CN108893454A - Polyethyleneglycol modified recombination glutathione peroxidase GPx1 mutant, preparation method and anti-oxidant application - Google Patents

Polyethyleneglycol modified recombination glutathione peroxidase GPx1 mutant, preparation method and anti-oxidant application Download PDF

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CN108893454A
CN108893454A CN201810823075.8A CN201810823075A CN108893454A CN 108893454 A CN108893454 A CN 108893454A CN 201810823075 A CN201810823075 A CN 201810823075A CN 108893454 A CN108893454 A CN 108893454A
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魏景艳
张广远
宋健
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Jilin University
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Abstract

A kind of polyethyleneglycol modified recombination glutathione peroxidase GPx1 mutant, preparation method and recombination glutathione peroxidase GPx1 mutant, in the application of anti-oxidant aspect, belong to field of biotechnology with polyethyleneglycol modified front and back.It is the straight-chain mono methoxy polyethylene glycol succinimidyl butanoate using activation as dressing agent, 10~60min is reacted under the conditions of 4~37 DEG C, pH=6.0~9.0 with recombination glutathione peroxidase GPx1 mutant, terminator glycine is added and terminates reaction, dialysis removal by-product, obtains mutant；Wherein, the dosage mass ratio for recombinating the straight-chain mono methoxy polyethylene glycol succinimidyl butanoate of glutathione peroxidase GPx1 mutant and activation is 1：10~120.Its amido modified rate reaches as high as 98.73% after measured, and when amido modified rate is up to 40% or more, into the antigen-antibody reaction that foreign protein is just completely eliminated after in vivo, therefore product of the present invention has the characteristic of elimination zymoprotein antigen-antibody reaction.

Description

Polyethyleneglycol modified recombination glutathione peroxidase GPx1 mutant, preparation Method and anti-oxidant application
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of polyethyleneglycol modified recombination glutathione peroxidase Enzyme GPx1 mutant, preparation method and recombination glutathione peroxidase GPx1 mutant are with polyethyleneglycol modified front and back anti- Aoxidize the application of aspect.
Background technique
Glutathione is the substrate of the glutathione peroxidase GPx1 containing selenium, and selenocysteine SeCys is that it is urged Change group.GPx with superoxide dismutase SOD, H2O2 enzyme CAT be present in living organism tissue three kinds it is important anti-oxidant Enzyme.GPx1 is important one of the hypotype of GPx containing selenium, is played a significant role in antioxidant system, can remove internal H2O2 and All kinds of hydroperoxides, blocking activity oxygen ROS prevent lipid peroxidation, and treatment aging as caused by active oxygen, is swollen at cataract Tumor and cardio-cerebrovascular diseases.GPx1 can not only remove ROS, can be using glutathione GSH as reducing agent degradation lipid peroxidation Object makes cell membrane and other body biological tissues from the damage of peroxidation, and this unique protective effect makes it anti- Play a significant role in oxidation enzyme system, is a kind of pharmaceutical enzyme with extensive market application prospect.But the enzyme is in room temperature item Its sulfydryl is oxidizable under part causes enzyme activity reduction, half-life short and stability relatively low, to limit its application.To this Enzyme is chemically modified, in its enzyme activity of holding while to improve its biological stability.
Chinese patent 201110122788.X discloses a kind of modified glutathione peroxidase and preparation method thereof, The preparation method provides a kind of method of modified pig glutathione peroxidase, does not include recombination glutathione peroxidating The mono methoxy polyethylene glycol (mPEG) of object enzyme GPx1 mutant and its GPx1 analogue enztme changes method of modifying.The preparation method is Single-stranded mono methoxy polyethylene glycol is reacted by the amino of dissociate chlorine atom and glutathione peroxidase, and amino Modification rate is 40~45%, and under the conditions of pH=10.0,1.5 days enzyme activities are 68.79%.The preparation method of this patent uses The amino of mono methoxy polyethylene glycol succinimidyl butanoate and glutathione peroxidase reacts, and amido modified Rate is 98.73%, and under the conditions of pH=10.0, enzyme activity is 77.93% after 15 days, is significantly higher than publication 201110122788.X。
Summary of the invention
The first purpose of this invention is to provide a kind of polyethyleneglycol modified recombination glutathione mistake that stability is strong Oxide enzyme GPx1 mutant (mPEG-GPx1).
A second object of the present invention is to provide above-mentioned polyethyleneglycol modified recombination glutathione peroxidase GPx1 The preparation method of mutant (mPEG-GPx1).
Third object of the present invention is to provide the poly- second two of above-mentioned recombination glutathione peroxidase GPx1 mutant Application of the alcohol modification front and back at anti-oxidant aspect.Before the antioxidation of GPx1 is better than modification after mPEG modification, it is specifically shown in this hair (6), (7), (8) and embodiment (5)~(10) of bright content beneficial effect, also see Figure 10~Figure 15.
The recombination glutathione peroxidase GPx1 mutant of mono methoxy polyethylene glycol modification disclosed by the invention, by The straight-chain mono methoxy polyethylene glycol succinyl of the amino and activation that recombinate glutathione peroxidase GPx1 mutant is sub- The connection of amine ester, structural formula are as follows：
Wherein GPx1 indicates recombination glutathione peroxidase GPx1 mutant, the recombination glutathione peroxidase GPx1 mutant is that (referring to Chinese patent 201310302778.3) is made through DNA recombinant expression, and mPEG indicates mono methoxy Polyethylene glycol.
It is a further object to provide the recombination glutathione of above-mentioned mono methoxy polyethylene glycol (mPEG) modification The preparation method of peroxidase GPx1 mutant (mPEG-GPx1) is the straight-chain mono methoxy polyethylene glycol using activation Succinimidyl butanoate is mutated precursor reactant as dressing agent, with recombination glutathione peroxidase GPx1, and reaction equation is：
Wherein GPx1 indicates recombination glutathione peroxidase GPx1 mutant.
The specific steps are：Using the straight-chain mono methoxy polyethylene glycol succinimidyl butanoate of activation as modification Agent, 10 are reacted under the conditions of 4~37 DEG C, pH=6.0~9.0 with recombination glutathione peroxidase GPx1 mutant~ 60min is added terminator glycine and terminates reaction, and dialysis removal by-product obtains mono methoxy polyethylene glycol (mPEG) modification Recombination glutathione peroxidase GPx1 mutant；Wherein, glutathione peroxidase GPx1 mutant and work are recombinated The dosage mass ratio of the straight-chain mono methoxy polyethylene glycol succinimidyl butanoate of change is 1：10~120；
Preferably：Recombinate the straight-chain mono methoxy polyethylene glycol of glutathione peroxidase GPx1 mutant and activation The dosage mass ratio of succinimidyl butanoate is 1：60；Reaction condition is 20 DEG C, reacts 30min under the conditions of pH=8.5.
The straight-chain mono methoxy polyethylene glycol succinimidyl butanoate wherein activated, is prepared by following steps It arrives：
(1) mono methoxy polyethylene glycol (mPEG) 1.0~3.0g of molecular weight 2000~12000 is weighed, 2~6mL is added N,N-Dimethylformamide sufficiently dissolves；
(2) 0.06~0.18g succinic anhydride is added into step (1) solution, mixes, sealing；
(3) step (2) mixture is reacted 2~4 hours under the conditions of 90~100 DEG C；
(4) step (3) reaction solution is cooled to room temperature, 0.233~0.699g n-hydroxysuccinimide, 0.413 is added ~1.238g 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride, heating make it dissolve；
(5) step (4) mixture is reacted 8~12 hours under the conditions of 25~30 DEG C, is cooled to room temperature；
(6) reaction solution after step (5) cooling is added dropwise 20~60mL ether, gained is sunk under the conditions of 0~8 DEG C It forms sediment and filters, vacuum drying, then desciccate is dissolved with methylene chloride, then precipitated again with ether；Repetition precipitated gained Filter, vacuum drying, desciccate are dissolved with methylene chloride, are used ether precipitation process 3~5 times again；
(7) step (6) products therefrom is dried in vacuo to get the straight-chain mono methoxy polyethylene glycol succinyl of activation is arrived Imines butyrate.
