CN1328156A - Process for preparing fusion protein used for glucose sensor - Google Patents

Abstract

A process for preparing the fusion protein used for glucose sensor includes such step as synthesizing DNA chain for coding polylysine, annealing it at 65 deg.C to become dual-chain short DNA fragment, gene splicing to create the expression carrier pPICGLT of fusion protein, linearizing it by restriction endonuclease, transferring it into yeast, screening yeast recon, extracting genom DNA of yeast for test, expressing and purifying fusion protein, culturing transformant in MM culture medium at 30 deg.C for 72 hr while adding methanol once per 24 hr, linking fusion protein with ceptor, preparing glucose sensor and electrochemical test. Resultant sensor features wide linear range up to 45 mmol/L, great amplitude of response signal and long storage period.

Description

A kind of preparation method who is used for the fusion rotein of glucose sensor

The present invention relates to biological technical field, more specifically relate to a kind of preparation method who is used for the fusion rotein of glucose sensor.

Glucose assays occupies considerable status in medical diagnosis, fermentation industry.Glucose electrode also be report the earliest, research and use at most, biosensor the most widely.Cass in 1984 etc. have at first reported a kind of with the glucose sensor of ferrocene deriv as electron mediator, have started enzyme-amboceptor biological respinse and amboceptor Study on Biosensor field thus, and this class biosensor is called as s-generation transmitter.Through combining, constitute disposable (disposable) that can produce in enormous quantities, excellent property enzyme electrodes with screen printing technique.Its leading product portable blood sugar enzyme electrodes has had about 50% world market share, is to develop the most successful biosensor so far.Yet there is a weakness in this class enzyme electrodes, and that is exactly that linearity range is narrow, and upper limit of detection can not satisfy hyperglycemia patient's needs generally at 20-25mmol/L.

The purpose of this invention is to provide a kind of preparation method who uses the fusion rotein of glucose sensor, linearity range is wide, and response signal is big, has solved the narrow problem of traditional glucose sensor linearity range.

In order to achieve the above object, the present invention adopts following technical measures: the glucose oxidase of said genetic modification is meant on gene level, can introduce the C-terminal of glucose oxidase with the special poly-bad sour peptide that engages of amboceptor ferrocenecarboxylic acid.Through expression, purifying, the fusion rotein that obtains is used to prepare glucose sensor.The steps include:

1, the DNA chain of composite coding polylysine (the needed restriction enzyme site of clone is contained at two ends):

5′-agctt，aag，aag，aag，aag，aaa，aag，aag，aaa，aag.aag，gc-3′

5′-ggccgc，ctt，ctt，ttt，ctt，ctt，ttt，ctt，ctt，ctt，ctt，a-3

2, pass through gene splicing, the expression vector pPICGLT of construction of fusion protein (GLT represents the fusion rotein that is constituted, and promptly contains the glucose oxidase of connection peptides and polylysine chain): above-mentioned synthetic DNA chain takes off fire at 65 ℃ and formed double-stranded short dna fragments (fragment 1) in 7 minutes; Plasmid pGEM-TGOL⁽²⁾DNA reclaims about 1.7Kb left and right sides fragment with restriction enzyme SnaBI and HindIII double digestion, agarose electrophoresis, isfragment 2; The fragment of plasmid pPIC9 DNA about SnaBI and NOtI double digestion recovery 8.0kb is fragment 3.Fragment 1,fragment 2,fragment 3 are used T₄Dna ligase connects, and (GLT represents GOD-(Ser-Gly) to obtain Expression of Fusion Protein carrier pPICGLT₅-(Lys)₁₀) (seeing accompanying drawing 1).

3, expression vector pPICGLT is after restriction enzyme Stul linearizing, transforms among the pichia pastoris GS115 with the protoplast transformation method.

4, the screening of yeast recon: extract the zymic genomic dna and do PCR (polymerase chain reaction) detection.

