CN101985478A - Fusion protein and application thereof to ischemic brain stroke - Google Patents
Fusion protein and application thereof to ischemic brain stroke Download PDFInfo
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Abstract
The invention relates to a glutathione S-transferase-tyrosin amino transferase-superoxide dimatase (GST-TAT-SOD) fusion protein, which is obtained by preparing fusion protein fermentation liquid and purifying the fermentation liquid, wherein the fusion protein can be used for treating ischemic brain stroke; the dosage range is 50 to 5,000SOD activity unit per kilogram of body weight; GST, TAT and SOD are linked by peptide bonds; any position which does not influence SOD activity obviously can be linked by the peptide bonds; and a medicament for treating the ischemic brain stroke can be injected or taken orally.
Description
Technical field
The invention belongs to biological field, more specifically relate to a kind of GST-TAT-SOD fusion rotein and the application in cerebral infarction thereof.
Background technology
Cerebral apoplexy is one of principal disease of the healthy and life of present serious harm the elderly, characteristics such as have sickness rate height, mortality ratio height, disability rate height, recurrence rate height, complication is many, curative ratio is low.On May 11st, 2008, on " first international cerebral apoplexy latest developments scientific seminar ", health ministry has announced that on April 29th, 2008, national for the third time resident's cause of death investigation result showed, cerebro-vascular diseases, malignant tumour are to occupy cause of death front two, account for 22.45% and 22.32% of dead sum respectively.China is one of district occurred frequently of cerebral apoplexy, along with the development of modern medicine, population is aging gradually, and the sickness rate of ischemic cerebrovascular disease raises year by year, lethality rate that it is high and disability rate cause serious threat for society and family, have become the hot subject of modern medicine study.
For the treatment of cerebral infarction, how to recover rapidly or the supply of blood flow that improves ischemic tissue of brain has become the center of gravity and the hot issue of treatment at present.In the regular hour, recover blood flow and supply with the brain cell that may save ischemic, but also increase the weight of cerebral lesion or causing bleeding property cerebral infarction simultaneously, cause reperfusion injury.
If relevant cerebral ischemia re-pouring injured mechanism has proposed at present in theory, as the free theory of oxygen, calcium overload theory, excitatory amino acid theory etc.The change of free radical (Free Radical) was recently paid close attention to by people more when wherein cerebral apoplexy took place, numerous studies show that, free radical occupies an important position in the pathogenesis of ischemic cerebrovascular, lipid peroxidation by free yl induction plays an important role in the pathologic, physiologic of many central nervous systems, and it may be one of cerebral infarction core pathology link.The oxyradical theory thinks that the mechanism of oxyradical (OFR) damage is to cause lipid peroxidation, and is because brain is the tissue that is rich in lipid, more more responsive to free radical than its hetero-organization.Because ATP reduces, cause the cytolemma pumping function malfunctioning, Ca during cerebral ischemia
2+Through going into the proteolytic ferment that cell-stimulating Ca relies on, make xanthine dehydrogenase (XD) become XOD (XO) in a large number, owing to releasing energy, decomposes ATP simultaneously, and ADP, AMP content are raise, and decompose a large amount of xanthoglobulin of generation successively.When perfusion, a large amount of molecular oxygens enter ischemic tissue of brain with blood, and XO produces a large amount of O when the catalysis xanthoglobulin is converted into xanthine and uric acid
2 -And H
2O
2, and H
2O
2In the presence of metal ion, form OH-, cause the explosive increase of OFR.