The present invention uses recombination glutathione peroxidase GPx1 mutant for objective for implementation, has compared with prior art There is following beneficial effect：
(1) present invention is connected to weight using the straight-chain mono methoxy polyethylene glycol succinimidyl butanoate of activation for the first time On group glutathione peroxidase GPx1 mutant, its amido modified rate reaches as high as 98.73% after measured；When amino is repaired When decorations rate is up to 40% or more, into the antigen-antibody reaction that foreign protein is just completely eliminated after in vivo, therefore list of the invention The recombination glutathione peroxidase GPx1 mutant of methoxy poly (ethylene glycol) modification has elimination zymoprotein antigen-antibody anti- The characteristic answered；
(2) the recombination glutathione peroxidase GPx1 of present invention mono methoxy polyethylene glycol modification obtained is prominent Variant, enzyme activity conservation rate is up to 100% under the conditions of pH=9.0, i.e., close with genetic recombination enzyme activity；
(3) the pH stability of enzyme improves：Under the conditions of pH 5.0 and 11.0 pH, glutathione peroxidase is recombinated It is 25.28% and 48.62% that GPx1 mutant, which keeps opposite enzyme activity,；And the recombination glutathione of mono methoxy polyethylene glycol modification It is 26.09% and 53.36% that peroxidase GPx1 mutant, which keeps opposite enzyme activity, is shown in low pH 5.0 and high pH 11.0 Under the conditions of its stability it is more preferable；
(4) thermal stability of enzyme improves：At 55 DEG C, recombination glutathione peroxidase GPx1 mutant keeps opposite Enzyme activity is 51.44%；And the recombination glutathione peroxidase GPx1 mutant of mono methoxy polyethylene glycol modification keeps phase It is 59.04% to enzyme activity, shows that its thermal stability is good；
(5) long-time stability of enzyme improve：It is stored 120 days under the conditions of 4 DEG C, the recombination of mono methoxy polyethylene glycol modification Glutathione peroxidase GPx1 mutant can keep 63.69% with respect to Rate activity, and GPx1 mutation vitality of subject maintains 47.77%；It is stored 90 days under the conditions of -20 DEG C, the recombination glutathione peroxidase of mono methoxy polyethylene glycol modification GPx1 mutant can keep 77.39% with respect to Rate activity, and GPx1 mutation vitality of subject maintains 60.42%；Under lyophilisation condition The recombination glutathione peroxidase GPx1 mutant of storage 12 months, mono methoxy polyethylene glycol modification can with respect to Rate activity To keep 91.22%, and GPx1 mutation vitality of subject maintains 89.50%.Illustrate that enzyme can be improved in the GPx1 after mPEG is modified Stability living.
(6) GPx1 and mPEG-GPx1 has antioxidant stress injury ability, but the latter is better than the former：30 μm ol/L Ah In the Human Embryonic Kidney HEK 293T cellular oxidation Stress model of mycin induction, model group cell survival rate is 55.05%, containing 2mM/L sun Property medicine N-acetylcystein prevention group cell survival rate is 65.02%, treatment group 61.04%, compared to model group cell Survival rate is respectively increased 18.11%, 10.88%；Be 69.03% containing 0.8 U/mL mPEG-GPx1 prevention group cell survival rate, Treatment group is 66.20%, is respectively increased 25.40%, 20.25% compared to model group cell survival rate；GPx1 containing 0.8U/mL Prevention group cell survival rate is 64.54%, treatment group 57.18%, is respectively increased compared to model group cell survival rate 17.24%, 3.87%.
It is established in oxidative stress model in 2.5 μm of ol/L adriamycin induction H9c2 rat myocardial cells, model group cell is deposited Motility rate is 60.13%, and the N-acetylcystein prevention group cell survival rate of positive drug containing 2mM/L is 74.57%, treatment group is 72.42%, 24.01%, 20.44% is respectively increased compared to model group cell survival rate；MPEG-GPx1 containing 0.08U/mL is pre- Anti- group of cell survival rate be 85.80%, treatment group 79.58%, is respectively increased compared to model group cell survival rate 42.69%, 20.44%；The prevention group cell survival rate of GPx1 containing 0.08U/mL is 83.84%, treatment group 76.93%, phase 39.43%, 27.94% is respectively increased compared with model group cell survival rate.
In 600 μm of ol/L hydrogen peroxide (H2O2) Human Embryonic Kidney HEK 293T cell is induced to establish in oxidative stress model, model Group cell survival rate is 53.84%, and the N-acetylcystein prevention group cell survival rate of positive drug containing 2mM/L is 70.87%, controls Treatment group is 68.76%, is respectively increased 31.39%, 27.48% compared to model group cell survival rate；MPEG- containing 0.1U/mL GPx1 prevention group cell survival rate is 93.67%, treatment group 83.46%, is respectively increased compared to model group cell survival rate 72.65%, 54.72%；The prevention group cell survival rate of GPx1 containing 0.1U/mL is 90.06%, treatment group 81.19%, is compared 66.96%, 50.51% is respectively increased in model group cell survival rate.
In 400 μm of ol/L H2O2Induction H9c2 rat myocardial cell is established in oxidative stress model, model group cell survival Rate is 52.78%, and the N-acetylcystein prevention group cell survival rate of positive drug containing 2mM/L is 73.95%, treatment group is 65.89%, 40.11%, 24.84% is respectively increased compared to model group cell survival rate；MPEG-GPx1 containing 0.08U/mL is pre- Anti- group of cell survival rate be 95.11%, treatment group 84.69%, is respectively increased compared to model group cell survival rate 80.19%, 60.45%；The prevention group cell survival rate of GPx1 containing 0.08U/mL is 92.60%, treatment group 83.54%, is compared 75.45%, 58.27% is respectively increased in model group cell survival rate.
(7) before GPx1 and mPEG-GPx1 can reduce the generation that lipid peroxide is reacted on cell membrane, but the latter is better than Person.It is established in oxidative stress model in 30 μm of ol/L induced by doxorubicin in human embryonic kidney HEK293T cells, the intracellular lactic acid of model group is de- Hydrogen enzyme content is 240.17 ± 5.35U/g, the intracellular lactic dehydrogenase of the N- acetylcysteine prevention group of positive drug containing 2mM/L Content is 287.62 ± 11.75U/g, 286.42 ± 3.64U/g for the treatment of group, compared to model group cell Dehydrogenase Content It is respectively increased 19.75 ± 4.89%, 19.25 ± 1.51%；The intracellular lactic dehydrogenase of the prevention group of mPEG-GPx1 containing 0.8U/mL Enzyme content is 295.02 ± 0.59U/g, 287.01 ± 6.17U/g for the treatment of group, compared to model group cell Dehydrogenase Content It is respectively increased 22.84 ± 0.25%, 19.50 ± 2.57%；The intracellular Dehydrogenase Content of the prevention group of GPx1 containing 0.8U/mL For 296.95 ± 8.87U/g, 288.74 ± 1.96U/g for the treatment of group, mentioned respectively compared to model group cell Dehydrogenase Content High 23.64 ± 3.69%, 19.25 ± 1.51%.
It is established in oxidative stress model in 2.5 μm of ol/L adriamycin induction H9c2 rat myocardial cells, model group is intracellular Dehydrogenase Content is 538.41 ± 4.59U/g, and the intracellular lactic acid of N-acetylcystein prevention group of positive drug containing 2mM/L is de- Hydrogen enzyme content is 577.08 ± 4.86U/g, 572.45 ± 4.45U/g for the treatment of group, is contained compared to model group cell lactic dehydrogenase Amount is respectively increased 7.18 ± 0.90%, 6.32 ± 0.83%；The intracellular lactic dehydrogenase of the prevention group of mPEG-GPx1 containing 0.08U/mL Enzyme content is 698.68 ± 9.50U/g, 647.05 ± 2.11U/g for the treatment of group, compared to model group cell Dehydrogenase Content It is respectively increased 29.77 ± 1.76%, 20.18 ± 0.39%；The intracellular lactic dehydrogenase of the prevention group of GPx1 containing 0.08U/mL contains Amount is 647.76 ± 3.92 U/g, 612.42 ± 2.82U/g for the treatment of group, is distinguished compared to model group cell Dehydrogenase Content Improve 20.31 ± 0.73%, 13.75 ± 0.52%.The damaged degree that can reduce cell membrane reduces releasing for lactic dehydrogenase It puts.
In 600 μm of ol/L H2O2Induction Human Embryonic Kidney HEK 293T cell is established in oxidative stress model, and model group is newborn into the cell Acidohydrogenase content is 240.18 ± 0.1U/g, the intracellular lactic dehydrogenase of the N-acetylcystein prevention group of positive drug containing 2mM/L Enzyme content is 308.60 ± 7.58U/g, 286.65 ± 0.81U/g for the treatment of group, compared to model group cell Dehydrogenase Content It is respectively increased 28.49 ± 3.16%, 19.35 ± 0.34%；The intracellular lactic dehydrogenase of the prevention group of mPEG-GPx1 containing 0.1U/mL Content is 400.56 ± 1.87U/g, 350.67 ± 0.22U/g for the treatment of group, compared to model group cell Dehydrogenase Content point Indescribably high 66.78 ± 0.78%, 46.01 ± 0.09%；The intracellular Dehydrogenase Content of the prevention group of GPx1 containing 0.1U/mL is 383.44 ± 2.43 U/g, 325.69 ± 5.59U/g for the treatment of group are respectively increased compared to model group cell Dehydrogenase Content 59.65 ± 1.01%, 35.61 ± 2.33%.
