CN100531795C

CN100531795C - Omega-conotoxin M VII A mutant and its preparation and uses

Info

Publication number: CN100531795C
Application number: CNB2006100524002A
Authority: CN
Inventors: 詹金彪; 夏铮; 王峰; 徐小蓉; 黄建松
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2006-07-11
Filing date: 2006-07-11
Publication date: 2009-08-26
Anticipated expiration: 2026-07-11
Also published as: CN1876176A

Abstract

The invention relates the Omega-M VII A(CTX M VIIA) mutant, possessing SEQ ID NO:1 amino acid sequence and adopting glutathione S- transferase(GST) fusion expression method. The method comprises the following steps: on the base of pGEX-2T/CTX M VIIA pronucleus expression plasmid, using IPTG to induce and express GST-CTX M VIIA fusion protein in coli bacillus, purifying fusion protein with GST affinity chromatography, using thromboses to produce CTX M VIIA mutant, separating and purifying product; opening the disulfide bridge of mutant with reducing agent, then carrying out dialysis with PBS, carrying out air oxidation, and getting final product. The invention has the advantages of simple operation, stable quality, and low cost.

Description

Omega-conotoxin M VII A mutant and preparation and application

Technical field

The invention belongs to genetic engineering, relate to omega-conotoxin M VII A (M VII A) mutant and amalgamation and expression and production method, and the application in the preparation analgesic.

Background technology

Conotoxin (Conotoxin is the peptide toxoid of the class biologically active that obtains from ocean gasteropod cone shell (Conus) CTX), estimate at 50000 surplus kind.These polypeptide structure novelties, the function uniqueness.By its target site can be divided into α-, ω-, μ-, δ-etc. type.Because they optionally act on different ion channels and hypotype thereof, play an important role at aspects such as neuro pharmacology research and disease treatments.

ω type CTX is the important research direction of conotoxin in recent years, has wide drug development prospect.Wherein, omega-conotoxin M VII A (CTX M VII A) is the inhibitor of voltage-sensitive type calcium channel, and its composition sequence is Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH ₂, it is made up of 25 aminoacid, contains 6 cysteine (Cys) residue, forms three pairs of full disulfide bond (be that the disulfide bond connected mode is Cys1-Cys16, Cys8-Cys20, Cys15-Cys25, C-hold and be amidatioon Cys) that intersect.They have very high analgesic activities and do not have addiction, can be used as the alternative medicine of addiction analgesic such as morphine; In recent years, the omega-conotoxin M VII A of chemosynthesis is gone on the market by the FDA approval in the U.S..

At present, the omega-conotoxin M VII A that has gone on the market uses the method for chemosynthesis to obtain, and forms disulfide bond owing to there are 6 cysteine to need accurately to match in its molecule, need 20 multistep reactions, yield is very low, and technology is very difficult, the quality instability, and cost is higher.Therefore, seek a kind of suitable method acquisition omega-conotoxin M VII A and become needs.

Summary of the invention

The purpose of this invention is to provide polypeptide organic compound omega-conotoxin M VII A mutant, have the aminoacid sequence of SEQ ID NO:1:

Gly-Ser-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys27

Form three pairs of disulfide bond in this polypeptide organic adduct molecule between Cys3 and Cys18, Cys10 and Cys22, Cys17 and the Cys27 respectively; The C-end is non-amidated Cys, and has the encoding mutant body DNA sequence of SEQ ID NO:2:

ggatcctgca?aaggtaaagg?tgcgaaatgc?tctcgtctga?tgtacgactg?ctgcaccggt?60

tcttgccgtt?ctggtaaatg?c?81

Another object of the present invention is to overcome the deficiency that prior art exists, and the preparation method of this omega-conotoxin M VII A mutant is provided, and realizes by following steps:

1, induce recombinant expression carrier pGEX-2T/CTXM VII A to express the e. coli bl21 (DE) (referring to CN03142052.4) of fusion rotein (the GST-CTX M VII A) conversion of glutathione S-transferase (GST) and omega-conotoxin M VII A with isopropylthio-(IPTG);

2, the method with the GST affinity chromatograph adopts the Glutathione-Agarose affinity column to come purified fusion protein, to obtain highly purified GST-CTX M VII A fusion rotein;

3, carry out enzyme action with thrombin (Thrombin) after GST-CTX M VII A fusion rotein is dialysed behind the purification in phosphate buffer (PBS), enzyme action afterproduct process Sephacryl S-100 HR gel column separates, purification, the CTX M VII A mutant that purification obtains is opened the disulfide bond of CTX M VII A mutant earlier with 0.1% beta-mercaptoethanol, reuse phosphate buffer (PBS) dialysed overnight, make its eremacausis in air, thereby form correct disulfide bond pairing.