Though also do not have the cerebral ischemia re-pouring injured medicine of ideal treatment at present, the antioxidant of clinical application and free-radical scavengers can be prevented and treated reperfusion injury.Be used to remove free radical at present clinically and lipid peroxidation inhibitor has VitE, VitC, GSH, MLT, rt-PA, Edaravone, SOD etc., test finds that they all can alleviate ischemic, particularly the brain injury after the perfusion again.(Superoxide dimatase is a kind of important cell defendance enzyme in the organism SOD) to superoxide dismutase, can remove the ultra-oxygen anion free radical in the human body, and the protective inner lipid is avoided the attack of free radical.But because SOD molecular weight big (32000 dalton) is difficult to break through smoothly hemato encephalic barrier and permeate through cell membranes, arrive the main source region of body inner cell oxyradical---tenuigenin and plastosome, thereby hindered exogenous biologically active substance and in cell, brought into play therapeutic action.Give SOD merely, oxyradical that can only the scavenger cell outside, and can not effectively eliminate the oxyradical of cell interior, make SOD still can not produce a desired effect at the treatment cerebral infarction.(protein transduction domain, PTD) Jie Dao protein transduction The Application of Technology has solved this difficult problem along with protein transduction domain.PTD can transduce self and enter nearly all tissue and cell with polypeptide, albumen and the dna molecular of its fusion, even blood-CSF barrier, transduction efficiency height and pair cell do not have obvious impairment.The membrane property of wearing of PTD provides effective methods of treatment for the intervention of nerve cell apoptosis in the cerebral apoplexy and nerve degenerative diseases.TAT be among the PTD one section have the small peptide of efficiently striding membrane interaction, constitute by 11 amino acid.Glutathione S-transferase (GST) can the catalysis nucleophilicity gsh and the association reaction of various electrophilic external source chemicals.Many external source chemicals very easily form for example free radical of some biological activity intermediate product in the bio-transformation first-phase reaction, they can with cell biological macromole important component generation covalent attachment, body is caused damage.Gsh can prevent this kind covalent attachment with after it combines, and plays detoxification.TAT stride membrane interaction and GST katalysis and SOD remove the free radical function combined utilization provide a new application approach for bioactive macromolecule treatment cerebral infarction.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of GST-TAT-SOD fusion rotein, and this fusion rotein is used for the treatment of cerebral infarction.This preparation method's simple possible, and have good pharmacological activity.
A kind of GST-TAT-SOD fusion rotein preparation method's concrete steps are: fusion rotein preparation of fermentation liquid and purifying:
1) preparation of fusion rotein GST-TAT-SOD fermentation broth sample:
The inoculation that contains the GST-TAT-SOD gene that builds is contained in the LB substratum of 30-70ug/mL Amp in 50-150mL, and 37 ℃ are shaken training and spend the night; Bacterium liquid is inoculated in the fresh LB substratum of 500-1500mL by the 1-3%v/v inoculum size, and this substratum contains 30-80ug/mL Amp, and 37 ℃ are shaken when training OD600=0.6-1.0, add CuSO respectively
4200-800uM, ZnSO
450-150uM and IPTG are 125-500uM to final concentration, 22-28 ℃ of jolting inducing culture 5-7h; Collect bacterium liquid, the centrifugal 10-25min of 5000rpm collects thalline, and is resuspended with 100mLPBS, ultrasonication 30min in the ice bath, and the centrifugal 10min of 12000rpm, the fermented liquid of supernatant liquor behind the 0.22um membrane filtration, standby;
2) fusion rotein GST-TAT-SOD purification process is an affinity chromatography
A. fermented liquid ultrasonication 12000rpm discards precipitation behind the centrifugal 20min, and supernatant liquor is got in repetitive operation three times;
B. diluent is added in the GST fusion rotein affinity chromatographic column of using the PBS pre-equilibration, stirs gently; Make the abundant mixing of affinity matrix and fusion rotein, chromatographic column is positioned in the ice bath, natural subsidence;
C. open constant flow pump, drain the liquid of chromatographic column;
D. add pH7.4 PBS, ten column volumes of balance are drained the liquid of chromatographic column;
E. the 0.01mol/L gsh and the 0.02mol/L Tris-HCl solution that add one times of column volume, pH=8.0 stirs gently and makes solution and the abundant mixing of glue, and chromatographic column is positioned in the ice bath, and natural subsidence is opened constant flow pump and is received elutriant;
F. repeating step E is twice;
G. mix the Affi-Gel elutriant three times ,-20 ℃ of preservations are standby.
The GST-TAT-SOD fusion rotein that makes can be applicable to treat cerebral infarction, and the dosage range of its use is 50～5000 SOD unit of activity/Kg body weight, and this protein vigor detection method is an xanthine oxidase; GST, TAT are connected with peptide bond with SOD three, and the peptide bond connection occurs in the active position of any not obvious SOD of influence.This fusion rotein be used for the treatment of the cerebral infarction product to give with mode be drug administration by injection and oral administration.