In 400 μm of ol/L H2O2Induction H9c2 cell is established in oxidative stress model, the intracellular lactic dehydrogenase of model group Content is 536.05 ± 5.02U/g, and the intracellular Dehydrogenase Content of the N-acetylcystein prevention group of positive drug containing 2mM/L is 603.75 ± 5.81U/g, 577.12 ± 5.03U/g for the treatment of group are respectively increased compared to model group cell Dehydrogenase Content 12.63 ± 1.08%, 7.66 ± 0.94%；The intracellular Dehydrogenase Content of the prevention group of mPEG-GPx1 containing 0.08U/mL is 801.97 ± 2.91U/g, 708.61 ± 7.60U/g for the treatment of group are respectively increased compared to model group cell Dehydrogenase Content 49.61 ± 0.54%, 32.19 ± 1.42%；The intracellular Dehydrogenase Content of the prevention group of GPx1 containing 0.08U/mL is 773.67 ± 5.98 U/g, 680.66 ± 0.11U/g for the treatment of group are respectively increased compared to model group cell Dehydrogenase Content 49.61 ± 0.54%, 26.98 ± 0.02%.
(8) GPx1 and mPEG-GPx1 can block the generation of lipid peroxide chain reaction, reduce containing for malonaldehyde Amount, but the latter is better than the former.It is established in oxidative stress model in 30 μm of ol/L induced by doxorubicin in human embryonic kidney HEK293T cells, mould The intracellular mda content of type group is 0.904 ± 0.11nmol/mg, thin containing 2 mM/L positive drug N-acetylcystein prevention groups Mda content intracellular is 0.452 ± 0.02nmol/mg, 0.442 ± 0.02nmol/mg for the treatment of group, compared to model group cell Mda content reduces by 49.96 ± 2.41%, 51.11 ± 0.16% respectively；The prevention group cell of mPEG-GPx1 containing 0.8U/mL Interior mda content is 0.369 ± 0.17nmol/mg, 0.468 ± 0.19nmol/mg for the treatment of group, compared to model group cell third Dialdehyde content reduces by 59.16 ± 18.72%, 48.26 ± 21.56% respectively；The prevention group of GPx1 containing 0.8U/mL intracellular the third two Aldehyde is 0.389 ± 0.13nmol/mg, 0.401 ± 0.07nmol/mg for the treatment of group, is contained compared to model group cell malonaldehyde Amount reduces by 57.01 ± 14.33%, 55.65 ± 7.68% respectively.
It is established in oxidative stress model in 2.5 μm of ol/L adriamycin induction H9c2 rat myocardial cells, model group is intracellular Mda content is 8.22 ± 0.19nmol/mg, and the intracellular malonaldehyde of the N-acetylcystein prevention group of positive drug containing 2mM/L contains Amount is 2.73 ± 0.02nmol/mg, 3.57 ± 0.02nmol/mg for the treatment of group, is distinguished compared to model group cell mda content Reduce by 66.79 ± 0.28%, 56.53 ± 0.29%；The intracellular mda content of the prevention group of mPEG-GPx1 containing 0.08U/mL is 2.34 ± 0.90nmol/mg, 3.32 ± 0.06 nmol/mg for the treatment of group, reduce respectively compared to model group cell mda content 71.49 ± 1.10%, 59.66 ± 0.67%；The intracellular mda content of the prevention group of GPx1 containing 0.08U/mL be 2.55 ± 0.06nmol/mg, 3.45 ± 0.25nmol/mg for the treatment of group reduce by 69.01 compared to model group cell mda content respectively ± 0.70%, 57.97 ± 3.07%.The damage that can effectively prevent or treat adriamycin to cell.
In 600 μm of ol/L H2O2Induction Human Embryonic Kidney HEK 293T cell is established in oxidative stress model, model group intracellular third Dialdehyde content is 0.911 ± 0.01nmol/mg, and the intracellular malonaldehyde of the N-acetylcystein prevention group of positive drug containing 2mM/L contains Amount is 0.437 ± 0.02nmol/mg, 0.435 ± 0.01nmol/mg for the treatment of group, compared to model group cell mda content point It Jiang Di by 52.08 ± 1.52%, 52.23 ± 1.36%；The intracellular mda content of the prevention group of mPEG-GPx1 containing 0.1U/mL is 0.303 ± 0.17nmol/mg, 0.332 ± 0.01 nmol/mg for the treatment of group, drop respectively compared to model group cell mda content Low 66.72 ± 0.17%, 63.57 ± 1.33%；The intracellular mda content of the prevention group of GPx1 containing 0.1U/mL be 0.326 ± 0.13nmol/mg, 0.387 ± 0.07nmol/mg for the treatment of group reduce by 64.19 compared to model group cell mda content respectively ± 0.42%, 57.53 ± 0.11%.
In 400 μm of ol/L H2O2Induction H9c2 cell is established in oxidative stress model, the intracellular mda content of model group For 8.313 ± 0.13nmol/mg, the intracellular mda content of N-acetylcystein prevention group of positive drug containing 2mM/L is 2.67 ± 0.01nmol/mg, 3.25 ± 0.03nmol/mg for the treatment of group reduce by 67.88 compared to model group cell mda content respectively ± 0.12%, 60.95 ± 0.37%；The intracellular mda content of the prevention group of mPEG-GPx1 containing 0.08U/mL be 1.67 ± 0.02nmol/mg, 2.73 ± 0.24nmol/mg for the treatment of group, 79.91 are reduced respectively compared to model group cell mda content ± 0.21%, 67.12 ± 2.85%；The intracellular mda content of the prevention group of GPx1 containing 0.08U/mL is 2.03 ± 0.05nmol/ Mg, 3.03 ± 0.03nmol/mg for the treatment of group reduce by 75.62 ± 0.54% compared to model group cell mda content respectively, 63.59 ± 0.37%.
Detailed description of the invention
Fig. 1 is mono methoxy polyethylene glycol succinimidyl butanoate hydrogen nuclear magnetic resonance wave spectrogram of the present invention.
Fig. 2 be the present invention modification result SDS-PAGE electrophoresis, 1 for molecular weight 14.3kD, 20.1kD, 29.0kD, 44.3kD, 66.4kD, 97.2kD, 2 be GPx1 mutant, and 3 be dressing agent (mono methoxy polyethylene glycol succinimidyl butanoic acid Ester), 4 be GPx1 and dressing agent mass ratio is 1:10 modified outcome, 5 be GPx1 and dressing agent mass ratio is 1:20 modification Product, 6 be GPx1 and dressing agent mass ratio is 1:30 modified outcome, 7 be GPx1 and dressing agent mass ratio is 1:60 modification Product, 8 be GPx1 and dressing agent mass ratio is 1:90 modified outcome, 9 be GPx1 and dressing agent mass ratio is 1:120 modification Product.
Fig. 3 be the present invention modification result iodine dye figure, 1 for molecular weight 14.3kD, 20.1kD, 29.0kD, 44.3kD, 66.4kD, 97.2kD, 2 be GPx1 mutant, and 3 be dressing agent, and 4 be GPx1 and dressing agent mass ratio is 1:10 modified outcome, 5 be GPx1 and dressing agent mass ratio is 1:20 modified outcome, 6 be GPx1 and dressing agent mass ratio is 1:30 modified outcome, 7 It is GPx1 and dressing agent mass ratio is 1:60 modified outcome, 8 be GPx1 and dressing agent mass ratio is 1:90 modified outcome, 9 It is GPx1 and dressing agent mass ratio is 1:120 modified outcome.
Fig. 4 is GPx1 of the present invention and dressing agent mass ratio is 1:10,1:20,1:30,1:60,1:90,1:Under the conditions of 120, Single lysine sites modification, the modification of multiple lysine sites and unmodified GPx1 comparative result figure.
Fig. 5 is GPx1 of the present invention and mPEG-GPx1 (wherein, GPx1 and dressing agent mass ratio are 1：60) 19,30,37, 45, the enzyme activity storage rate comparison diagram under the conditions of 50,55 DEG C.
Fig. 6 is GPx1 of the present invention and mPEG-GPx1 (wherein, GPx1 and dressing agent mass ratio are 1：60) pH5.0, 6.0, the enzyme activity storage rate comparison diagram under the conditions of 7.4,8.0,9.0,10.0,11.0
Fig. 7 is GPx1 of the present invention and mPEG-GPx1 (wherein, GPx1 and dressing agent mass ratio are 1：60) under the conditions of 4 DEG C Store the stability diagram of different time.