5, the expression of fusion rotein GLT, purifying, transformant is cultured to OD in 30 ℃ of following MD substratum (the basic chlorine of yeast source substratum (YNB) 1.7g/L, ammonium sulfate 5g/L, glucose 20g/L,vitamin H 400 μ g/L) substratum₆₀₀=1.2-1.5,2000 rev/mins of centrifugal collection yeast cell.With cell be resuspended in 10ml liquid MM (YNB 1.7g/L, ammonium sulfate 5g/L, methyl alcohol 12ml/L,vitamin H 400 μ g/L, casein hydrolysate 10g/L pH5.6) in the substratum, added 2ml methyl alcohol in per 24 hours in culture, cultivated about 72 hours.The centrifugal supernatant liquor of collecting.With reinforcing yin essence ion exchange column Q-Sephorose Fast Flow purification column purifying.

6, being connected of fusion rotein GLT and amboceptor, the amino on the polylysine chain in the fusion rotein with the amboceptor ferrocenecarboxylic acid on carboxyl be connected by the reaction of shrinking.

7, the preparation of glucose sensor: each the 2 μ l of mediator-modified enzyme that learn from else's experience drip on the working area of working electrode.At room temperature after the drying, cover one deck cellulose acetate film, be cut into one electrode, place 4 ℃ of moisture eliminators standby at electrode surface.

8, Electrochemical Detection: cyclic voltammetric detects and the timing current detecting is determined experimentation with reference to Model 270/250 electrochemical software user manual.The amboceptor enzyme electrodes for preparing is connected with electro-chemical systems, gets 20 μ l testing samples with microsyringe and drip, start electro-chemical systems immediately and carry out cyclic voltammetry scan in electrode surface.Sweep parameter is as follows: starting voltage (E_Ii=-0.5V, final voltage (E_λThe 0.5V of)=+, sweep velocity is 20mV/S.Contain 0.1mol/L Repone K in the sample, play supporting electrolyte.Chronoamperometry is used for the recording responses electric current, under fixed voltage (+0.45V is with respect to the Ag/AgCl reference electrode), add 20 μ l sample solutions after, Electrochemical Detection just begins to start, and can obtain the background current value with damping fluid.Current value at the 30th second experiment Analysis that goes on record.

The present invention compared with prior art, have the following advantages and effect: linearity range is wide, can reach 45mmol/L, and response signal is big, long preservative period.

Fig. 1 is the structure schema of fusion protein expression vector pPICGLT.

Fig. 2 detects the GLT gene synoptic diagram of pcr amplification from the genome of recombination yeast for the fusion rotein agarose electrophoresis.M:1Kb dna ladder degree molecular weight standard; 1: positive control; 2, the 3:PCR product; 4: negative control.

Fig. 3 is the cyclic voltammetric detection figure of three kinds of enzyme electrodess.A) Fc-GLT electrode; B) Fc-GOD_cElectrode; C) Fc-GOD_wElectrode.(starting voltage, final voltage (E_I0.5V (the E of)=-_λ), sweep velocity=+ 0.5VmV/s).

Fig. 4 is the linearity range of three kinds of enzyme electrodess.A) Fc-GLT electrode; B) Fc-GOD_cElectrode; C) Fc-GOD_wElectrode.(operating voltage=450mV)

Below in conjunction with accompanying drawing the present invention is described in further detail:

According to Fig. 1, Fig. 2, Fig. 3, Fig. 4 as can be known, its concrete steps are:

1, the DNA chain (the needed restriction enzyme site of clone is contained at two ends) of synthetic (worker company is given birth in Shanghai) coding polylysine:

5′-agctt，aag，aag，aag，aag，aaa，aag，aag，aaa，aag.aag，gc-3′

5′-ggccgc，ctt，ctt，ttt，ctt，ctt，ttt，ctt，ctt，ctt，ctt，a-3

2, by gene splicing, the expression vector pPICGLT of construction of fusion protein: above-mentioned synthetic DNA chain takes off fire at 65 ℃ and formed double-stranded short dna fragment (fragment 1) in 7 minutes; Plasmid pGEM-TGOL DNA reclaims about 1.7Kb left and right sides fragment with restriction enzyme SnaBI and HindIII double digestion, agarose electrophoresis, isfragment 2; The fragment of plasmid pPIC9 DNA about SnaBI and NOtI double digestion recovery 8.0kb is fragment 3.Enzyme is cut system (60 μ l): 10 * b μ ffer, 6 μ l, plasmid DNA 45 μ l, 0.1%BSA 6 μ l,restriction enzyme 3 μ l.