Affinity chromatograph is adopted in the preparation of described GST-CTX MVII A fusion rotein, selects filler Glutathione-Agarose for use, and reduced glutathion and 50mM Tris-HCl (pH8.0) carry out eluting (referring to CN03142052.4).

Described thrombin (Thrombin) enzyme action GST-CTX MVII A fusion rotein, be with thrombin under the PBS buffer system at 22 ℃ with different enzyme concentrations, the different enzyme action working times make GST-CTX MVII A fusion rotein through enzyme action, obtain CTX M VII A mutant small fragment.The suitableeest endonuclease reaction condition is: in PBS solution, 100 μ g fusion rotein are added the thrombin of 2 units (unit), 22 ℃ of enzyme action 20 hours obtain CTX M VII A mutant.

Described enzyme action product C TX M VII A mutant sieve chromatography purification CTX M VII A mutant polypeptide fragment, filler is selected Sephacryl S-100HR for use, with through the filterable deionized water of 22 μ m sterile filters with the flow velocity of 1ml/min with Sephacryl S-100HR prepacked column on AKTA Purifier protein purification instrument, to mixture behind the enzyme action separate, purification and desalination.

The correct pairing of the disulfide bond of the CTX M VII A mutant of described purification, be earlier the disulfide bond of CTX M VII A mutant to be opened with 0.1% beta-mercaptoethanol earlier, reuse PBS dialysed overnight makes its eremacausis in air, thereby forms correct disulfide bond pairing.

Another purpose of the present invention provides the application of omega-conotoxin M VII A mutant in the preparation analgesic.

Come production gene recombinaton CTX M VII A mutant in engineered mode.This method success with CTX M VII A gene directed cloning in prokaryotic expression carrier pGEX-2T.The characteristics of this prokaryotic expression system are: P _TacPromoter is subjected to the control of lac operon.Its downstream is glutathione transferase (GST) gene, has the restriction enzyme site of thrombin between GST and multiple clone site.Exogenous genetic fragment is cloned into the proper reading frame frame on the multiple clone site in strong promoter downstream, can express the fusion rotein that foreign protein and GST form under the inducing of IPTG, and can significantly improve the Expression of Fusion Protein productive rate.Fusion rotein can be cut to obtain the little peptide of purpose by site-specific protease such as thrombin.Gst fusion protein is solvable in aqueous solution, under the condition of invariance, obtains by affinity chromatograph.Thereby adopt this prokaryotic expression system to make CTX M VII A be able to high yield expressing with the form of GST-CTX M VII A fusion rotein earlier, and by GST adopt affinity chromatograph quick, easy obtain highly purified fusion rotein, using the special thrombin of this expression system to carry out enzyme action then separates GST, obtain purer CTX M VII A mutant, simplified the work that is further purified this polypeptide fragment greatly.Though on the CTX M VII A mutant band that finally obtains two unnecessary amino acid residues but have very strong analgesic effect equally, can be used as neuroscience and pharmaceutical research, also can be developed further into analgesic into novel no addiction type.

The production method of antigen-4 fusion protein gene engineering of the present invention, simple to operate, steady quality, cost are lower, obtain active omega-conotoxin M VII A mutant and can carry out drug development.

Description of drawings

Fig. 1 is a CTX MVIIA partial dna sequence measurement result in the expression plasmid.

Fig. 2 is the abduction delivering of fusion rotein GST-CTX MVIIA.

Fig. 3 is the purification result of fusion rotein GST-CTX MVIIA.

Fig. 4 is the result of AKTA purifier purification GST-CTX M VII A.

Fig. 5 adds thrombin at 22 ℃ of following enzyme action with 100 μ g fusion rotein/1unit thrombin ratio, the enzyme action result of different time.

Fig. 6 adds thrombin at 22 ℃ of following enzyme action with 100 μ g fusion rotein/2unit thrombin ratio, the enzyme action result of different time.