The invention has the advantages that: by GST, the fusion of TAT and SOD solves SOD and breaks through brain blood barrier and stride the problem that film enters cell interior.These means adopt intracellular natural mechanism; effectively avoid the infringement of allogenic material to target cell; and this method can be sent the SOD molecule in a large number and be entered cell; the chain reaction of blocking-up oxyradical; help to protect cell to avoid oxidative damage, and the symptom of cerebral apoplexy is alleviated and even recover.TAT-SOD itself also can stride film and enter in the cell, make the preparation method easy on the one hand after adding GST, only need just can obtain GST-TAT-SOD through affinity chromatography, GST itself also has the effect that suppresses free radical on the other hand, combined utilization with TAT, GST and SOD, solved SOD and entered the difficult problem that plays a role in the cell, the adding of GST has simultaneously also reduced the detrimental effect of radical pair cell.
The contrast experiment of GST-TAT-SOD and TAT-SOD
Cell model:, set up the cell hypoxia model with 7.5mmol/L V-Brite B treatment S H-SY5Y (human neuroblastoma cell) 2 hours.
One. GST-TAT-SOD and TAT-SOD are to the prophylactic effect of anoxia model cell injury
1. the prevention provide protection of different concns GST-TAT-SOD and TAT-SOD pair cell damage:
Experimental technique: before setting up the cell hypoxia model, the GST-TAT-SOD and the TAT-SOD(concentration of different concns is respectively: 125U/ml, 250 U/ml, 500 U/ml, 1000 U/ml, 2000 U/ml) places 37 ℃, 5%CO with the SH-SY5Y cell
2Hatched altogether in the incubator 2 hours, 2 hours the sample with in the culture plate is poured out, clean 3 times with PBS, change the processing of 7.5mmol/L V-Brite B into and set up the cell hypoxia model in 2 hours, pour out the V-Brite B treatment solution after 2 hours and change serum-free medium continuation cultivation 24 hours, observation of cell survival condition into.
Experimental result: two kinds of equal pair cell anoxia-induced apoptosis of albumen have certain prophylactic effect.Wherein concentration is that the preventive effect of GST-TAT-SOD of 250 U/ml and 500 U/ml is more effective than the protection of the TAT-SOD under the same concentrations.With respect to the survival rate of model group, the optimum concn survival rate of GST-TAT-SOD has improved 15.2%, and the optimum concn survival rate of TAT-SOD has improved 11.6%, sees Fig. 1.
2. the provide protection of different incubation time pair cell damages:
Experimental technique: before setting up the cell hypoxia model, respectively the GST-TAT-SOD of 500 U/ml and TAT-SOD and SH-SY5Y cell are placed 37 ℃, 5%CO
2Hatch altogether in the incubator after 0.5,1,1.5,2,2.5,3,3.5 hour and remove sample, clean 3 times with PBS, change the processing of 7.5mmol/L V-Brite B into and set up the cell hypoxia model in 2 hours, pour out the V-Brite B treatment solution after 2 hours and change serum-free medium continuation cultivation 24 hours, observation of cell survival condition into.
Experimental result: along with the prolongation of incubation time, the preventive effect of GST-TAT-SOD is better than the preventive effect of the TAT-SOD under the identical incubation time, sees Fig. 2.
