CN107400167A

CN107400167A - GST SOD1 X R9 fusion proteins and preparation method and application

Info

Publication number: CN107400167A
Application number: CN201710572456.9A
Authority: CN
Inventors: 潘剑茹; 李玲玲
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2017-07-14
Filing date: 2017-07-14
Publication date: 2017-11-28
Anticipated expiration: 2037-07-14
Also published as: CN107400167B

Abstract

The present invention relates to fusion protein field, particularly provide a kind of GST SOD1 X R9 fusion proteins and preparation method and application, using gene recombination technology, according to the gene order of human copper zinc superoxide dismutase in Genbank, fusion connection 1-9Nac MBP and cell-penetrating peptide R9, SOD1 X R9 are obtained, the SOD1 X R9 genes of synthesis are inserted into the plasmids of pGEX4T 1 containing GST sequences, the SOD1 X R9 expression plasmids of pGEX4T 1 are obtained, then in e. coli bl21（DE3）Middle expression obtains the soluble protein GST SOD1 X R9 of high expression quantity.Fusion protein GST SOD1 X R9 not only have GST and SOD vigor in the present invention, and GST SOD1 X R9 can be worn film by R9 and enter cell in normal cellular environment, removed the unnecessary free radical of intracellular, protected cells from oxidative damage.

Description

GST-SOD1-X-R9 fusion proteins and preparation method and application

Technical field

The present invention relates to fusion protein field, relates more specifically to GST-SOD1-X-R9 fusion proteins and preparation method thereof With application.

Background technology

Malignant tumour is that current serious influences one of human health, the principal disease for threatening human life, cancer, heart and brain blood Pipe disease and contingency, it is the three big causes of death of world today's All Countries.Radiotherapy is having for the row of killing tumour The means of effect, carry the treatment of 2/3 tumour patient.But radiation injury side effect is big caused by free radical caused by radiotherapy, The local and overall resistance of body is seriously undermined, patient is often forced interruption and put because of the generation of these side effects Treat；Radiation injury side effect can also produce some anaphase effects, influence the life quality even threat to life of patient.

The effect of ionising radiation has two kinds, be respectively directly act on and indirectly-acting.Directly effect refers to that ray directly will Energy transmission causes ionization and excited, cause the change of molecular structure and the forfeiture of bioactivity to biomolecule.Indirectly-acting Then refer to ray and act on water first, first pass through the radiolysis activation oxidizing ferment and nitric oxide synthase of cell reclaimed water, cause moisture The activation of son and the generation of free radical, are then remake for biomolecule by free radical, cause their damage.Ionising radiation Mitochondrial function may also be disturbed, so that lipid, protein, core DNA（nDNA）And mitochondrial DNA（mtDNA）Produce notable Change.

Because the direct effect of ray needs dry environment, the water content of organism is big, therefore body radiation insult master To be caused by the indirectly-acting radiated.The indirectly-acting of radiation, which passes through, produces free radical outside the ionization in the cell of water, then certainly By the further damaging cells of base and tissue, so as to cause body injury.Radioprotectant and various radiation therapy medicines pass through work Played a role for one or several symptoms of radiotherapy side effect, be such as used for anti-inflammatory, anti-infectious medicine.Since radiation damage The main reason for hindering produces the synthesis result of primary reaction and the secondary reaction of active oxygen radical for radiation, removes free radical Polyphenoils is maximally effective radioprotector naturally.

Natural, such as superoxide dismutase（SOD）, catalase（CAT）, glutathione peroxidase （GSH-Px）, glutathione reductase etc., by being acted on from different free radicals such as superoxide anion etc., free radical can be taken precautions against and drawn The damage of hair.Low molecular mass antioxidants such as ascorbic acid, tocopherol, Tea Polyphenols, mercaptan（Such as glutathione）Deng can also provide Anti-oxidative defense.In this case, oxidant by the hindrance blocks of hydrogen donors or terminates free radical chain reactionses chain, Cause damage so as to prevent and suppress oxygen radical.