Fig. 7 adds thrombin at 22 ℃ of following enzyme action with 100 μ g fusion rotein/3unit thrombin ratio, the enzyme action result of different time.

Fig. 8 adds thrombin at 22 ℃ of following enzyme action with 100 μ g fusion rotein/4unit thrombin ratio, the enzyme action result of different time.

Fig. 9 is the enzyme action efficient of fusion rotein GST-CTX M VII A under conditions such as different concentration of thrombin, different thrombin enzyme action times.

Figure 10 is the response rate of CTX M VII A mutant under conditions such as different concentration of thrombin, different thrombin enzyme action times.

Figure 11 is a sieve chromatography purification CTX M VII A mutant elution profile.

Figure 12 identifies recombinant C TX M VII A mutant for the MALDI-TOF-MS mass spectrum.

Figure 13 is the analgesic activities that the hot tail method of rat is measured CTX M VII A mutant.

The specific embodiment

The present invention is further described in conjunction with the accompanying drawings and embodiments.

Embodiment one

1 materials and methods

1.1 material and reagent

PGEX-2T/CTX M VII A plasmid is made up by this chamber; Isopropylthio-(IPTG) and standard molecular weight marker, reduced glutathion purchase in Shanghai life worker bio-engineering corporation; Glutathione-Agarose, Sephacryl S-100HR purchase the company in Parmacia; Thrombin (Thrombin) is purchased the company in Parmacia; E. coli bl21 (DE) strain is purchased the company in Sigma; Other reagent are all purchased in domestic each company.

1.2 method

1.2.1 the abduction delivering of GST-CTX M VII A fusion rotein

GST-CTX M VII A plasmid is imported in the e. coli bl21 (DE), is the positive and negative contrasts with the empty bacterium of BL21 (DE) with the BL21 (DE) that contains the plasmid that does not insert genes of interest.The picking reorganization successful clone be inoculated in the LB culture medium that contains ampicillin, and 37 ℃ of joltings are spent the night, and 2% ratio is inoculated fresh LB culture medium, is cultured to mid-log phase.Take out 1 milliliter, making negative control, all the other add IPTG is 0.1mM to final concentration, 37 ℃ of abduction deliverings 4 hours, (referring to CN 03142052.4).

1.2.2 the affinity purification of GST-CTX M VII A fusion rotein

1.2.2.1 measure the adsorption capacity of Glutathione-Agarose filler

The Glutathione-Agarose filler that is soaked among 20% ethanol and the 0.5M NaCl is loaded on a simple and easy pillar, changed sterilization PBS balance after 1 hour 1 hour with 20% ethanol with the form balance of drop, the gst fusion protein that then adds abduction delivering, with sterilization PBS balance, collect effluent, treat to regather behind 10 column length volumes, then change affinity elution liquid into, reduced glutathion and 50mM Tris-HCl (pH8.0) carry out eluting and collect effluent, collecting effluent behind 10 column length volumes of eluting, change 20% ethanol equilibrate overnight then, unloaded 4 ℃ of preservations in the solution that filler is immersed in 20% ethanol and 0.5M NaCl in second day.

1.2.2.2 the installation of Glutathione-Agarose affinity column

Dismounting Tricorn5/20column also is soaked in 20% the ethanol, utilizes iron stand to build holder.The Glutathione-Agarose filler that will be soaked in subsequently among 20% ethanol and the 0.5M NaCl adds centrifuge tube and adds 20% ethanol, soft mixing.Fixing void column on holder, set up asbestos filter plug in the bottom to hold back filler, fill with 20% ethanol subsequently in pillar, treat that liquid level flow to the Glutathione-Agarose that mixes from the 1/3 column length place adding of pillar bottom, the speed that control adds makes it roughly the same with effusive speed.Treat that filler adds to from 9/10 column length place of pillar bottom, the tap hole of sealing pillar bottom is carefully set up top cover, avoids gas to enter.4 ℃ fully deposited filler in static pillar 1-2 hour.Subsequently pillar is installed on the AKTA Purifier protein purification instrument, the ethanol with 20% is walked flat with the flow velocity balance pillar of 1ml/min until OD215, OD280 and electric conductance isobase.