Two,GST-TAT-SOD and TAT-SOD are to the repair of anoxia model cell injury
1. the repair of different concns GTS/TS pair cell damage:
Experimental technique: the 7.5mmol/L V-Brite B is handled and was set up the cell hypoxia model in 2 hours, pour out the V-Brite B treatment solution after 2 hours, clean 3 times with PBS, GST-TAT-SOD and TAT-SOD(concentration with different concns is respectively respectively: 125U/ml, 250 U/ml, 500 U/ml, 1000 U/ml, 2000 U/ml) place 37 ℃, 5%CO with the SH-SY5Y cell
2Hatch altogether in the incubator change serum-free medium after 2 hours into and continue to cultivate 24 after, the observation of cell survival condition.
Experimental result: two kinds of equal pair cell anoxia-induced apoptosis of albumen have certain repair.Wherein concentration is that the repairing effect of GST-TAT-SOD of 250 U/ml and 500 U/ml is better than the repairing effect of the TAT-SOD under the same concentrations.Concentration is that the repairing effect of TAT-SOD of 2000 U/ml is better than the repairing effect of GST-TAT-SOD, sees Fig. 3.
2. the repair of different incubation time pair cell damages:
Experimental technique: the 7.5mmol/L V-Brite B is handled and was set up the cell hypoxia model in 2 hours, pour out the V-Brite B treatment solution after 2 hours, clean 3 times with PBS, respectively with GST-TAT-SOD and the TAT-SOD and 37 ℃ of SH-SY5Y cell placements, 5%CO of 500 U/ml
2Hatch altogether in the incubator change serum-free medium after 2 hours into and continue to cultivate 24 after, the observation of cell survival condition.
Experimental result: two kinds of equal pair cell anoxia-induced apoptosis of albumen have certain repair and repairing effect suitable, see Fig. 4.
Three, GST-TAT-SOD and TAT-SOD pair cell damage prevention is compared with repairing effect
From four groups of above data analyses as can be known: from prevention and the effect of repairing; two kinds of proteic repairs are better than its prevention provide protection; compare with model group; the best repair activity survival rate of GST-TAT-SOD and TAT-SOD has improved 42.0% and 43.9% respectively, and best prophylactic effect concentration survival rate only is 15.2% and 11.6%.With regard to preventive effect, the preventive effect of GST-TAT-SOD has certain advantage, and with regard to repairing effect, both effects are suitable.See Fig. 5
The test of GST-TAT-SOD fusion rotein using dosage
1. experimental technique:
Select 30 of healthy kunming mices for use, experiment one week of preadaptation.Mouse is divided into blank group (n=10) at random by body weight, does not take any measure; Drug administration by injection group (n=10), dosage are 2000 unit of activity/Kg body weight; Gastric infusion group (n=10), dosage are 25000 unit of activity/Kg body weight.To execution in 4 hours behind mouse enforcement drug administration by injection and the gastric infusion, low temperature is got brain immediately, makes 10% tissue homogenate, measures SOD content in the brain.
2. experimental result:
SOD content in mouse administrated by injection and gastric infusion GST-TAT-SOD4 hour hindbrain has increased by 20.10% and 21.76% respectively than blank group SOD content; Statistical analysis shows that there is significance (P＜0.05) in these data.
3. experiment conclusion: this experiment shows, no matter normal mouse is to adopt drug administration by injection or the mode of gastric infusion GST-TAT-SOD all can effectively increase the content of SOD in the brain, and ischemic causes in brain intracerebral oxidation level is unbalance to provide effective experimental basis for GST-TAT-SOD can repair for this.This experimental result is also pointed out and is taked the required dosage of drug administration by injection only to need 8.66% of oral administration dosage just can reach the effect of the raising SOD level identical with oral administration.