So far, in existing anti-oxidative radioprotector, be applied to clinical radioprotector have Ah Meter Fu Ting and superoxide dismutase（SOD）.Amifostine（AMFT）Also known as WR-2721, it is artificial synthesized sulfydryl class synthesis Thing, it is approved for dry caused by head and neck cancer radiotherapy within 99 years, but its is expensive, domestic parenteral solution every（0.4 mg）Price Up to 800 ~ 1200 yuans, and there is larger toxicity, most of patient can be made dizziness, Nausea and vomiting etc. occur bad Reaction, these drawbacks cause its Clinical practice to gradually decrease.

SOD is a kind of natural anti-oxidation enzyme, is the radioprotector being most worth mentioning.Because ionising radiation can generate super oxygen Free radical, this free radical activity is high, extremely harmful to cell, and SOD is that can uniquely removing of being found so far is this The enzyme of free radical.

SOD is a kind of highly important endocellular enzyme, the superoxide anion of separate sources in its energy catalytic body（O^2-）Generate oxygen Gas（O₂）And hydrogen peroxide（H₂O₂）, hydrogen peroxide further can generate water in the presence of peroxidase or catalase （H₂O）And oxygen, have the function that to protect body from ultra-oxygen anion free radical toxicity, but by its molecular size and other spies The strict limitation of property, can not pass through cell membrane.Early in 70 mid-nineties 90s, people have found that Cu, and Zn-SOD radiation proof is made With, but hydrone is by free radical caused by ionising radiation or macrophage respiratory burst etc., be not only present in it is extracellular, carefully The content of intracellular is quite a few, the free radical outside a scavenger-cell, can not effect a radical cure radiation insult naturally.

Existing transgene method can improve superoxide dismutase in tissue (SOD) level, the knot of zoopery Fruit shows, after SOD enters cell, can prevent or reduce catarrh, esophagitis and the pulmonary fibrosis of radiation induction.Although with Upper research all confirms that entering cell by SOD by transgene method has good preventive effect to a variety of radiation insults, but Gene therapy is implemented also relatively difficult on mankind.

The transmembrane peptides such as TAT and R9, cell itself can be not only entered with cross-film, and pass through chemical method crosslinking or genetic engineering The mode of method fusion, the albumen and other external source medicines that can carry more than 50 kinds bring cell into, or even the external source that can carry it Medicine passes through blood-brain barrier, and into brain tissue, many external source macromolecule proteins of what is more important remain in that its bioactivity.

We once use technique for gene engineering in early days, and transmembrane polypeptide TAT-PTD and people Cu is merged with genetic engineering means, Zn superoxide dismutases（SOD1）, obtain SOD1-TAT fusion proteins, it was demonstrated that, without acute toxicity, stability is strong, has excellent for it Different cross-film performance, or even blood-brain barrier is may span across, to being had good radioactive protection effect by photo cell and mouse.This basis On, we are with TAT-PTD SOD1 and glutathione s-transferase（GST）Two kinds of antioxidases are merged, and have been obtained simultaneously GST-TAT-SOD1 with SOD1 and GST activity.GST is that vivo biodistribution converts one of most important II phase metabolic enzyme, and it can It is cell antibody Monoclonal, the main detoxification system of anticancer change to be catalyzed the association reaction of glutathione and a variety of free radicals.In cell In zoopery, it has been found that there is the GST-TAT-SOD1 of two kinds of oxidation resistances to being made by the protection according to normal liver cell It is better than the SOD1-TAT of single-action with radioactive protection ability, defeats clinical application amifostine completely.But for all radioprotectors, One query that can not be ignored is：The protective agent, can or can not be to tumor group while protecting normal structure from radiation insult Knit and also produce protective effect, so as to influence the effect of radiotherapy.The famous small molecule antioxidant such as vitamin E, although can subtract The damage of light radioactive normal structure, but they also have very strong protective effect to tumor tissues simultaneously, so that they Application in terms of radioactive protection is restricted.