Fig. 8 is GPx1 of the present invention and mPEG-GPx1 (wherein, GPx1 and dressing agent mass ratio are 1：60) in -20 DEG C of conditions The stability diagram of lower storage different time.
Fig. 9 is GPx1 of the present invention and mPEG-GPx1 (wherein, GPx1 and dressing agent mass ratio are 1：60) after being freeze-dried The stability diagram of different time is stored under the conditions of -20 DEG C.
Figure 10 is that GPx1 of the present invention and mPEG-GPx1 causes the prevention of HEK293T cellular damage and treatment to be made adriamycin With cell survival rate figure.
Figure 11 is that GPx1 of the present invention and mPEG-GPx1 causes the prevention of H9c2 cellular damage and therapeutic effect thin adriamycin Born of the same parents' survival rate figure.
Figure 12 is that GPx1 of the present invention and mPEG-GPx1 causes the prevention of HEK293T cellular damage and treatment to be made adriamycin With intracellular Dehydrogenase Content figure.
Figure 13 is that GPx1 of the present invention and mPEG-GPx1 causes the prevention of H9c2 cellular damage and therapeutic effect thin adriamycin Dehydrogenase Content figure intracellular.
Figure 14 is that GPx1 of the present invention and mPEG-GPx1 causes the prevention of HEK293T cellular damage and treatment to be made adriamycin With intracellular mda content figure.
Figure 15 is that GPx1 of the present invention and mPEG-GPx1 causes the prevention of H9c2 cellular damage and therapeutic effect thin adriamycin Mda content figure intracellular.
Figure 16 is GPx1 of the present invention and mPEG-GPx1 to hydrogen peroxide (H2O2) cause HEK293T cellular damage prevention and Therapeutic effect cell survival rate figure.
Figure 17 is GPx1 of the present invention and mPEG-GPx1 to H2O2Cause prevention and the therapeutic effect cell of H9c2 cellular damage Survival rate figure.
Figure 18 is GPx1 of the present invention and mPEG-GPx1 to H2O2Cause prevention and the therapeutic effect of HEK293T cellular damage thin Dehydrogenase Content figure intracellular.
Figure 19 is GPx1 of the present invention and mPEG-GPx1 to H2O2Cause prevention and the therapeutic effect cell of H9c2 cellular damage Interior Dehydrogenase Content figure.
Figure 20 is GPx1 of the present invention and mPEG-GPx1 to H2O2Cause prevention and the therapeutic effect of HEK293T cellular damage thin Mda content figure intracellular.
Figure 21 is GPx1 of the present invention and mPEG-GPx1 to H2O2Cause prevention and the therapeutic effect cell of H9c2 cellular damage Interior mda content figure.
Specific embodiment
The straight-chain mono methoxy polyethylene glycol succinimidyl butanoate synthesis that embodiment 1 activates.
1. being prepared by the following：
(1) mono methoxy polyethylene glycol (mPEG) 2.0g for weighing molecular weight 5000, is added 4 mL N, N- bis- in flask Methylformamide sufficiently dissolves.
(2) 0.12g succinic anhydride is added, mixes, sealing.
(3) mixture reacts 3 hours under the conditions of 100 DEG C.
(4) after being cooled to room temperature above-mentioned reaction solution, 0.466g n-hydroxysuccinimide, 0.825g 1- second is added Base-(3- dimethylaminopropyl) carbodiimide hydrochloride, heating make it dissolve.
(5) reaction overnight 12 hours under the conditions of 30 DEG C, are cooled to room temperature.
(6) by reaction solution after cooling at 4 DEG C, 40mL ether is added dropwise, gained is precipitated and is filtered, vacuum drying. Dried product is dissolved with methylene chloride again, is then precipitated again with ether.It repeats this process 4 times.
(7) products therefrom is dried in vacuo, is confirmed by hydrogen nuclear magnetic resonance wave spectrum (Fig. 1), that is, obtains the straight-chain of activation Mono methoxy polyethylene glycol succinimidyl butanoate.
2. recombinating the acquisition of glutathione peroxidase GPx1 mutant：
It is obtained by 201310302778.3 method of the granted patent preparation of this seminar.
3. recombinating the mPEGization modification of glutathione peroxidase GPx1 mutant
In 20 DEG C of reaction temperature, pH 8.5, recombination glutathione peroxidase GPx1 mutant and straight-chain list methoxy Base polyethylene glycol succinimidyl butanoate mass ratio is respectively 1:10,1:20,1:30,1:60,1:90, 1:120, reaction After 30min, glycine is added to terminate reaction, the recombination glutathione peroxidase GPx1 for obtaining the mPEGization modification respectively is prominent Variant.
The recombination glutathione peroxidase GPx1 mutant mPEGization modified outcome that embodiment 1 obtains, using poly- third Acrylamide gel electrophoresis SDS-PAGE (Fig. 2), coomassie brilliant blue staining, coloration result proves, mPEG is connected on GPx1.
The recombination glutathione peroxidase GPx1 mutant mPEGization modified outcome that embodiment 1 obtains, using poly- third Acrylamide gel electrophoresis SDS-PAGE, iodine contaminate (Fig. 3), and coloration result proves, mPEG is connected on GPx1.
As shown in figure 4, by investigating single lysine sites modification, the modification of multiple lysine sites and unmodified GPx1, It is preferred that recombination glutathione peroxidase GPx1 mutant and straight-chain mono methoxy polyethylene glycol succinimidyl butanoate Mass ratio is 1:60, as target product.
Embodiment 2
The modified outcome performance evaluation of mPEGization recombination glutathione peroxidase GPx1 mutant：
(1) recombination glutathione peroxidase GPx1 mutant and straight-chain mono methoxy polyethylene glycol succinimide Butyrate mass ratio is 1:The thermal stability of obtained enzyme improves when 60.
Reaction system is 500 μ L, wherein including PBS, 1mmol/L EDTA, the 1mmol/L of 50mmol/L pH 7.4 GSH, 0.25mmol/L NADPH, 1U glutathione reductase and 20-50nM protein sample.19~55 DEG C of reaction mixture pre- 5min is kept the temperature, final concentration 0.5mmol/L H is then added2O2Starting reaction.It is monitored at 340nm by ultraviolet specrophotometer The situation of change of NADPH absorbance value.Enzyme activity is defined as at 37 DEG C, enzyme needed for which aoxidizes 1 μm of ol NADPH per minute Amount be a unit of activity, and indicated with 100% enzyme activity.As shown in figure 5, recombinating glutathione peroxidating at 55 DEG C It is 51.44% that object enzyme GPx1 mutant, which keeps opposite enzyme activity,；And the recombination glutathione peroxidase GPx1 of mPEG modification is prominent It is 59.04% that variant, which keeps opposite enzyme activity,.The result shows that the recombination glutathione peroxidase GPx1 mutant of mPEG modification Thermal stability is good.
(2) the pH stability of enzyme improves.Reaction system is 500 μ L, wherein comprising 50mmol/L pH 5.0~11.0 PBS, 1mmol/L EDTA, 1mmol/L GSH, 0.25mmol/L NADPH, 1U glutathione reductase and 20-50nM albumen Sample.Then final concentration 0.5mmol/L H is added in 19-55 DEG C of pre-incubation 5min of reaction mixture2O2Starting reaction.By ultraviolet Spectrophotometer monitors the situation of change of NADPH absorbance value at 340nm.Enzyme activity is defined as at 37 DEG C, every point of the albumen The amount of enzyme needed for clock aoxidizes 1 μm of ol NADPH is a unit of activity, and is indicated with 100% enzyme activity.As shown in fig. 6, in pH Under the conditions of 5.0 and pH11.0, it is 25.28% He that recombination glutathione peroxidase GPx1 mutant, which keeps opposite enzyme activity, 48.62%；And it is 26.09% He that the recombination glutathione peroxidase GPx1 mutant of mPEG modification, which keeps opposite enzyme activity, 53.36%.The recombination glutathione peroxidase GPx1 mutant stability of mPEG modification is slightly good.
Embodiment 3
Modified outcome (mPEG-GPx1) frozen-dried protective of mPEGization recombination glutathione peroxidase GPx1 mutant Agent screening：
In 4 degree of chromatography freezers, GPx1 and mPEG-GPx1 is frozen containing 10% mannitol, 5% glycine (W/V) respectively Dialysis 4 hours in protectant solution (200mL) are done, replacement 4 dialyzates in centre are pre- under the conditions of -20 DEG C after the completion of dialysing Freeze 12 hours, is lyophilized in 5 freeze dryer of Zirbus Voco；GPx1 and mPEG-GPx1 does not add the sample of freeze drying protectant -20 Pre-freeze 12 hours under the conditions of DEG C are lyophilized in 5 freeze dryer of Zirbus Voco；According to 2 enzyme activity determination method of embodiment, 37 DEG C are measured When enzyme activity.Sample enzyme activity is in terms of 100% before being lyophilized.