Enzyme is cut product and is detected through agarose electrophoresis (8%), and (Sangon Uniq-10) reclaims the purpose fragment to reclaim test kit withglue.Fragment 1,fragment 2,fragment 3 are used T₄DNA ligase connects, and (GLT represents GOD-(Ser-Gly) to obtain Expression of Fusion Protein carrier pPICGLT₅-(Lys)₁₀) (seeing accompanyingdrawing 1).The ratio of the dna content offragment 1,fragment 2,fragment 3 is 3: 1: 1 in the linked system.

Linked system (20 μ l):

1.7Kb?DNA 8μl，

8.0kb?DNA 9μl，

10 * damping fluid, 2 μ l

I₄Dna ligase 1 μ l

3, expression vector pPICGLT is after restriction enzyme StuI linearizing, transforms among the yeast Pichia pastoris GS115 with the protoplast transformation method.

The StuI enzyme is cut system:

pPICGLTDNA 44μl

10 * damping fluid, 6 μ l

StuI 4μl

The distilled water 6 μ l of sterilization

The step of protoplast transformation method: 50ml GS115 culture is cultured to cell to OD₆₀₀=0.2, in 2000 rev/mins of centrifugal 5 minutes collecting cells, use 10ml water, 10ml SCE solution (1mol/L sorbyl alcohol successively, 1mmol/L EDTA, 10mmmol/L lemon sodium) washing, re-suspended cell is in 10ml SK solution (1mol/L sorbyl alcohol, 67mmol/L potassium phosphate buffer, pH7.5) in, add 30 ℃ of effects of 20 μ l lyase (Lyticase) 30 minutes.Protoplastis uses 10ml sorbyl alcohol (1mol/L) to wash twice successively, 10ml CaS (10mmol/L CaCl₂, the 1mol/L sorbyl alcohol) to wash once, resuspended protoplastis is in the CaS of 0.6ml.Get 100ul protoplastis, 10ul plasmid DNA, 5ul salmon sperm dna and mixed atroom temperature incubation 20 minutes, then add 1.2ml PEG (3350) and continuedincubation 15 minutes, in 3000g/min centrifugal 4 minutes, collect protoplastis, add 150ul SOS (1mol/L sorbyl alcohol, 10mmol/LCaCl₂, 0.3*YPD), incubation is 30 minutes under the room temperature, makes cell walls regeneration.Be diluted to 0.5ml with the 1mol/L sorbyl alcohol, add the top-layer agar substratum that is incubated in 56 ℃, be poured on then on the RD flat board, with the full whole flat board of mixed solution lid, placed 4 minutes under the room temperature rapidly, allow agarose harden, the surface should be very flat, do not have projection.Under room temperature, be inverted dull and stereotyped 2-3 hours, and allowed redundant moisture volatilize, then in 28-30 ℃ of incubators, hatched 4-7 days, screen.

4, the screening of yeast recon

Extract the genomic dna of yeast transformant and do the PCR detection.The extracting method concrete operations step of yeast genes group is as follows: at first prepare protoplastis, method is the same, resuspended protoplastis (contains 1%SDS in 8ml lysis damping fluid, 10mmol/L pH7.4 Tris-HCl, 10mmol/L EDTA, 0.05mol/LNaCl), add 70 μ l Proteinase Ks (15mg/ml) and 80 μ l RNA enzymes, 37 ℃ of followingincubations 2 hours, and in 70 ℃ following 10 minutes with termination reaction, then add the ice-cold Potassium ethanoate damping fluid of the 5mol/L of 1/10 volume.Placed 30 minutes on ice.In 4 ℃ following 16,000 rev/min of centrifugal removal white precipitate, add isopyknic phenol-chloroformic solution (phenol: chloroform: DNA extraction primary isoamyl alcohol=25: 24: 1), supernatant is transferred to a centrifuge tube, the 100% cold ethanol precipitation DNA that adds 2 times, centrifugal to reclaim DNA and with its TE damping fluid that is dissolved in 2 times, add deposit D NA in the cold 3mol/L NaAc of 1/10 volume and the 0.6 times of Virahol, the centrifugal supernatant that goes.DNA is dissolved in the damping fluid of 100-200ul at last.