Using economic benefits and social benefits antioxidase substitution single-action antioxidase, on the basis of lifting its radioactive protection effect, Wo Menjin One step uses the stronger TAT derivative R9 transmembrane peptides of transmembrane ability（RRRRRRRRR）Substitute TAT, build and express to obtain cross-film The more preferable economic benefits and social benefits antioxidase GST-SOD1-R9 of function.

In order to further eliminate the protective effect that economic benefits and social benefits antioxidase may have to tumour cell, targeting protection is normal Cell, we obtain it is new can be after cross-film economic benefits and social benefits antioxidase GST-SOD1-R9, for the microenvironment of tumour cell, again It is transformed.

MMP2（MMP 2）It is to participate in the protease that extracellular matrix components decompose, is that tumour growth must Need, and be all over-expressed in almost all kinds of tumour, therefore be the mark of malignant tumour.R. Tsien is ground Study carefully group and be directed to this tumor microenvironment at first, design tumour-specific transmembrane peptides delivery vehicles ACPP, ACPP is by positively charged The cell-penetrating peptide R9 of lotus, can be by matrix metalloproteinase（MMPs）The connection 1-9Nac MBP of enzymolysis（PLGLAG）And with negative electrical charge 9 Asp composition anionic peptides D9 form.ACCP makes cell-penetrating peptide by the positive charge in anionic peptides D9 with cell-penetrating peptide R9 R9, which loses, wears film activity, cannot be introduced into normal cell, but when it reaches tumour cell, can pass through the MMP in tumor stroma 2 enzymolysis cleavage of peptide, remove anionic peptides, regain R9 cell-penetrating peptides and wear film activity so as to which selectivity enters tumour cell.Make In this way, specificity fluorescent mark successfully is carried out to tumour cell or conveyed cancer therapy drug specificity thin into tumour Born of the same parents.

Using identifications and enzymolysis property of the MMP 2 of high expression in tumor microenvironment to cracking 1-9Nac MBP, we construct brand-new melt Hop protein GST-SOD1-X-R9.New albumen not only has GST and SOD vigor, also by economic benefits and social benefits antioxidase GST-SOD1 with The connection 1-9Nac MBP inserted between transmembrane peptides R9 is provided with targeting and wears film ability.GST-SOD1-X-R9 can in normal cellular environment Film is worn by R9 and enters cell, but its connection 1-9Nac MBP loses R9 cell-penetrating peptides by MMP2 enzymolysis in tumour cell environment, it is remaining GST-SOD1 cannot be introduced into tumour cell, be achieved in the radioprotective effect of targeting.

The content of the invention

Radiation damage is prevented and treated it is an object of the invention to provide GST-SOD1-X-R9 fusion proteins and preparation method and its preparing Application in vulnerary thing, to make up the defects of GST-SOD1-R9 is without targeting cross-film.GST-SOD1-X-R9 in the present invention melts Enter to hop protein energy targeting normal structure protection normal structure, without into tumor tissues.

The technical scheme that the present invention takes is as follows：

GST-SOD1-X-R9 fusion proteins, comprising human copper zinc superoxide dismutase SOD1, can be by matrix metalloproteinase MMP- Connection 1-9Nac MBP that 2 and MMP-9 is identified and degraded, with nine arginic cell-penetrating peptide R9, glutathione s-transferase GST.Connection The amino acid sequence of 1-9Nac MBP is PLGLAG；Cell-penetrating peptide R9 amino acid sequence is RRRRRRRRR.