After GPx1 and mPEG-GPx1 are directly lyophilized, enzyme activity keeps 75.36%, 82.42% respectively；When use 10% When mannitol, 5% glycine cook protective agent, after GPx1 and mPEG-GPx1 freeze-drying enzyme activity keep 82.42% respectively, 86.19%；Illustrate that the stability of mPEGization recombination glutathione peroxidase GPx1 mutant is good compared with GPx1 mutant, preferably 10% mannitol, 5% glycine are as freeze drying protectant.
Embodiment 4
Long-time stability evaluation：
(1) GPx1 and mPEG-GPx are separately added into 40% glycerol, are stored under the conditions of 4 DEG C, respectively at 0,15,30, 45,60,75,90,105 and 120 days when, according to enzyme activity determination method shown in embodiment 2, measure its 37 DEG C of enzyme activities.As a result such as Shown in Fig. 7, at 120 days, mPEG-GPx1 can keep 63.69% opposite Rate activity, and GPx1 only keeps 47.77%, GPx1 after mPEG modification is compared to GPx1 and 33.33% enzyme activity can be improved, and the GPx1 enzyme activity after illustrating mPEG modification is steady It is qualitative good.
(2) GPx1 and mPEG-GPx1 are separately added into 40% glycerol, are stored under the conditions of -20 DEG C, respectively at 0,15,30, At 60 and 90 days, according to enzyme activity determination method shown in embodiment 2, its 37 DEG C of enzyme activities are measured.As a result as shown in figure 8, at 90 days When, mPEG-GPx1 can keep 77.39% opposite Rate activity, and GPx1 only keeps the GPx1 phase after 60.42%, mPEG modification Being compared to GPx1 can be improved 28.09% enzyme activity, and enzyme activity stability can be improved in the GPx1 after illustrating mPEG modification.
(3) 10% mannitol, 5% glycine is added for freeze drying protectant, in -20 after freeze-drying in GPx1 and mPEG-GPx1 It is stored under the conditions of DEG C, respectively at 1~12 month, according to enzyme activity determination method shown in embodiment 2, measures its 37 DEG C of enzyme activity.As a result As shown in figure 9, mPEG-GPx1 can keep 91.22% opposite Rate activity, and GPx1 keeps 89.50% at 12 months, GPx1 after mPEG modification is compared to GPx1 and 1.92% enzyme activity can be improved.Even if illustrating the GPx1 freeze-drying after mPEG modification Under the conditions of storage enzyme activity stability also slightly improve, only not as good as obvious under liquid condition.
Embodiment 5 GPx1 and mPEG-GPx1 is improved by preventing or treating oxidativestress damage caused by adriamycin HEK293T cell survival rate
HEK293T cell is with 1 × 105A/hole is inoculated in 96 orifice plates of the DMEM in high glucose culture solution containing 10% fetal calf serum In, it is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e., normally Control group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into dense eventually Spend 30 μm of ol/L adriamycins；In prevention group, be separately added into final concentration 2mmol/L positive control drug N-acetylcystein, eventually it is dense The GPx1 and mPEG-GPx1 of 0.8U/mL are spent, 30 μm of ol/L adriamycins of final concentration are added after effect 1 hour and act on 24 hours；Treatment In group, it is separately added into 30 μm of ol/L adriamycins of final concentration and acts on 12 hours, final concentration 0.8U/mL is then separately added into cell GPx1 and mPEG-GPx1 act on 12 hours.The 20 μ L of MTT solution of 5mg/ml is added in every hole, continues to cultivate 4h, terminates culture It inhales afterwards and abandons culture supernatant in hole, and every hole adds 150 μ L dimethyl Asias to mock (DMSO) termination reaction, vibrates 5min, makes crystal Matter sufficiently dissolves.492nm wavelength is selected, each hole light absorption value is measured in microplate reader.Calculate different groups of cell survival rate.
As shown in Figure 10, it is established in oxidative stress model in 30 μm of ol/L induced by doxorubicin in human embryonic kidney HEK293T cells, mould Type group cell survival rate be 55.05%, the N-acetylcystein prevention group cell survival rate of positive drug containing 2mM/L be 65.02%, Treatment group is 61.04%, is respectively increased 18.11%, 10.88% compared to model group cell survival rate；Containing 0.8U/mL MPEG-GPx1 prevention group cell survival rate is 69.03%, treatment group 66.20%, is distinguished compared to model group cell survival rate Improve 25.40%, 20.25%；The prevention group cell survival rate of GPx1 containing 0.8U/mL is 64.54%, treatment group 57.18%, 17.24%, 3.87% is respectively increased compared to model group cell survival rate.Illustrate, GPx1 and mPEG-GPx1 all have anti-oxidant Stress damage ability, and the GPx1 antioxidant stress injury ability after mPEG is modified showed in HEK293T cell it is stronger.
Embodiment 6 GPx1 and mPEG-GPx1 is improved by preventing or treating oxidativestress damage caused by adriamycin H9c2 cell survival rate
H9c2 cell is with 1 × 105A/hole is inoculated in 96 orifice plates of the DMEM in high glucose culture solution containing 10% fetal calf serum, It is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e. normal control Group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into final concentration 2.5 μm ol/L adriamycin；In prevention group, it is separately added into final concentration 2mmol/L positive control drug N-acetylcystein, final concentration The GPx1 and mPEG-GPx1 of 0.08U/mL is added 2.5 μm of ol/L adriamycins of final concentration and acts on 24 hours after effect 1 hour；It controls In treatment group, 2.5 μm of ol/L adriamycins of final concentration are separately added into, after effect 12 hours, then are separately added into 0.08 U/mL's of final concentration GPx1 and mPEG-GPx1 is acted on 12 hours.The 20 μ L of MTT solution of 5mg/ml is added in every hole, continues to cultivate 4h, inhale after terminating culture Culture supernatant in hole is abandoned, and every hole adds 150 μ L dimethyl Asias to mock (DMSO) termination reaction, vibrates 5min, fills crystalline material Divide dissolution.492nm wavelength is selected, each hole light absorption value is measured in microplate reader.Calculate different groups of cell survival rate.
As shown in figure 11, it is established in oxidative stress model in 2.5 μm of ol/L adriamycin induction H9c2 rat myocardial cells, mould Type group cell survival rate be 60.13%, the N-acetylcystein prevention group cell survival rate of positive drug containing 2mM/L be 74.57%, Treatment group is 72.42%, is respectively increased 24.01%, 20.44% compared to model group cell survival rate；Containing 0.08U/mL MPEG-GPx1 prevention group cell survival rate is 85.80%, treatment group 79.58%, is distinguished compared to model group cell survival rate Improve 42.69%, 20.44%；The prevention group cell survival rate of GPx1 containing 0.08U/mL is 83.84%, treatment group 76.93%, 39.43%, 27.94% is respectively increased compared to model group cell survival rate.Illustrate, GPx1 and mPEG-GPx1 all have antioxygen Change stress damage ability, and the GPx1 antioxidant stress injury ability after mPEG is modified showed in H9c2 cell it is stronger.
Embodiment 7 GPx1 and mPEG-GPx1 by reduce HEK293T cell membrane on lipid peroxide response prevention and Treat oxidativestress damage caused by adriamycin
HEK293T cell is with 1 × 105A/hole is inoculated in the culture of the DMEM in high glucose culture solution of the fetal calf serum containing 8mL10% In bottle, it is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e., just Normal control group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into end 30 μm of ol/L adriamycins of concentration；In prevention group, it is separately added into final concentration 2mmol/L positive control drug N-acetylcystein, end The GPx1 and mPEG-GPx1 of concentration 0.8U/mL is added 30 μm of ol/L adriamycins of final concentration and acts on 24 hours after effect 1 hour； In treatment group, it is separately added into 30 μm of ol/L adriamycins of final concentration and acts on 12 hours, final concentration is then separately added into cell The GPx1 and mPEG-GPx1 of 0.8U/mL is acted on 12 hours.Lactic dehydrogenase enzyme reagent kit (A020-2) is built up by Nanjing, according to Step described in specification evaluates the lactic dehydrogenase index in damaging cells.