Experiment 2:
1. experimental technique:
Select 50 of healthy male mouse SD rats for use, experiment one week of preadaptation.Rat is divided into model group (n=10) at random by body weight; GST-TAT-SOD high dosage administration group (oral 25000 unit of activity/Kg body weight, n=10); Dosed administration group among the GST-TAT-SOD (oral 12500 unit of activity/Kg body weight, n=10); GST-TAT-SOD low dosage administration group (oral 6250 unit of activity/Kg body weight, n=10); Sham operated rats (n=10), totally 5 groups.Rat is implemented operation, set up cerebral ischemia re-pouring model (MCAO).Its neuroethology feature is observed in the clear-headed back of rat operation.Ischemic poured into after 24 hours in 1 hour again puts to death rat and gets brain, calculates the rat cerebral infarction area, and brain tissue slice HE dyeing is measured GSH-PX in the brain tissue homogenate, SOD, CAT, MDA, NO content.
2. result:
(1) neuroethology observation of characteristics: the result shows that after the rat operation was clear-headed, operation branch hole ball appears in the model group rat to cave in, and non-operation branch hole ball is outstanding slightly; Carry tail when unsettled, fore paw elongation in right side is better than the left side, occurs knocking into the back and levies phenomenon, and body rotates to the left during motion.Compare with model group, each dosage group neurologic impairment is light, and there is significant difference (P＜0.05) in high dose group, and there is utmost point significant difference (P＜0.01) in middle low dose group, and the neurologic impairment of middle low dose group is compared little with high dose group; Sham operated rats animal impassivity functional impairment.
(2) brain infarction area is measured: behind the ischemia-reperfusion, sham operated rats is not seen the infarct kitchen range; Model group brain sheet is made coronal section can see bigger infarct kitchen range; Each dosage group infarct size of GST-TAT-SOD and the apparent in view minimizing of model group (P＜0.01).
(3) oral administration gavage GST-TAT-SOD is to GSH-PX in the ischemia-reperfusion big rat brain tissue homogenate, SOD, and CAT, MDA, the influence of NO content: the result shows that the SOD of model group, GSH-PX, CAT level are compared with sham operated rats has utmost point significant difference; The SOD of senior middle school's low dose group, GSH-PX, CAT level are apparently higher than model group; The MDA of each dosage group, NO level are starkly lower than model group; The GSH-PX of high dose group, CAT level will be lower than middle low dose group, and MDA, NO level will be higher than middle low dose group.
(4) brain tissue slice HE dyeing is observed: the cellular form of sham operated rats is normal, and kernel is clear, and neurocyte and capillary vessel peripheral clearance are normal, and a matter does not have necrosis and oedema phenomenon (Figure 10 E); The right side cerebrum ischemia scope inner cortex oedema of model group is obvious, and neurocyte periphery crack enlarges, perivascular canal broadening, and at cortex and the visible mass mortality neurone of striatum, interstitial edema is (Figure 10 A) obviously; The neurocyte and the capillary vessel peripheral clearance of GST-TAT-SOD administration group are big slightly, but are significantly less than model group, and cortex and interstitial edema also are lighter than model group; Wherein the neurocyte of high dosage administration group and capillary vessel peripheral clearance is greater than middle low dosage administration group, cortex and interstitial edema also greater than in low administration group, see Figure 10.
3. experiment conclusion: this experimental study shows that the recovery of the neural function of each dosage group rat of oral administration gavage GST-TAT-SOD will obviously be better than model group, and the infarct size of brain also is less than model group.See that from the result of HE dyeing electron microscopic observation each GST-TAT-SOD dosage group brain inner cell degree of injury also is less than model group.From intravital oxidation index, SOD, GSH-PX, CAT content in each dosage group body brain all increase than model group and normal group, and MDA, NO level fall after rise to some extent, this points out ectogenic GST-TAT-SOD can improve the interior antioxidant levels of cerebral ischemia re-pouring rat brain, reduce the generation of brain inner lipid oxide compound, effectively improve brain inner cell degree of injury, recover neural function.Find also in experiment that simultaneously during the antioxidant levels of high dose group will be lower than, low dose group, the GST-TAT-SOD of this prompting high dosage might suppress the activity of GSH-PX, CAT, middle low dosage GST-TAT-SOD is more effective in prevention ischemia brain injury.