Its protein sequence of GST-SOD1-X-R9 fusion proteins is as shown in SEQ ID NO.1：

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIR YIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTH PDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSP EFPGRLERPHRDMATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHF NPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAG SRLACGVIGIAQPLGLAGRRRRRRRRR。

The preparation method of the GST-SOD1-X-R9 fusion proteins is：

Using gene recombination technology, according to human copper zinc superoxide dismutase in Genbank (SOD1) gene order, fusion can The connection 1-9Nac MBP for being identified and being degraded by matrix metalloproteinase MMP-2 and MMP-9（PLGLAG）And arginic worn with nine Film peptide R9（RRRRRRRRR）, and the SOD1-X-R9 genes of synthesis are inserted into pGEX4T-1 plasmids, obtain pGEX4T-1-SOD1- X-R9 expression plasmids, then in e. coli bl21（DE3）In obtain the solvable GST-SOD1-X-R9 of high expression quantity.Pass through sulphur Sour ammonium precipitation and GST affinity chromatographys extract the GST-SOD1-X-R9 fusion proteins of high-purity from bacterial cell disruption liquid.The GST- SOD1-X-R9 fusion proteins can be used for preparing preventing and treating radiation insult medicine.

The GST-SOD1-X-R9 fusion proteins are 0.5-1.5 mg/ml using protein concentration；By what is be sterile filtered GST-SOD1-X-R9 fusion proteins are with sterile PBS or normal saline dilution to concentration.Application method is 2h- before radiation 3h carries out intramuscular injection or intravenous injection.

The advantage of the invention is that：

（1）Present invention structure obtains GST-SOD1-X-R9 fusion proteins, it was demonstrated that GST-SOD1-X-R9 can be by rich in matrix metal The tumor microenvironment matrix metalloproteinase digestion of protease, it is impossible to which cross-film enters inside tumor cells, so as to play targeting Property protection normal structure effect.

(2) structure for connecting two kind antioxidases of the 1-9Nac MBP on being merged generates influence, improves two kinds of antioxygens of fusion The ratio for changing enzyme is lived, and makes the GST-SOD1-X-R9 of same dose have preferably protection to make than GST-SOD1-R9 normal tissue With.

The particular use of medicine of the present invention：

（1）Available for preventing or weakening cancer patient's side effect caused by radiotherapy, such as induced lung injury, radioactivity hematopoiesis system System damage and hepatic radiation-induced injury etc..

（2）Available for protection nuclear explosion instantaneous radiation and the radiation injury of fission product, in atomic energy usually and flat interest The pre- antiradiation injury in, aerospace personnel is set to prevent the universe injury of space, prevention long-time, the modern communication instrument of low dosage Electromagnetic radiation damage etc..

（3）Available for preventing or mitigate cancer patient's radical damage caused by chemotherapeutics in chemotherapy.

（4）Available for preventing or mitigate other oxidative damages caused by free radical.

The pharmacological action of medicine of the present invention：

Fusion protein GST-SOD1-X-R9 not only has GST and SOD vigor, the GST- in normal cellular environment in the present invention SOD1-X-R9 can be worn film by R9 and enter cell, removed the unnecessary free radical of intracellular, protected cells from oxidative damage.Swollen In oncocyte environment, due to being all overexpressed MMP2 in almost all kinds of tumour（MMP 2）, GST- X connection peptides in SOD1-X-R9 are digested by MMP-2 to be broken, and so as to lose R9 cell-penetrating peptides, remaining GST-SOD1 cannot be introduced into Tumour cell, it is achieved in the protective action of targeting.

Brief description of the drawings

Fig. 1 recombinant plasmids pGEX-4T-1-SOD1-X-R9 structure.M：DNA Marker；1:Recombinant plasmid pGEX4T- 1-SOD1-X-R9 ；2:EcoR I and the digestion pGEX4T-1-SOD1-X-R9 of Xho I product.

The purifying of Fig. 2 GST-SOD1-X-R9 fusion proteins.1：0-80% ammonium sulfate precipitations dissolve supernatant；2−3：GST is affine Chromatography penetrates peak 1 and 2；4−9：The eluting peak 1-6 of GST affinity chromatographys；M：Marker.

The digestion result of Fig. 3 GST-SOD1-X-R9 fusion proteins.M：Marker；1：Clostridiopetidase A；2：Clostridiopetidase A IV+ APMA； 3：Simple GST-SOD1-X-R9 is not incubated；4：Simple GST-SOD1-X-R9 is incubated 3h；5：GST-SOD1-X-R9+ Clostridiopetidase A IV+APMA.