PCR system (50 μ l):

10 * damping fluid, 5 μ l

Mg²⁺ 3μl

Upstream primer^*1 μ l

Upstream primer^*1 μ l

dNTP 4μl

Pastorisgenomic dna 1 μ l

Taq archaeal dna polymerase 0.3 μ l

DDW 34.7μl

(* upstream primer: 5 ' TACGTAAGCAATGGCATTGAAGCCAGC-3 '

* swim primer: 5 '-GGCCGCCTTCTTTTTCTTCTTTTTCTTCTTCTTCTTA-3 ')

PCR thermal cycle conditions: be reflected on the PE480 thermal cycler and carry out.94 ℃, 3 minutes, 1 circulation, 94 ℃, 1 minute, 57 ℃, 1 minute, 72 ℃, 2 minutes, 30 circulations.The gel electrophoresis condition: 0.5 * tbe buffer liquid (prepare 0.7% sepharose, 0.5 μ g/ μ L ethidium bromide pre-staining, 60V voltage stabilizing electrophoresis 1-2 hours, the nucleic acid molecular weight contrast is 1kb dna ladder degree molecular weight standard.Ultraviolet lamp is observed down and record result's (seeing accompanying drawing 2).

5, Expression of Fusion Protein, purifying

Transformant 30 ℃ of following MM substratum (basic chlorine source substratum YNB0.17g, (NH4)₂SO₄0.5g, 5% methyl alcohol) cultivated 72 hours, added methyl alcohol one time in per 24 hours, the GLT fusion gene is expressed in PichiapastriesGS115 under the effect of AOX1 promotor.The protein purification step is as follows:

Dress post → wash post → wash post → with 0.02mol/L citric acid cushioning balance chromatography column → the go up sample (about 100ml) that enzyme is alive and protein content → collection has enzymic activity of sample in sample → gradient elution (elutriant is 0-0.1mol/L or 0-0.2mol/LNaCl solution, and elution speed is 1.8ml/min) → substep collection (every pipe 7ml) → detection collection tube with distilled water with 20% ethanol.The protein of collecting is standby through ultrafiltration and concentration-20 ℃ preservation.When crossing the post wash-out, detect (Shanghai 61 instrument plants) online detection protein concn with UV700 albumen/detection of nucleic acids instrument.It is as follows to detect GOD enzyme method alive: make typical curve with Sigma company commodity GOD (A.niger).With pH5.6,0.1mol/L sodium citrate buffer solution preparation 2mg/ml glucose, adjacent biphenyl two methyl oxyanilines of 2mg/ml and 100U/ml horseradish peroxidase solution.In the 5ml centrifuge tube, add each 100 μ l of 1ml glucose solution, 2ml glycerine and adjacent biphenyl two methyl oxyanilines and horseradish peroxidase solution respectively.30 ℃ of insulations added 20 μ lGOD standardized solution or samples after 10 minutes, shook up rapidly.Reaction is after 30 minutes down at 30 ℃, and adding 2ml 5mol/L hydrochloric acid termination reaction is measured 0D₅₂₅

6, fusion rotein and amboceptor is connected

Albumen and amboceptor to be connected the concrete operations step as follows: (N-(2-carboxylic propyloic)-piperazine-N-2 (propanesulfonic acid)) that 80mg GOD is dissolved in 4ml (0.15mol/L) forms solution about little mixed pH7.3 in the damping fluid (if be necessary, can be suitably regulate pH value with the Na-HEPES of 0.1mol/LHCl or 0.15mol/L) carbodiimide (EDC) and the 480mg urea of adding 100mg, the pH value readjusts and is 7.2-7.3, the glucose oxidase that adds 60mg subsequently, solution is packed in the vial, seal to place on ice with paraffin and spend the night.Use 0.1mol/L, the citrate buffer solution dialysis of pH6.0 48 hours, dialyzate is changed 4-8 times in this process, to remove responseless ferrocenecarboxylic acid and other small-molecule substance.To be divided Fc-GLT, Fc-GOD by the fusion rotein of ferrocenecarboxylic acid modified, wild-type enzyme, commercial enzyme_wFc-GOD_c

7, the preparation of glucose sensor

Screen printing electrode comprises a working electrode and an Ag/AgCl reference electrode in the experiment.On the PVC of 15 * 15CM (polyvinyl chloride (PVC) sheets), print 30 electrodes, the process that prints electrode of electrode.Electrode is before use successively with alcohol and water flushing.Get three kinds and drip on the working area of working electrode, at room temperature after the drying, cover one deck cellulose acetate film, be cut into one electrode, place 4 ℃ of moisture eliminators standby at electrode surface through each 2 μ l of mediator-modified enzyme.