Metabolic rate according to rat is 6 times of conversions of human body, and this experimental result prompting human body oral administration dosage range is 1042-4167 unit of activity/Kg body weight.Drug administration by injection required dosage according to experiment 1 has only 8.66% result of oral administration dosage to convert, and the drug administration by injection dosage range is 90-360 unit of activity/Kg body weight.Comprehensively oral and drug administration by injection situation determines that finally the human body dosage scope of GST-TAT-SOD is 50-5000 unit of activity/Kg body weight.
Description of drawings
The prevention provide protection of Fig. 1 different concns GST-TAT-SOD and the damage of TAT-SOD pair cell;
The provide protection of different incubation time GST-TAT-SOD of Fig. 2 and the damage of TAT-SOD pair cell;
The repair of Fig. 3 different concns GST-TAT-SOD and the damage of TAT-SOD pair cell;
The repair of different incubation time GST-TAT-SOD of Fig. 4 and the damage of TAT-SOD pair cell;
Fig. 5 GST-TAT-SOD and TAT-SOD pair cell damage prevention are compared with repairing effect.
Fig. 6 fusion rotein GST-TAT-SOD purification result;
Fig. 7 oral administration gavage GST-TAT-SOD compares with model group to the influence of ischemia-reperfusion rat neural function,
﹡P＜0.05,
﹡ ﹡P＜0.01;
Fig. 8 oral administration gavage GST-TAT-SOD compares with model group to the influence of ischemia-reperfusion rat cerebral infarction area,
﹡ ﹡P＜0.01;
Fig. 9 oral administration gavage GST-TAT-SOD compares with model group to the influence of MCAO rat cerebral tissue biochemical indicator,
﹡P＜0.05,
﹡ ﹡P＜0.01; Compare with sham operated rats,
△P＜0.05,
△ △P＜0.01;
The brain HE dyeing (* 640) of each dosage group rat of Figure 10 oral administration gavage GST-TAT-SOD, wherein A is a model group, and B is a high dose group, and C is middle dosage group, and D is a low dose group, E is a sham operated rats.
Embodiment
A kind of GST-TAT-SOD fusion rotein preparation method's concrete steps are: fusion rotein preparation of fermentation liquid and purifying:
1) preparation of fusion rotein GST-TAT-SOD fermentation broth sample:
The inoculation that contains the GST-TAT-SOD gene that builds is contained in the LB substratum of 30-70ug/mL Amp in 50-150mL, and 37 ℃ are shaken training and spend the night; Bacterium liquid still is the quality percentage amounts by 1-3%(volume percentage amounts) inoculum size is inoculated in the fresh LB substratum of 500-1500mL, and this substratum contains 30-80ug/mL Amp, and 37 ℃ are shaken and train OD
600During=0.6-0.8, add CuSO respectively
4500uM, ZnSO
4100uM and IPTG are 250uM to final concentration, 22-28 ℃ of jolting inducing culture 5-7h; Collect bacterium liquid, the centrifugal 10-25min of 5000rpm collects thalline, and is resuspended with 100mLPBS, ultrasonication 30min in the ice bath, and the centrifugal 10min of 12000rpm, the fermented liquid of supernatant liquor behind the 0.22um membrane filtration, standby;
2) fusion rotein GST-TAT-SOD purification process is an affinity chromatography
A. fermented liquid ultrasonication 12000rpm discards precipitation behind the centrifugal 20min, and supernatant liquor is got in repetitive operation three times;
B. diluent is added in the GST fusion rotein affinity chromatographic column of using the PBS pre-equilibration, stirs gently; Make the abundant mixing of affinity matrix and fusion rotein, chromatographic column is positioned in the ice bath, natural subsidence;
C. open constant flow pump, drain the liquid of chromatographic column;
D. add pH7.4 PBS, ten column volumes of balance are drained the liquid of chromatographic column;
E. the 0.01mol/L gsh and the 0.02mol/L Tris-HCl solution that add one times of column volume, pH=8.0 stirs gently and makes solution and the abundant mixing of glue, and chromatographic column is positioned in the ice bath, and natural subsidence is opened constant flow pump and is received elutriant;