The cross-film effect of Fig. 4 GST-SOD1-X-R9 fusion proteins.

Two kinds of fusion proteins of Fig. 5 to by being influenceed according to mouse body weight,（A）Various dose GST-SOD1-R9 is to by according to mouse body weight Influence；(B) various dose GST-SOD1-X-R9 by according to mouse body weight on being influenceed.

Two kinds of fusion proteins of Fig. 6 by according to mouse peripheral white blood cells to being influenceed.N=5, compared with CON groups, ##P ＜ 0.01, Compared with XRT groups, * P ＜ 0.05.

Two kinds of fusion proteins of Fig. 7 by according to mouse thymus index to being influenceed, n=5, compared with CON groups, ##P ＜ 0.01, with XRT groups are compared, * P ＜ 0.05.

Two kinds of fusion proteins of Fig. 8 by according to mouse index and spleen index to being influenceed.N=5, compared with CON groups, ##P ＜ 0.01, with XRT groups are compared, * P ＜ 0.05.

Fig. 9 is cut into slices by according to mouse spleen pathology.A：CON；B：XRT；C：AMFT+XRT；D：GSXR-1+XRT；E：GSXR-2 +XRT；F：GSXR-3+XRT；G：GSR-1+XRT；H：GSR-2+XRT；I：GSR-3+XRT.

Figure 10-A：Two kinds of fusion proteins by according to mouse liver organization Antioxidant Indexes MDA levels to being influenceed.

Figure 10-B：Two kinds of fusion proteins by according to mouse liver organization Antioxidant Indexes SOD vigor to being influenceed.

Figure 10-C：Two kinds of fusion proteins by according to mouse liver organization Antioxidant Indexes GST vigor to being influenceed.

Figure 10-D：Two kinds of fusion proteins by according to mouse liver organization Antioxidant Indexes CAT levels to being influenceed.

Figure 10-E：Two kinds of fusion proteins by according to mouse liver organization Antioxidant Indexes GSH-PX vigor to being influenceed.

Figure 10-F：Two kinds of fusion proteins by according to mouse liver organization Antioxidant Indexes T-AOC vigor to being influenceed.

Two kinds of fusion proteins of Figure 11 by according to tumor weight to being influenceed.

Embodiment

Embodiment one：GST-SOD1-X-R9 builds characteristic

（One）, GST-SOD1-X-R9 structure

Pass through raw work bioengineering Shanghai（Share）Co., Ltd synthesizes to obtain SOD1-X-R9 complete genome sequence, upper and lower at its Trip introduces EcoR I and the restriction enzyme sites of Xho I respectively, by itself and pGEX-4T-1 plasmids（Contain GST sequences in complete sequence）Use EcoR I and Xho I carry out double digestion, then with T4 DNA ligases by SOD1-X-R9 purify after digestion through glue reclaim with PGEX-4T-1 overnight, conversion e. coli bl21 (DE3), takes out plasmid and carries out EcoR I in 4 DEG C of connections after picking single bacterium colony culture Identified with the double digestions of Xho I, and EcoR I and Xho I are identified that correct positive colony passes through raw work bioengineering Shanghai（Share） Co., Ltd carries out DNA sequencing identification.

（Two）, GST-SOD1-X-R9 purifying and identification

The GST-SOD1-X-R9 fusion proteins that high-purity is extracted in liquid are crushed from bacterium mud by ammonium sulfate precipitation and affinity chromatography. Determine the SOD and GST of GST-SOD1-X-R9 fusion proteins after purification respectively by SOD and GST viability detection kits specification Vigor, with reference to the molecular weight of albumen of SDS-PAGE electrophoretic determinations, identify whether be target protein.