As shown in figure 12, it is established in oxidative stress model in 30 μm of ol/L induced by doxorubicin in human embryonic kidney HEK293T cells, mould The intracellular Dehydrogenase Content of type group is 240.17 ± 5.35U/g, the N-acetylcystein prevention group of positive drug containing 2mM/L Intracellular Dehydrogenase Content is 287.62 ± 11.75U/g, 286.42 ± 3.64U/g for the treatment of group, compared to model group cell Dehydrogenase Content is respectively increased 19.75 ± 4.89%, 19.25 ± 1.51%；The prevention group of mPEG-GPx1 containing 0.8U/mL Intracellular Dehydrogenase Content is 295.02 ± 0.59U/g, 287.01 ± 6.17U/g for the treatment of group, compared to model group cell Dehydrogenase Content is respectively increased 22.84 ± 0.25%, 19.50 ± 2.57%；The prevention group of GPx1 containing 0.8U/mL is intracellular Dehydrogenase Content is 296.95 ± 8.87U/g, 288.74 ± 1.96U/g for the treatment of group, is taken off compared to model group cell lactic acid Hydrogen enzyme content is respectively increased 23.64 ± 3.69%, 19.25 ± 1.51%.It can reduce the damaged degree of cell membrane, reduce lactic acid The release of dehydrogenase.And mPEG modification after GPx1 reduce cell membrane damage ability showed in HEK293T cell it is stronger.
Embodiment 8 GPx1 and mPEG-GPx1 is by reducing lipid peroxide response prevention and treatment on H9c2 cell membrane Oxidativestress damage caused by adriamycin
H9c2 cell is with 1 × 105A/mL is inoculated in the culture bottle of the DMEM in high glucose culture solution of the fetal calf serum containing 8mL10% In, it is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e., normally Control group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into dense eventually Spend 2.5 μm of ol/L adriamycins；In prevention group, be separately added into final concentration 2mmol/L positive control drug N-acetylcystein, eventually it is dense The GPx1 and mPEG-GPx1 of 0.08U/mL are spent, 2.5 μm of ol/L adriamycins of final concentration are added after effect 1 hour and act on 24 hours；It controls In treatment group, 2.5 μm of ol/L adriamycins of final concentration are separately added into, after effect 12 hours, then are separately added into 0.08 U/mL's of final concentration GPx1 and mPEG-GPx1 is acted on 12 hours.Lactic dehydrogenase enzyme reagent kit (A020-2) is built up by Nanjing, to specifications institute Step is stated to evaluate the lactic dehydrogenase index in damaging cells.
As shown in figure 13, it is established in oxidative stress model in 2.5 μm of ol/L adriamycin induction H9c2 rat myocardial cells, mould The intracellular Dehydrogenase Content of type group is 538.41 ± 4.59U/g, the N-acetylcystein prevention group of positive drug containing 2mM/L Intracellular Dehydrogenase Content is 577.08 ± 4.86U/g, 572.45 ± 4.45U/g for the treatment of group, compared to model group cell Dehydrogenase Content is respectively increased 7.18 ± 0.90%, 6.32 ± 0.83%；The prevention group of mPEG-GPx1 containing 0.08U/mL is thin Dehydrogenase Content intracellular is 698.68 ± 9.50U/g, 647.05 ± 2.11U/g for the treatment of group, compared to model group cell cream Acidohydrogenase content is respectively increased 29.77 ± 1.76%, 20.18 ± 0.39%；The prevention group of GPx1 containing 0.08U/mL is newborn into the cell Acidohydrogenase content is 647.76 ± 3.92U/g, 612.42 ± 2.82U/g for the treatment of group, compared to model group cell lactic dehydrogenase Enzyme content is respectively increased 20.31 ± 0.73%, 13.75 ± 0.52%.It can reduce the damaged degree of cell membrane, it is de- to reduce lactic acid The release of hydrogen enzyme.And the GPx1 reduction cell membrane damage ability after mPEG modification is stronger in H9c2 cell.
The generation that embodiment 9 GPx1 and mPEG-GPx1 passes through blocking HEK293T cytolipin peroxide chain reaction Prevent and treat oxidativestress damage caused by adriamycin
HEK293T cell is with 1 × 105A/hole is inoculated in the culture of the DMEM in high glucose culture solution of the fetal calf serum containing 8mL10% In bottle, it is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e., just Normal control group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into end 30 μm of ol/L adriamycins of concentration；In prevention group, it is separately added into final concentration 2mmol/L positive control drug N-acetylcystein, end The GPx1 and mPEG-GPx1 of concentration 0.8U/mL is added 30 μm of ol/L adriamycins of final concentration and acts on 24 hours after effect 1 hour； In treatment group, it is separately added into 30 μm of ol/L adriamycins of final concentration and acts on 12 hours, final concentration is then separately added into cell The GPx1 and mPEG-GPx1 of 0.8U/mL is acted on 12 hours.Malondialdehyde Kit (A003-1) is built up by Nanjing, according to explanation Step described in book evaluates the malonaldehyde index in damaging cells.
As shown in figure 14, it is established in oxidative stress model in 30 μm of ol/L induced by doxorubicin in human embryonic kidney HEK293T cells, mould The intracellular mda content of type group is 0.904 ± 0.11nmol/mg, and the N- acetylcysteine prevention group of positive drug containing 2mM/L is thin Mda content intracellular is 0.452 ± 0.02nmol/mg, 0.442 ± 0.02nmol/mg for the treatment of group, compared to model group cell Mda content reduces by 49.96 ± 2.41%, 51.11 ± 0.16% respectively；The prevention group cell of mPEG-GPx1 containing 0.8U/mL Interior mda content is 0.369 ± 0.17 nmol/mg, 0.468 ± 0.19nmol/mg for the treatment of group, compared to model group cell third Dialdehyde content reduces by 59.16 ± 18.72%, 48.26 ± 21.56% respectively；The prevention group of GPx1 containing 0.8U/mL intracellular the third two Aldehyde is 0.389 ± 0.13nmol/mg, 0.401 ± 0.07nmol/mg for the treatment of group, is contained compared to model group cell malonaldehyde Amount reduces by 57.01 ± 14.33%, 55.65 ± 7.68% respectively.The generation of lipid peroxide chain reaction can be blocked, is dropped The content of low malonaldehyde, and it is stronger in the GPx1 reduction lipid peroxide ability HEK293T cell after mPEG modification.
Embodiment 10 GPx1 and mPEG-GPx1 is pre- by blocking the generation of H9c2 cytolipin peroxide chain reaction Oxidativestress damage caused by anti-and treatment adriamycin
H9c2 cell is with 1 × 105A/mL is inoculated in the culture bottle of the DMEM in high glucose culture solution of the fetal calf serum containing 8mL10% In, it is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e., normally Control group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into dense eventually Spend 2.5 μm of ol/L adriamycins；In prevention group, be separately added into final concentration 2mmol/L positive control drug N-acetylcystein, eventually it is dense The GPx1 and mPEG-GPx1 of 0.08U/mL are spent, 2.5 μm of ol/L adriamycins of final concentration are added after effect 1 hour and act on 24 hours； In treatment group, 2.5 μm of ol/L adriamycins of final concentration are separately added into, after effect 12 hours, then are separately added into 0.08 U/mL of final concentration GPx1 and mPEG-GPx1 act on 12 hours.Malondialdehyde Kit (A003-1) is built up by Nanjing, it is described to specifications Step evaluates the malonaldehyde index in damaging cells.
As shown in figure 15, it is established in oxidative stress model in 2.5 μm of ol/L adriamycin induction H9c2 rat myocardial cells, mould The intracellular mda content of type group is 8.22 ± 0.19nmol/mg, and the N- acetylcysteine prevention group of positive drug containing 2mM/L is thin Mda content intracellular is 2.73 ± 0.02nmol/mg, 3.57 ± 0.02 nmol/mg for the treatment of group, compared to model group cell third Dialdehyde content reduces by 66.79 ± 0.28%, 56.53 ± 0.29% respectively；The prevention group of mPEG-GPx1 containing 0.08U/mL is intracellular Mda content is 2.34 ± 0.90nmol/mg, 3.32 ± 0.06nmol/mg for the treatment of group, compared to model group cell malonaldehyde Content reduces by 71.49 ± 1.10%, 59.66 ± 0.67% respectively；The intracellular malonaldehyde of the prevention group of GPx1 containing 0.08U/mL contains Amount is 2.55 ± 0.06nmol/mg, 3.45 ± 0.25nmol/mg for the treatment of group, is distinguished compared to model group cell mda content Reduce by 69.01 ± 0.70%, 57.97 ± 3.07%.GPx1 and mPEG-GPx1 can effectively prevent or treat adriamycin to thin The damage of born of the same parents.The generation that lipid peroxide chain reaction can be blocked reduces the content of malonaldehyde, and after mPEG modification GPx1 reduces stronger in lipid peroxide ability H9c2 cell.