F. repeating step E is twice;
G. mix the Affi-Gel elutriant three times ,-20 ℃ of preservations are standby.
A kind of GST-TAT-SOD fusion rotein preparation method's concrete steps are: fusion rotein preparation of fermentation liquid and purifying:
1) preparation of fusion rotein GST-TAT-SOD fermentation broth sample:
The inoculation that contains the GST-TAT-SOD gene that builds is contained in the LB substratum of 30ug/mL Amp in 50mL, and 37 ℃ are shaken training and spend the night; Bacterium liquid still is the quality percentage amounts by 1%(volume percentage amounts) inoculum size is inoculated in the fresh LB substratum of 500mL, and this substratum contains 30ug/mL Amp, and 37 ℃ are shaken and train OD
600, add CuSO respectively at=0.6 o'clock
4200uM, ZnSO
450uM and IPTG are 125uM to final concentration, 22 ℃ of jolting inducing culture 7h; Collect bacterium liquid, the centrifugal 10min of 5000rpm collects thalline, and is resuspended with 100mLPBS, ultrasonication 30min in the ice bath, and the centrifugal 10min of 12000rpm, the fermented liquid of supernatant liquor behind the 0.22um membrane filtration, standby; The same specific embodiment of all the other steps.
A kind of GST-TAT-SOD fusion rotein preparation method's concrete steps are: fusion rotein preparation of fermentation liquid and purifying:
1) preparation of fusion rotein GST-TAT-SOD fermentation broth sample:
The inoculation that contains the GST-TAT-SOD gene that builds is contained in the LB substratum of 70ug/mL Amp in 150mL, and 37 ℃ are shaken training and spend the night; Bacterium liquid still is the quality percentage amounts by 3%(volume percentage amounts) inoculum size is inoculated in the fresh LB substratum of 500mL, and this substratum contains 80ug/mL Amp, and 37 ℃ are shaken and train OD
600, add CuSO respectively at=1.0 o'clock
4800uM, ZnSO
4150uM and IPTG are 500uM to final concentration, 28 ℃ of jolting inducing culture 5h; Collect bacterium liquid, the centrifugal 25min of 5000rpm collects thalline, and is resuspended with 100mLPBS, ultrasonication 30min in the ice bath, and the centrifugal 10min of 12000rpm, the fermented liquid of supernatant liquor behind the 0.22um membrane filtration, standby; The same specific embodiment of all the other steps.
A kind of GST-TAT-SOD fusion rotein preparation method's concrete steps are: fusion rotein preparation of fermentation liquid and purifying:
1) preparation of fusion rotein GST-TAT-SOD fermentation broth sample:
The inoculation that contains the GST-TAT-SOD gene that builds is contained in the LB substratum of 65ug/mL Amp in 100mL, and 37 ℃ are shaken training and spend the night; Bacterium liquid still is the quality percentage amounts by 2%(volume percentage amounts) inoculum size is inoculated in the fresh LB substratum of 1000mL, and this substratum contains 50ug/mL Amp, and 37 ℃ are shaken and train OD
600, add CuSO respectively at=0.8 o'clock
4500uM, ZnSO
4100uM and IPTG are 250uM to final concentration, 25 ℃ of jolting inducing culture 6h; Collect bacterium liquid, the centrifugal 20min of 5000rpm collects thalline, and is resuspended with 100mLPBS, ultrasonication 30min in the ice bath, and the centrifugal 10min of 12000rpm, the fermented liquid of supernatant liquor behind the 0.22um membrane filtration, standby; The same specific embodiment of all the other steps.
The above only is preferred embodiment of the present invention, and all equalizations of being done according to the present patent application claim change and modify, and all should belong to covering scope of the present invention.