1.2.2.3 Glutathione-Agarose affinity purification gst fusion protein

The successful reorganization bacterium of picking clone is inoculated in the LB culture medium that contains ampicillin, and 37 ℃ of joltings are spent the night, and bacterium liquid is inoculated in the fresh LB culture medium that contains ampicillin in the ratio of 1:50, and 37 ℃ are cultured to OD ₆₀₀Be about 0.8, adding IPTG is 0.1mM to final concentration, and continuation was cultivated 4 hours.5000rpm/4 ℃ of centrifugal 10min after the supernatant discarded, is resuspended in precipitation among the 3ml sterilization PBS, and carrying out ultrasonic bacteria breaking is clarified until solution, with ℃ centrifugal 15min of the solution 15000rpm/4 after the fragmentation, gets 4 ℃ of preservations of supernatant.

With walking flat with the flow velocity balance affinity column of 1ml/min until OD215, OD280 and electric conductance isobase through the filterable sterilization of 22 μ sterile filters PBS.The supernatant of ultrasonic centrifugal back 4 ℃ of preservations after filtering, 22 μ sterile filters is gone up sample, with carrying out balance with the flow velocity of 1ml/min through the filterable sterilization of 22 μ m sterile filters PBS, treat OD215, OD280 and electric conductance isobase are taken each other's daughter as daughters-in-law and eluent after walking to put down again, reduced glutathion and 50mM Tris-HCl (pH8.0) carry out eluting, elution flow rate still is 1ml/min, and according to OD215, the variation of numerical value such as OD280 and electric conductance is collected eluting peak and is also surveyed absorbance calculate protein concentration under ultraviolet 280nm wavelength, change when waiting not have other materials by eluting 20% ethanol with the flow velocity balance pillar of 1ml/min until OD215, OD280 and electric conductance isobase are walked flat once more.Unload pillar and be placed on 4 ℃ of preservations.The sample of collecting behind the purification is identified with 12%SDS-PAGE.

1.2.3 separate and purification CTX M VII A polypeptide fragment

1.2.3.1 the work efficiency of assaying blood clotting enzyme Thrombin

To be transferred to molecular cut off through the GST-CTX of affinity purification M VII A fusion rotein is to be immersed in the PBS buffer 4 ℃ of dialysis in 8000 the bag filter and to bring in constant renewal in PBS (PBS is the working buffer solution of this thrombin).Dialyse and reclaim GST-CTX M VII A fusion rotein after 20 hours, the absorbance of surveying under the ultraviolet 280nm wavelength is estimated protein concentration.100 μ g fusion rotein are used the thrombin of 1,2,3 and 4 units (unit) respectively, at 22 ℃ of

enzyme action

4,8,12,16,20 and 24 hours respectively; Each working time point takes out corresponding reaction tube immediately, and adding 10 μ l, 5 * SDS PAGE Loading Buffer boils 5min, and the centrifugal 5min of 10000rpm is positioned over-20 ℃ and waits for SDS-PAGE electrophoretic analysis evaluation subsequently.

1.2.3.2 enzyme action separation of C TX M VII A polypeptide fragment

GST-CTX M VII A fusion rotein behind the affinity purification is replaced with the PBS buffer system through dialysis, and calculates the concentration of fusion rotein with ultraviolet spectrophotometry.Ratio with 100 μ g fusion rotein/2unit enzymes adds thrombin, enzyme action work is after 20 hours under the condition of 22 ℃ of water-baths, it is in 1000 the bag filter that the enzyme action product is transferred to molecular cut off, concentrates with PEG 20000, collects sample after concentrating and is positioned over 4 ℃ and preserves etc. to be purified.

1.2.3.3 sieve chromatography separation of C TX M VII A polypeptide fragment

Sephacryl S-100HR prepacked column is installed on the AKTA Purifier protein purification instrument, and the ethanol with 20% is walked flat with the flow velocity balance pillar of 1ml/min until OD215, OD280 and electric conductance isobase.With walking flat with the flow velocity balance affinity column of 1ml/min until OD215, OD280 and electric conductance isobase through the filterable deionized water of 22 μ m sterile filters.Carry out molecular sieving, purification and desalination with concentrating good sample, still carry out eluting with filtering deionized water with the flow velocity of 1ml/min through 22 μ m sterile filters, and collect eluting peak according to the variation of numerical value such as OD215, OD280 and electric conductance, when waiting not have other materials by eluting, the ethanol of change 20% is walked flat with the flow velocity balance pillar of 1ml/min until OD215, OD280 and electric conductance isobase once more.Unload pillar and be placed on 4 ℃ of preservations.Appearance time according to eluting peak is roughly estimated molecular weight ranges, preserves the eluting peak of molecular weight in the 2000-4000 scope, as CTX M VII A mutant, carries out structure and identifies and pharmaceutical research.