8, Electrochemical Detection

Cyclic voltammetric detects and the timing current detecting is determined experimentation with reference to Model 270/250 electrochemical software user manual.The amboceptor enzyme electrodes for preparing is connected with electro-chemical systems, gets 20 μ l testing samples with microsyringe and drip, start electro-chemical systems immediately and carry out cyclic voltammetry scan in electrode surface.Sweep parameter is as follows: starting voltage (E_iThe 0.5V of)=-, final voltage (E_λThe 0.5V of)=+, sweep velocity is 20mV/S.Contain 0.1mol/L Repone K in the sample, play supporting electrolyte (the results are shown in accompanyingdrawing 3).Chronoamperometry is used for the recording responses electric current, under fixed voltage (+0.45V is with respect to the Ag/AgCl reference electrode), add 20 μ l sample solutions after, Electrochemical Detection just begins to start, and can obtain the background current value with damping fluid.Current value at the 30th second experiment Analysis that goes on record.Measurement is three electrode replication results' mean value.

Claims (2)

1, a kind of preparation method who is used for the fusion rotein of glucose sensor, it comprises the following steps:

The needed restriction enzyme site of clone is contained at the DNA chain of A, composite coding polylysine or two ends:

5′-agctt，aag，aag，aag，aag，aaa，aag，aag，aaa，aag.aag，gc-3′

5′-ggccgc，ctt，ctt，ttt，ctt，ctt，ttt，ctt，ctt，ctt，ctt，a-3；

B, pass through gene splicing, the expression vector pPICGLT of construction of fusion protein, above-mentioned synthetic DNA chain takes off fire at 65 ℃ and forms double-stranded short dna fragment, plasmid pGEM-TGOL DNA restriction enzyme SnaBI and HinaIII double digestion, agarose electrophoresis reclaims fragment, and plasmid pPIC9 DNA reclaims fragment with SnaBI and NOtI double digestion;

C, expression vector pPICGLT transform among the yeast Pichia pastoris GS115 with the protoplast transformation method after restriction enzyme Stul linearizing;

The screening of D, yeast recon is extracted the zymic genomic dna and is done the PCR detection;

The expression of E, fusion rotein GLT, purifying, transformant is being cultivated in the MD substratum under 30 ℃, centrifugal collection yeast cell, cell is resuspended in the 10ml liquid MM substratum, in culture, added 2ml methyl alcohol in per 24 hours, cultivated about 72 hours, the centrifugal supernatant liquor of collecting is with reinforcing yin essence ion exchange column Q-Sephorose Fast Flow purification column purifying;

F, fusion rotein GLT are connected with amboceptor, and the carboxyl on the amino acid on the polylysine chain in the fusion rotein and the amboceptor ferrocenecarboxylic acid is connected by the reaction of shrinking;

The preparation of G, glucose sensor, each the 2 μ l of mediator-modified enzyme that learn from else's experience drip on the working area of working electrode, at room temperature after the drying, cover one deck cellulose acetate film at electrode surface, are cut into one electrode, place 4 ℃ of moisture eliminators standby;

H, Electrochemical Detection, the amboceptor enzyme electrodes for preparing is connected with electro-chemical systems, getting 20 μ l testing samples with microsyringe drips in electrode surface, start electro-chemical systems and carry out cyclic voltammetry scan, Electrochemical Detection just begins to start, with damping fluid in 30 seconds the current value experiment Analysis that goes on record.

2, a kind of preparation method who uses the fusion rotein of glucose sensor according to claim 1, it is characterized in that being connected of fusion rotein and amboceptor, at first 80mgGOD is dissolved in the solution that forms little mixed pH7.3 in the NaHEPES damping fluid of 4ml, next adds carbodiimide and the 480mg urea of 100mg, pH7.2-7.3, the 3rd is the glucose oxidase that adds 60ml, solution is packed in the vial, seal with paraffin, the 4th is to use 0.1mol/L, the citrate buffer solution dialysis of pH6.0 48 hours, dialysis is changed 4-8 times.

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