Embodiment 11 GPx1 and mPEG-GPx1 is by preventing or treating H2O2Caused oxidativestress damage improves HEK293T cell survival rate
HEK293T cell is with 1 × 105A/hole is inoculated in 96 orifice plates of the DMEM in high glucose culture solution containing 10% fetal calf serum In, it is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e., normally Control group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into dense eventually Spend 600 μm of ol/L H2O2；In prevention group, it is separately added into final concentration 2mmol/L positive control drug N-acetylcystein, final concentration 600 μm of ol/L H of final concentration are added after effect 1 hour in the GPx1 and mPEG-GPx1 of 0.1U/mL2O2Effect 12 hours；Treatment group In, it is separately added into 600 μm of ol/L H of final concentration2O2Effect 6 hours, is then separately added into final concentration 0.1U/mL's in cell GPx1 and mPEG-GPx1 is acted on 6 hours.The 20 μ L of MTT solution of 5mg/ml is added in every hole, continues to cultivate 4h, inhale after terminating culture Culture supernatant in hole is abandoned, and every hole adds 150 μ L dimethyl Asias to mock (DMSO) termination reaction, vibrates 5min, fills crystalline material Divide dissolution.492nm wavelength is selected, each hole light absorption value is measured in microplate reader.Calculate different groups of cell survival rate.
As shown in figure 16, in 600 μm of ol/L H2O2Induction Human Embryonic Kidney HEK 293T cell is established in oxidative stress model, mould Type group cell survival rate be 53.84%, the N-acetylcystein prevention group cell survival rate of positive drug containing 2mM/L be 70.87%, Treatment group is 68.76%, is respectively increased 31.39%, 27.48% compared to model group cell survival rate；Containing 0.1U/mL MPEG-GPx1 prevention group cell survival rate is 93.67%, treatment group 83.46%, is distinguished compared to model group cell survival rate Improve 72.65%, 54.72%；The prevention group cell survival rate of GPx1 containing 0.1U/mL is 90.06%, treatment group 81.19%, 66.96%, 50.51% is respectively increased compared to model group cell survival rate.Illustrate, GPx1 and mPEG-GPx1 all have antioxygen Change stress damage ability, and the GPx1 antioxidant stress injury ability after mPEG is modified shows more in HEK293T cell By force.
Embodiment 12 GPx1 and mPEG-GPx1 is by preventing or treating H2O2Caused oxidativestress damage improves H9c2 Cell survival rate
H9c2 cell is with 1 × 105A/hole is inoculated in 96 orifice plates of the DMEM in high glucose culture solution containing 10% fetal calf serum, It is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e. normal control Group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into final concentration 400 μmol/L H2O2；In prevention group, it is separately added into 2 mmol/L positive control drug N-acetylcystein of final concentration, final concentration 400 μm of ol/LH of final concentration are added after effect 1 hour in the GPx1 and mPEG-GPx1 of 0.08U/mL2O2Effect 12 hours；Treatment group In, it is separately added into 400 μm of ol/L H of final concentration2O2Effect 6 hours, is then separately added into final concentration 0.08U/mL's in cell GPx1 and mPEG-GPx1 is acted on 6 hours.The 20 μ L of MTT solution of 5mg/ml is added in every hole, continues to cultivate 4h, inhale after terminating culture Culture supernatant in hole is abandoned, and every hole adds 150 μ L dimethyl Asias to mock (DMSO) termination reaction, vibrates 5min, fills crystalline material Divide dissolution.492nm wavelength is selected, each hole light absorption value is measured in microplate reader.Calculate different groups of cell survival rate.
As shown in figure 17, in 400 μm of ol/L H2O2Induction H9c2 rat myocardial cell is established in oxidative stress model, model Group cell survival rate is 52.78%, and the N-acetylcystein prevention group cell survival rate of positive drug containing 2mM/L is 73.95%, controls Treatment group is 65.89%, is respectively increased 40.11%, 24.84% compared to model group cell survival rate；MPEG- containing 0.08U/mL GPx1 prevention group cell survival rate is 95.11%, treatment group 84.69%, is respectively increased compared to model group cell survival rate 80.19%, 60.45%；The prevention group cell survival rate of GPx1 containing 0.08U/mL is 92.60%, treatment group 83.54%, is compared 75.45%, 58.27% is respectively increased in model group cell survival rate.Illustrate, GPx1 and mPEG-GPx1 all have anti-oxidant answer Swash lesion capability, and the GPx1 antioxidant stress injury ability after mPEG is modified showed in H9c2 cell it is stronger.
Embodiment 13 GPx1 and mPEG-GPx1 by reduce HEK293T cell membrane on lipid peroxide response prevention and Treat H2O2Caused oxidativestress damage
HEK293T cell is with 1 × 105A/hole is inoculated in 96 orifice plates of the DMEM in high glucose culture solution containing 10% fetal calf serum In, it is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e., normally Control group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into dense eventually Spend 600 μm of ol/L H2O2；In prevention group, it is separately added into final concentration 2mmol/L positive control drug N-acetylcystein, final concentration 600 μm of ol/L H of final concentration are added after effect 1 hour in the GPx1 and mPEG-GPx1 of 0.1U/mL2O2Effect 12 hours；Treatment group In, it is separately added into 600 μm of ol/L H of final concentration2O2Effect 6 hours, is then separately added into final concentration 0.1U/mL's in cell GPx1 and mPEG-GPx1 is acted on 6 hours.Lactic dehydrogenase enzyme reagent kit (A020-2) is built up by Nanjing, it is described to specifications Step evaluates the lactic dehydrogenase index in damaging cells.
As shown in figure 18, in 600 μm of ol/L H2O2Induction Human Embryonic Kidney HEK 293T cell is established in oxidative stress model, mould The intracellular Dehydrogenase Content of type group is 240.18 ± 0.1U/g, and the N- acetylcysteine prevention group of positive drug containing 2mM/L is thin Dehydrogenase Content intracellular is 308.60 ± 7.58U/g, 286.65 ± 0.81U/g for the treatment of group, compared to model group cell cream Acidohydrogenase content is respectively increased 28.49 ± 3.16%, 19.35 ± 0.34%；The prevention group of mPEG-GPx1 containing 0.1U/mL is thin Dehydrogenase Content intracellular is 400.56 ± 1.87U/g, 350.67 ± 0.22U/g for the treatment of group, compared to model group cell cream Acidohydrogenase content is respectively increased 66.78 ± 0.78%, 46.01 ± 0.09%；The prevention group of GPx1 containing 0.1U/mL is newborn into the cell Acidohydrogenase content is 383.44 ± 2.43U/g, 325.69 ± 5.59U/g for the treatment of group, compared to model group cell lactic dehydrogenase Enzyme content is respectively increased 59.65 ± 1.01%, 35.61 ± 2.33%.It can reduce the damaged degree of cell membrane, it is de- to reduce lactic acid The release of hydrogen enzyme.And mPEG modification after GPx1 reduce cell membrane damage ability showed in HEK293T cell it is stronger.
Embodiment 14 GPx1 and mPEG-GPx1 is by reducing lipid peroxide response prevention on H9c2 cell membrane and controlling Treat H2O2Caused oxidativestress damage
H9c2 cell is with 1 × 105A/hole is inoculated in 96 orifice plates of the DMEM in high glucose culture solution containing 10% fetal calf serum, It is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e. normal control Group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into final concentration 400 μmol/L H2O2；In prevention group, it is separately added into 2 mmol/L positive control drug N-acetylcystein of final concentration, final concentration 400 μm of ol/L H of final concentration are added after effect 1 hour in the GPx1 and mPEG-GPx1 of 0.08U/mL2O2Effect 12 hours；Treatment group In, it is separately added into 400 μm of ol/L H of final concentration2O2Effect 6 hours, is then separately added into final concentration 0.08U/mL's in cell GPx1 and mPEG-GPx1 is acted on 6 hours.Lactic dehydrogenase enzyme reagent kit (A020-2) is built up by Nanjing, it is described to specifications Step evaluates the lactic dehydrogenase index in damaging cells.
As shown in figure 19, in 400 μm of ol/L H2O2Induction H9c2 cell is established in oxidative stress model, and model group is intracellular Dehydrogenase Content is 536.05 ± 5.02U/g, and the intracellular lactic acid of N-acetylcystein prevention group of positive drug containing 2mM/L is de- Hydrogen enzyme content is 603.75 ± 5.81U/g, 577.12 ± 5.03U/g for the treatment of group, is contained compared to model group cell lactic dehydrogenase Amount is respectively increased 12.63 ± 1.08%, 7.66 ± 0.94%；It is de- containing the 0.08 intracellular lactic acid of U/mL mPEG-GPx1 prevention group Hydrogen enzyme content is 801.97 ± 2.91U/g, 708.61 ± 7.60U/g for the treatment of group, is contained compared to model group cell lactic dehydrogenase Amount is respectively increased 49.61 ± 0.54%, 32.19 ± 1.42%；The intracellular lactic dehydrogenase of the prevention group of GPx1 containing 0.08U/mL Content is 773.67 ± 5.98 U/g, 680.66 ± 0.11U/g for the treatment of group, compared to model group cell Dehydrogenase Content point Indescribably high 49.61 ± 0.54%, 26.98 ± 0.02%.The damaged degree that can reduce cell membrane, reduces lactic dehydrogenase Release.And mPEG modification after GPx1 reduce cell membrane damage ability showed in H9c2 cell it is stronger.