2 results

2.1 the IPTG abduction delivering of GST-CTX M VII A fusion rotein in the host bacterium

Recombinant expression plasmid pGEX-2T/CTX M VII A transformed into escherichia coli BL (DE), LB culture plate (Amp ⁺) last well-grown monoclonal, amplification back extracting plasmid DNA, give birth to worker bio-engineering corporation by Shanghai and measure DNA sequence, partial sequence the results are shown in Figure 1, wherein nucleotide sequence is from 33～108 coding CTX M VII A, its sequence is tgc aaa ggt aaa ggt gcg aaa tgc tct cgt ctg atg tacgac tgc tgc acc ggt tct tgc cgt tct ggt aaa tgc, shows that CTX M VII A gene order is correct.

The monoclonal bacterium colony that transforms the e. coli bl21 (DE) of recombinant expression plasmid pGEX-2T/CTX M VII A is increased and abduction delivering, respectively get 20 μ l IPTG abduction delivering bacterium liquid, do not add the resuspended precipitation in culture medium supernatant, carrying out ultrasonic bacteria breaking, centrifugal back behind the inductive bacterium liquid of IPTG, the IPTG abduction delivering and carrying out ultrasonic bacteria breaking, the supernatant after centrifugal, add 5 μ l, 5 * SDS PAGE Loading Buffer and boil 5min, get 10 μ l behind the centrifugal 5min of 10000rpm and carry out the evaluation of SDS-PAGE electrophoresis, whether observe gst fusion protein matter by abduction delivering.SDS-PAGE result is referring to Fig. 2, wherein 1: be standard molecular weight marker, 2: be the inductive IPTG abduction delivering of IPTG bacterium liquid, 3: for not using the inductive expression of IPTG bacterium liquid, 4: be the culture medium supernatant behind the IPTG abduction delivering, 5: with IPTG inductive expression bacterium, ultrasonication, the supernatant after centrifugal, 6: with IPTG inductive expression bacterium, ultrasonication, the precipitation after centrifugal.According near the concentration and the proteic content of shared whole swimming lane of the band this band in more ultrasonic centrifuged deposit and the supernatant, the gst fusion protein of abduction delivering mainly concentrates on brokenly in the supernatant solution of bacterium after centrifugal.The culture medium supernatant carries out electrophoresis and identifies that the back finds not have obvious band at molecular weight about 31000 after to abduction delivering, illustrates that GST-CTX M VII A fusion rotein is not by secreting, expressing.

2.2 the purification of GST-CTX M VII A fusion rotein

2.2.1 measure the adsorption capacity of Glutathione-Agarose filler

With above-mentioned transformed bacteria through abduction delivering, ultrasonication, the supernatant of centrifugal gained is through the Glutathione-Agarose affinity column chromatography, the effluent that each link is collected carries out the 12%SDS-PAGE Analysis and Identification, the result is referring to Fig. 3, wherein 1: standard molecular weight marker, 2: not with the inductive expression of IPTG bacterium liquid, 3: with the inductive expression of IPTG bacterium, ultrasonication, supernatant after centrifugal, 4: with the inductive expression of IPTG bacterium, ultrasonication, precipitation after centrifugal, 5: eluting composition at first behind the sample on the supernatant, 6: with the composition of the PBS eluent eluting of 10 times of Glutathione-Agarose column volumes, the composition of 7:10mM reduced glutathion and 50mM Tris-HCl (pH8.0) eluent eluting, the composition behind the 10mM reduced glutathion of 8:10 times of column volume and 50mM Tris-HCl (pH8.0) the eluent eluting.See at molecular weight about 31000 in the water outlet peak of behind last sample, collecting that a spot of band is arranged, illustrate that Glutathione-Agarose is by saturated; Except that a dense band is arranged at molecular weight about 31000, do not have other band in the sample of when eluting, collecting, show that Glutathione-Agarose has very strong specificity; And do not see basically in the sample of behind 10 bed volumes of eluting, collecting that the bar carrying means adopts Glutathione-Agarose to carry out affinity elution and has extremely strong efficient, and collect proteic content when calculating eluting and estimate that the maximal absorptive capacity of Glutathione-Agarose is 7mg/ml.

