ml+PBS 1ml+10ul sample (about 10mg/ml albumen), 37 ℃ of water-bath 2h add 1ml stop buffer termination reaction, add 3ml distilled water, mixing before measuring.

Undecomposed pipe: substrate 1ml+PBS 1ml+10ul sample (about 10mg/ml albumen)+1ml stop buffer, 37 ℃ of water-bath 2h add 3ml distilled water, mixing before measuring.

Measure pipe: substrate 1ml+PBS 1ml+10ul sample (about 10mg/ml albumen), 37 ℃ of water-bath 10min add 1ml stop buffer termination reaction, add 3ml distilled water, mixing before measuring.

Calculation formula: U sample=(A not-A sample) M/ (A not-A is complete) T, U is complete=V is total * U sample/V sample U sample is for measuring the vigor of enzyme sample, and U be total enzyme activity entirely, and A is not, A sample, A be respectively undecomposed pipe, the full absorbance that decomposes pipe, mensuration pipe entirely.M is a 1ml substrate volumetric molar concentration, and T is for measuring the warm bath time of pipe, and the V sample is for adding the volume of measuring sample in the pipe, and V always is the gross sample volume.

With blank pipe is contrast, measures OD ₂₃₃The time A not, A sample, absorbancy that A is complete, calculate total enzyme activity by top calculation formula.

Claims (5)

1. the expression and purification and the fermentation process in high density thereof of a recombined beta lactamase in superspectrum (ESBL) is characterized in that: may further comprise the steps:

(1) adopting round pcr, is template with the total DNA of intestinal bacteria of the anti-super wide spectrum beta-lactam antibiotics of screening, the design universal primer, and the ESBL gene is obtained in amplification, inserts on the pMD18T cloning vector, changes E.coliJM109 over to.

(2) the SHV-4 type ESBL of order-checking affirmation (being called for short the SHV-4 enzyme) with NcoI, Xho double digestion, inserts secretor type pET26b expression vector, makes up the pET26-SHV-4 recombinant expression plasmid, changes E.coliBL21 (DE3) over to, carries out the expression of SHV-4 enzyme.

(3) E.coliBL21 (DE3) that contains recombinant plasmid carries out high density fermentation, and the bacterium that obtains is dissolved in the sterile distilled water of 3 times of volumes and carries out ultrasonication, obtains ultrasonic supernatant and inclusion body thereof precipitation respectively.

(4) purifying of SHV-4 enzyme adopts affinity chromatography in the ultrasonic supernatant, and the His tail that Ni affinity chromatography resin-bonded reorganization SHV-4 enzyme merges obtains purity＞95%, height ratio SHV-4 enzyme alive.

(5) sedimentary renaturation of inclusion body and purifying carry out simultaneously, and with the urea dissolving inclusion body of 4～10mol/L, sex change liquid is directly gone up sample sieve chromatography column purification, obtains purity＞95%, height ratio live-weight group SHV-4 enzyme.

2. according to expression and purification and the fermentation process in high density thereof of right 1 described ESBL, it is characterized in that: upstream primer is: 5 '-GG CCATGGGGATGCGTTATATTCGC-3 ' line sequence cuts the position for the NcoI enzyme and downstream primer is: 5 '-TA CTCGAGGCGTTGCC AGTGCTCGA-3 ' line sequence is the XhoI restriction enzyme site.

3. according to expression and purification and the fermentation process in high density thereof of right 1 described ESBL, it is characterized in that: applicable all ESBL expression of gene purifying and high density fermentations thereof

4. according to expression and purification and the fermentation process in high density thereof of right 1 described ESBL, it is characterized in that: high density fermentation culture medium is based on the LB substratum, adds to form behind the kantlex of 1%～6% glucose, 1%～4% inorganic salt solution and 0.01%～0.05%.

5. high density fermentation culture medium as claimed in claim 4 is characterized in that: described inorganic salt solution is made up of 0.2%～0.5% SODIUM PHOSPHATE, MONOBASIC, 0.6%～0.8% dipotassium hydrogen phosphate, 0.01%～0.05% zinc sulfate, 0.01%～0.05% sal epsom, 0.2%～0.8% ammonium phosphate.

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