The generation that embodiment 15 GPx1 and mPEG-GPx1 passes through blocking HEK293T cytolipin peroxide chain reaction Prevent and treat H2O2Caused oxidativestress damage
HEK293T cell is with 1 × 105A/hole is inoculated in 96 orifice plates of the DMEM in high glucose culture solution containing 10% fetal calf serum In, it is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e., normally Control group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into dense eventually Spend 600 μm of ol/L H2O2；In prevention group, it is separately added into final concentration 2mmol/L positive control drug N-acetylcystein, final concentration 600 μm of ol/L H of final concentration are added after effect 1 hour in the GPx1 and mPEG-GPx1 of 0.1U/mL2O2Effect 12 hours；Treatment group In, it is separately added into 600 μm of ol/L H of final concentration2O2Effect 6 hours, is then separately added into final concentration 0.1U/mL's in cell GPx1 and mPEG-GPx1 is acted on 6 hours.Malondialdehyde Kit (A003-1) is built up by Nanjing, to specifications the step Malonaldehyde index in damaging cells is evaluated.
As shown in figure 20, in 600 μm of ol/L H2O2Induction Human Embryonic Kidney HEK 293T cell is established in oxidative stress model, mould The intracellular mda content of type group is 0.911 ± 0.01nmol/mg, and the N- acetylcysteine prevention group of positive drug containing 2mM/L is thin Mda content intracellular is 0.437 ± 0.02nmol/mg, 0.435 ± 0.01nmol/mg for the treatment of group, compared to model group cell Mda content reduces by 52.08 ± 1.52%, 52.23 ± 1.36% respectively；The prevention group cell of mPEG-GPx1 containing 0.1U/mL Interior mda content is 0.303 ± 0.17 nmol/mg, 0.332 ± 0.01nmol/mg for the treatment of group, compared to model group cell third Dialdehyde content reduces by 66.72 ± 0.17%, 63.57 ± 1.33% respectively；The intracellular malonaldehyde of the prevention group of GPx1 containing 0.1U/mL Content is 0.326 ± 0.13nmol/mg, 0.387 ± 0.07nmol/mg for the treatment of group, compared to model group cell mda content 64.19 ± 0.42%, 57.53 ± 0.11% are reduced respectively.The generation of lipid peroxide chain reaction can be blocked, reduces by third The content of dialdehyde, and it is stronger in the GPx1 reduction lipid peroxide ability HEK293T cell after mPEG modification.
Embodiment 16 GPx1 and mPEG-GPx1 is pre- by blocking the generation of H9c2 cytolipin peroxide chain reaction Anti- and treatment H2O2Caused oxidativestress damage
H9c2 cell is with 1 × 105A/hole is inoculated in 96 orifice plates of the DMEM in high glucose culture solution containing 10% fetal calf serum, It is placed in 37 DEG C, 5%CO2It is cultivated in saturated humidity incubator, when cell grows to 80% density, is divided into 4 groups, i.e. normal control Group, model group, prevention group and treatment group.Normal group is added and model group equivalent culture medium；Model group is separately added into final concentration 400 μmol/L H2O2；In prevention group, it is separately added into 2 mmol/L positive control drug N-acetylcystein of final concentration, final concentration 400 μm of ol/L H of final concentration are added after effect 1 hour in the GPx1 and mPEG-GPx1 of 0.08U/mL2O2Effect 12 hours；Treatment group In, it is separately added into 400 μm of ol/L H of final concentration2O2Effect 6 hours, is then separately added into final concentration 0.08U/mL's in cell GPx1 and mPEG-GPx1 is acted on 6 hours.Malondialdehyde Kit (A003-1) is built up by Nanjing, to specifications the step Malonaldehyde index in damaging cells is evaluated.
As shown in figure 21, in 400 μm of ol/L H2O2Induction H9c2 cell is established in oxidative stress model, and model group is intracellular Mda content is 8.313 ± 0.13nmol/mg, the intracellular malonaldehyde of the N-acetylcystein prevention group of positive drug containing 2mM/L Content is 2.67 ± 0.01nmol/mg, 3.25 ± 0.03nmol/mg for the treatment of group, compared to model group cell mda content point It Jiang Di by 67.88 ± 0.12%, 60.95 ± 0.37%；The intracellular mda content of the prevention group of mPEG-GPx1 containing 0.08U/mL For 1.67 ± 0.02nmol/mg, 2.73 ± 0.24 nmol/mg for the treatment of group, dropped respectively compared to model group cell mda content Low 79.91 ± 0.21%, 67.12 ± 2.85%；The intracellular mda content of the prevention group of GPx1 containing 0.08U/mL be 2.03 ± 0.05nmol/mg, 3.03 ± 0.03nmol/mg for the treatment of group reduce by 75.62 compared to model group cell mda content respectively ± 0.54%, 63.59 ± 0.37%.The generation that lipid peroxide chain reaction can be blocked reduces the content of malonaldehyde, And it is stronger in the GPx1 reduction lipid peroxide ability H9c2 cell after mPEG modification.

Claims (6)

1. a kind of polyethyleneglycol modified recombination glutathione peroxidase GPx1 mutant, structural formula are as follows：
Wherein GPx1 indicates that recombination glutathione peroxidase GPx1 mutant, mPEG indicate mono methoxy polyethylene glycol.
2. the preparation of the polyethyleneglycol modified recombination glutathione peroxidase GPx1 mutant of one kind described in claim 1 Method, it is characterised in that：Using the straight-chain mono methoxy polyethylene glycol succinimidyl butanoate of activation as dressing agent, with Recombination glutathione peroxidase GPx1 mutant reacts 10~60min under the conditions of 4~37 DEG C, pH=6.0~9.0, adds Enter terminator glycine and terminate reaction, dialysis removal by-product obtains polyethyleneglycol modified recombination glutathione peroxidase Enzyme GPx1 mutant；Wherein, the poly- second of straight-chain mono methoxy of glutathione peroxidase GPx1 mutant and activation is recombinated The dosage mass ratio of glycol succinimidyl butanoate is 1：10~120.
3. a kind of system of polyethyleneglycol modified recombination glutathione peroxidase GPx1 mutant as claimed in claim 2 Preparation Method, it is characterised in that：Recombinate the poly- second of straight-chain mono methoxy of glutathione peroxidase GPx1 mutant and activation The dosage mass ratio of glycol succinimidyl butanoate is 1：60；Reaction condition is 20 DEG C, reacts 30min under the conditions of pH=8.5.
4. a kind of system of polyethyleneglycol modified recombination glutathione peroxidase GPx1 mutant as claimed in claim 2 Preparation Method, it is characterised in that：The straight-chain mono methoxy polyethylene glycol succinimidyl butanoate of activation through the following steps that It is prepared,
(1) 1.0~3.0g of mono methoxy polyethylene glycol of molecular weight 2000~12000 is weighed, 2~6mL N, N- dimethyl is added Formamide sufficiently dissolves；
(2) 0.06~0.18g succinic anhydride is added into step (1) solution, mixes, sealing；
(3) step (2) mixture is reacted 2~4 hours under the conditions of 90~100 DEG C；
(4) step (3) reaction solution is cooled to room temperature, be added 0.233~0.699g n-hydroxysuccinimide, 0.413~ 1.238g 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride, heating make it dissolve；
(5) step (4) mixture is reacted 8~12 hours under the conditions of 25~30 DEG C, is cooled to room temperature；
(6) reaction solution after step (5) cooling is added dropwise 20~60mL ether, gained was precipitated under the conditions of 0~8 DEG C Filter, vacuum drying, then desciccate is dissolved with methylene chloride, then precipitated again with ether；It repeats by the filtering of gained precipitating, very Empty drying, desciccate are dissolved with methylene chloride, are used ether precipitation process 3~5 times again；
(7) step (6) products therefrom is dried in vacuo to get the straight-chain mono methoxy polyethylene glycol succinimide of activation is arrived Butyrate.
5. application of the recombination glutathione peroxidase GPx1 mutant described in claim 1 in anti-oxidant.
6. polyethyleneglycol modified recombination glutathione peroxidase GPx1 mutant described in claim 1 is in anti-oxidant Application.
CN201810823075.8A 2018-07-25 2018-07-25 Polyethyleneglycol modified recombination glutathione peroxidase GPx1 mutant, preparation method and anti-oxidant application Pending CN108893454A (en)

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