2.2.2 Glutathione-Agarose affinity purification gst fusion protein

2ml Glutathione-Agarose is loaded on Tricorncolumn 5/20 pillar, with AKTA Purifier protein purification instrument purification gst fusion protein, immediately measure OD215, OD280 and electric conductance with automatic detector, collect sample according to testing result, SDS-PAGE the results are shown in Figure 4, and eluting goes out GST-CTX M VII A fusion rotein between the 35th fen kind to the 42 minutes.The sample of collecting identifies through 12%SDS-PAGE, and only finding has a band clearly at molecular weight about 31000.The result shows the fusion rotein that adopts this kind method can obtain highly purified GST-CTX M VII A easy, efficiently.

2.3 separate and purification CTX M VII A mutant polypeptide fragment

2.3.1 the work efficiency of assaying blood clotting enzyme Thrombin

The GST-CTX M VII A fusion rotein of 100 μ g is added different enzyme concentrations (1,2,3 and 4unit) at 22 ℃ of enzyme action 4,8,12,16,20 and 24 hours respectively down in the PBS buffer, sample carries out the SDS-PAGE electrophoretic analysis then and identifies that the thrombin work efficiency selects best enzyme action condition.The enzyme action result represents that adding thrombin with 100 μ g fusion rotein/1unit thrombin ratio (recommendation ratio) still has 26.2% fusion rotein not have digested at 22 ℃ of following enzyme action after 24 hours, SDS-PAGE the results are shown in Figure 5, wherein, 1: standard molecular weight marker, 2: without the GST-CTX M VII A fusion rotein of enzyme action, 3: 4 hours result of enzyme action, 4: 8 hours result of enzyme action, 5: 12 hours result of enzyme action, 6: 16 hours result of enzyme action, 7: 20 hours result of enzyme action, 8: 24 hours result of enzyme action.The small fragment of enzyme action gained has reached 15.7% at enzyme action in the time of 20 hours simultaneously, but has only 12.8% in the time of 24 hours, illustrates that the enzyme action condition may influence the stability of small fragment.

Add thrombin with 100 μ g fusion rotein/2unit thrombin ratio, there have 8.1% fusion rotein not have after 24 hours at 22 ℃ of following enzyme action to be digested, and SDS-PAGE the results are shown in Figure 6, wherein, 1: standard molecular weight marker, 2: without the GST-CTX M VII A fusion rotein of enzyme action, 3: 4 hours result of enzyme action, 4: 8 hours result of enzyme action, 5: 12 hours result of enzyme action, 6: 16 hours result of enzyme action, 7: 20 hours result of enzyme action, 8: 24 hours result of enzyme action.Wherein, small fragment enzyme action under this enzyme action condition of enzyme action gained reached maximum production in the time of 20 hours simultaneously, and 20.6%, pointing out this condition may be to reclaim the best enzyme action condition of enzyme action small fragment.

Add thrombin with 100 μ g fusion rotein/3unit thrombin ratio, at 22 ℃ of following enzyme action after 24 hours, have only 0.1% fusion rotein not have digested SDS-PAGE to the results are shown in Figure 7, wherein, 1: standard molecular weight marker, 2: without the GST-CTX M VII A fusion rotein of enzyme action, 3: 4 hours result of enzyme action, 4: 8 hours result of enzyme action, 5: 12 hours result of enzyme action, 6: 16 hours result of enzyme action, 7: 20 hours result of enzyme action, 8: 24 hours result of enzyme action.Show that fusion rotein can be digested complete with this understanding.But the output of enzyme action gained small fragment has been compared tangible reduction (enzyme action reaches maximum level 15.7% in the time of 24 hours) with gained output under preceding two kinds of enzyme action conditions, has illustrated that also this enzyme action condition may influence the stability of small fragment.