Claims (4)
1. fusion rotein, it is characterized in that: this albumen is a kind of GST-TAT-SOD fusion rotein, and this proteic preparation method's concrete steps are: fusion rotein preparation of fermentation liquid and purifying:
1) preparation of fusion rotein GST-TAT-SOD fermentation broth sample:
The inoculation that contains the GST-TAT-SOD gene that builds is contained in the LB substratum of 30-70ug/mL Amp in 50-150mL, and 37 ℃ are shaken training and spend the night; Bacterium liquid is inoculated in the fresh LB substratum of 500-1500mL by the 1-3%v/v inoculum size, and this substratum contains 30-80ug/mL Amp, and 37 ℃ are shaken and train OD
600During=0.6-1.0, add CuSO respectively
4200-800uM, ZnSO
450-150uM and IPTG are 125-500uM to final concentration, 22-28 ℃ of jolting inducing culture 5-7h; Collect bacterium liquid, the centrifugal 10-25min of 5000rpm collects thalline, and is resuspended with 100mLPBS, ultrasonication 30min in the ice bath, and the centrifugal 10min of 12000rpm, the fermented liquid of supernatant liquor behind the 0.22um membrane filtration, standby
2) fusion rotein GST-TAT-SOD purification process: affinity chromatography
A. fermented liquid ultrasonication 12000rpm discards precipitation behind the centrifugal 20min, and supernatant liquor is got in repetitive operation three times;
B. supernatant liquor is added in the GST fusion rotein affinity chromatographic column with the PBS pre-equilibration, stirs gently, make the abundant mixing of affinity matrix and fusion rotein, chromatographic column is positioned in the ice bath, natural subsidence;
C. open constant flow pump, drain the liquid of chromatographic column;
D. add pH7.4 PBS, ten column volumes of balance are drained the liquid of chromatographic column;
E. the 0.01mol/L gsh and the 0.02mol/L Tris-HCl solution that add one times of column volume, pH=8.0 stirs gently and makes solution and the abundant mixing of glue, and chromatographic column is positioned in the ice bath, and natural subsidence is opened constant flow pump and is received elutriant;
F. repeating step E is twice;
G. mix the Affi-Gel elutriant three times ,-20 ℃ of preservations are standby.
2. the application of a fusion rotein, it is characterized in that: this albumen is a kind of GST-TAT-SOD fusion rotein, described GST-TAT-SOD fusion rotein can be applicable to treat cerebral infarction, and the dosage range of its use is 50～5000 SOD unit of activity/Kg body weight.
3. the application of GST-TAT-SOD fusion rotein according to claim 1 in the treatment cerebral infarction, it is characterized in that: GST, TAT are connected with peptide bond with SOD three, and the peptide bond connection occurs in the active position of any not obvious SOD of influence.
4. the application of GST-TAT-SOD fusion rotein according to claim 3 is characterized in that: described treatment cerebral infarction product give with mode be inject, oral.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113243338A (en) * | 2021-05-14 | 2021-08-13 | 福州大学 | Construction and evaluation method of mouse ischemic stroke model |
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| CN1712415A (en) * | 2005-05-20 | 2005-12-28 | 福州大学 | Connexin transduction structural domain and bioactive protein of auxiliary protein, its production and use |
| CN101143901A (en) * | 2007-08-30 | 2008-03-19 | 福州大学 | Application of fusion protein GST-PTD-SOD in radioresistance |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712415A (en) * | 2005-05-20 | 2005-12-28 | 福州大学 | Connexin transduction structural domain and bioactive protein of auxiliary protein, its production and use |
| CN101143901A (en) * | 2007-08-30 | 2008-03-19 | 福州大学 | Application of fusion protein GST-PTD-SOD in radioresistance |
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| Title |
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| 王春娟 等，: "缺血性脑卒中与NO、SOD关系研究进展", 《脑与神经疾病杂志》, vol. 15, no. 5, 31 December 2007 (2007-12-31) * |
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| CN113243338A (en) * | 2021-05-14 | 2021-08-13 | 福州大学 | Construction and evaluation method of mouse ischemic stroke model |
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