Add thrombin with 100 μ g fusion rotein/4unit thrombin ratio, at 22 ℃ of following enzyme action after 24 hours, have 1.1% fusion rotein not have digested, SDS-PAGE the results are shown in Figure 8, wherein, 1: standard molecular weight marker, 2: without the GST-CTX M VII A fusion rotein of enzyme action, 3: 4 hours result of enzyme action, 4: 8 hours result of enzyme action, 5: 12 hours result of enzyme action, 6: 16 hours result of enzyme action, 7: 20 hours result of enzyme action, 8: 24 hours result of enzyme action., and the output of enzyme action gained small fragment is 18.4% with this understanding), this enzyme action condition of also supporting to know clearly may influence the saying of the stability of small fragment.

Comprehensive above enzyme action dynamic experiment result, the enzyme action efficient (with not enzyme action fusion rotein cubage) of fusion rotein GST-CTX M VII A under conditions such as different enzyme concentrations, different enzyme working time is referring to Fig. 9, shows that this thrombin can make GST-CTX MVIIA fusion rotein enzyme action more than 90% in 16 hours at 22 ℃ of enzyme concentration enzyme action with 100 μ g fusion rotein/3unit thrombins under the PBS buffer system.

And small fragment CTX M VII A mutant obtains the maximum response rate 20.6% (with the cubage of small fragment) at 22 ℃ of enzyme concentration enzyme action with 100 μ g fusion rotein/2unit thrombins in the time of 20 hours, referring to Figure 10.

2.3.2 molecular sieve purification CTX M VII A mutant polypeptide fragment

With through the filterable deionized water of 22 μ m sterile filters with the flow velocity of 1ml/min with Sephacryl S-100HR prepacked column on AKTA Purifier protein purification instrument, to mixture behind the enzyme action separate, purification and desalination.According to the variation of numerical value such as OD215, OD280 and electric conductance, referring to Figure 11.At elution volume 30-50ml and 90-105ml place two eluting peaks are arranged, according to eluting peak go out peak position standard of comparison elution curve, probably near 25000-30000, be GST albumen at the eluting peak molecular weight at elution volume 30-50ml place; And at the eluting peak molecular weight at elution volume 90-105ml place in the 2000-3000 scope, be the CTX M VII A mutant of purification.

Embodiment two

The mass spectrum of the CTX M VII A mutant of gene recombinaton identifies that we collect the liquid lyophilizing with the CTX M VII A mutant of purification, delivers to Chinese Academy of Sciences Shanghai biochemical cell institute, does molecular weight identification by MALDI-TOF-MS.It is 2783 that result's proof is held the molecular weight of the CTX M VII A mutant of unnecessary amino acid residue Gly and Ser with two N-, conforms to theoretical value; The result is referring to Figure 12.

Embodiment three

The hot tail method of rat (Hot-tail flick) is used to measure the analgesic activities of CTX M VII A mutant.Healthy male Sprague-Dawley rat, body weight 250 ± 20g, 10% chloral hydrate is anaesthetized, and the tricorn pipe laying is injected penicillin every day, carries out pharmacological testing after raising a week.Regulate 53 ± 0.5 ℃ of waters bath with thermostatic control, administration survey half an hour pain territory.Normal mice pain territory is generally 2s, and pain territory 5s is analgesia fully.Select 30 of qualified animals, be divided into 6 groups at random,, experimental results show that by the hot tail method of rat the CTX M VII A mutant that obtains has very strong analgesic activity (seeing Figure 13) through the sleeve pipe administration.Among Figure 13: PBS is the normal saline group; Morphine (L) is low dosage morphine group (25 μ g/kg); Morphine (H) is high dose morphine group (250 μ g/kg); GST-CTX is a GST-CTX M VII A fusion rotein group (25 μ g/kg); CTX (L) is a low dosage CTX M VII A mutant group (2.5 μ g/kg); CTX (H) is a high dose CTX MVII A mutant group (25 μ g/kg).Measure the average pain threshold of different time mice after the administration respectively, calculate the percentage rate that improve the threshold of pain as follows:

Percentage rate=(the preceding average pain threshold of average pain threshold-medication after the medication)/preceding average pain threshold of medication is improved in the threshold of pain.The result is referring to Figure 13: the result shows that the analgesic effect of the analgesic effect of CTX M VII A mutant (2.5 μ g/Kg) and GST-CTX M VII A fusion rotein group (25 μ g/Kg) or morphine (250 μ g/Kg) is suitable.Consider the molecular weight (2783) of recombinant C TX M VII A mutant, the molecular weight (about 29000) of GST-CTX M VII A, the molecular weight of morphine (285), by mole analgesic effect relatively, the analgesia of this CTX M VII A mutant is renderd a service with the analgesia of GST-CTX M VII A fusion rotein and is renderd a service quite, but is eager to excel 800 times than morphine.

In sum, we use technique for gene engineering, have successfully expressed GST-CTX MVII A fusion rotein in escherichia coli, and behind the thrombin enzyme action, but purification obtains CTX M VII A mutant; This method can be produced novel CTX M VII A mutant, and preparation technology is simple, and every liter of fermentation liquid can obtain about 50mgCTX M VII A mutant at last, and production cost is lower; Zoopery proves: this recombinant C TX MVII A mutant has very high analgesic activities, and its analgesia effectiveness is 800 times of morphine, can be used for the exploitation of analgesic.

More than the description of better embodiment of the present invention is not limited the present invention, those skilled in the art can make various changes and distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to scope of the present invention.

The sequence that the present invention relates to

＜110〉Zhejiang University

＜120〉omega-conotoxin M VII A mutant and preparation and application

<160>1

<210>1

<211>27

<212>PRT

＜213〉artificial sequence

<220>

＜223〉according to the aminoacid sequence design, be used for analgesia

<400>1

Gly?Ser?Cys?Lys?Gly?Lys?Gly?Ala?Lys?Cys?Ser?Arg?Leu?Met

1 5 10

Tyr?Asp?Cys?Cys?Thr?Gly?Ser?Cys?Arg?Ser?Gly?Lys?Cys

15 20 25

(C3 and C18, C10 and C22, C17 and C27 form three pairs of disulfide bond respectively in this peptide molecule, and the C-end is non-amidated Cys)

<210>2

<211>81

<212>DNA

＜213〉artificial sequence

<220>

＜223〉codon according to E.coli preference designs, the structural gene sequence of the omega-conotoxin M VII A mutant that is used to encode

<400>1

ggatcctgca?aaggtaaagg?tgcgaaatgc?tctcgtctga?tgtacgactg?ctgcaccggt 60

tcttgccgtt?ctggtaaatg?c?81

Claims

1. omega-conotoxin M VII A mutant, it is characterized in that: SEQ ID NO:1 is the aminoacid sequence of described mutant, form three pairs of disulfide bond in this mutant molecule between Cys3 and Cys18, Cys10 and Cys22, Cys17 and the Cys27 respectively, the C-end is non-amidated Cys, SEQ ID NO1:

Gly-Ser-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-

Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys。

2. the preparation method of omega-conotoxin M VII A mutant according to claim 1 is characterized in that: realize by following steps:

(1) has in the e. coli bl21 of expression plasmid pGEX-2T/CTX M VII A in conversion, come the fusion rotein of abduction delivering glutathione S-transferase and omega-conotoxin M VIIA with isopropylthio-;

(2) method with the GST affinity chromatograph adopts Glutathione-Agarose affinity column purified fusion protein, 50mM Tris-HCl with reduced glutathion and pH 8.0 carries out eluting, obtains the fusion rotein of highly purified glutathione S-transferase and omega-conotoxin M VII A;

(3) carry out enzyme action with thrombin after the fusion rotein of glutathione S-transferase behind the purification and omega-conotoxin M VII A is dialysed in PBS solution, enzyme action afterproduct process Sephacryl S-100HR gel column separates, purification;

(4) the omega-conotoxin M VII A mutant that purification is obtained is opened the disulfide bond of omega-conotoxin M VII A mutant with 0.1% beta-mercaptoethanol earlier, reuse phosphate buffer dialysed overnight, air oxidation, thus correct disulfide bond pairing formed, obtain the purpose product.

3. the preparation method of omega-conotoxin M VIIA mutant according to claim 2, it is characterized in that: step (3) is described to obtain omega-conotoxin M VII A mutant with thrombin enzyme action glutathione S-transferase and omega-conotoxin M VIIA fusion rotein, the suitableeest endonuclease reaction condition is: in PBS solution, the thrombin that 100 μ g fusion rotein is added 2 units, 22 ℃ of enzyme action 20 hours obtain omega-conotoxin M VII A mutant.

4. the application of omega-conotoxin M VII A mutant according to claim 1 in the preparation analgesic.