（Three）, GST-SOD1-X-R9 digestion

25ul clostridiopetidase As IV (1mg/ml) are taken, add 5ul APMA immediately, are mixed.1h is incubated at 37 DEG C.20ul concentration is added afterwards It is placed in 37 DEG C of water-baths for 0.5mg/ml GST-SOD1-X-R9 and is incubated 3h.Handled with reduction treatment liquid, 12.5% SDS- PAGE is verified.

（Four）, GST-SOD1-X-R9 cross-film experiment

GST-SOD1-X-R9 fusion proteins are marked with FITC.Take the logarithm growth period L-02 cells use it is thin containing 15% calf serum Born of the same parents' nutrient solution is made into individual cells suspension, is inoculated into 24 orifice plates, per 40 000, hole cell, cultivates 24 h.Suck culture Liquid, adds PBS 2 times, and the mg/ml of 400 μ L 0.125 FITC- albumen, 37 DEG C of effect 3h are separately added into per hole.Inhale Protein liquid removal, add 250 μ L cell pyrolysis liquids with PBS twice（0.5% Triton-X-100 + 0.1% SDS）, whirlpool Whirlpool vibrates 2 min, and after placing 4 DEG C of min of refrigerator 15,1 200 r/min centrifuge 5 min, take 200 μ L of supernatant liquid luciferases Instrument detection is marked, and with the protein content of BCA kit measurement supernatants.Compared with fluorescence intensity corresponding to every μ g cell proteins Compared with the cross-film efficiency of two kinds of fusion proteins.

3 parallel holes of every group of data, statistical disposition is carried out using Excel softwares, make standard deviation is calculated, t is examined etc..With P >0.05 is to differ without conspicuousness.

Experimental result

1st, GST-SOD1-X-R9 structure

The SOD1-X-R9 full genomes fragment of synthesis is returned through EcoRI/XhoI enzymes double zyme cuttings, agarose electrophoresis separation, glue Kit recovery is received, obtains purifying the SOD1-R9 genetic fragments after digestion.By SOD1-R9 genetic fragments with equally being limited through two kinds Expression vector pGEX-4T-1 plasmids after property inscribe ferment treatment processed, by T4 DNA ligases, so as to by SOD1-X-R9 genes Orientation is inserted into the multiple cloning sites of pGEX-4T-1 plasmids, and translation table reaches bacterial strain BL (DE3).Matter is taken out after choosing single bacterium colony culture Grain carries out EcoR I and the digestions of Xho I identification, as shown in figure 1, swimming lane 1 is recombinant plasmid pGEX4T-1-SOD1-X-R9, swimming lane 2 is Recon obtains two purpose bands, about 4946 bp and 510 bp after digestion is identified, respectively with pGEX-4T-1 matter Grain is consistent with SOD1-X-R9 expection molecular weight.Digestion is identified that correct plasmid carries out DNA sequencing identifications, sequencing result with it is pre- The sequence of phase is consistent, demonstrates in the transformant containing the expression vector pGEX-4T-1-SOD1-X-R9 successfully constructed, transformant PGEX-4T-1-SOD1-X-R9/BL21 (DE3) can be used as expression bacterial strain to use.

Digestion is identified that correct plasmid carries out DNA sequencing identifications, sequencing result is consistent with expected sequence, demonstrates Contain the expression vector pGEX-4T-1-SOD1-X-R9, transformant pGEX-4T-1-SOD1-X- successfully constructed in the transformant R9/BL21 (DE3) can be used as expression bacterial strain to use.

2nd, GST-SOD1-X-R9 purifying and identification

Being calculated by Quantity One, GST-SOD1-X-R9 fusion proteins actual molecular weight is about 47 kDa in Fig. 2, and GST-SOD1-X-R9 albumen theoretical molecular is 46 kDa or so, and the two is consistent substantially.Further detected using SOD and GST Kit determines the enzyme activity of the albumen respectively, it was demonstrated that and the SOD of the GST-SOD1-X-R9 albumen of purifying is 2954 U/mg than work, GST is 328 U/mg than work.Comprehensive molecular weight and the result of activity analysis, it may be determined that GST-SOD1-X-R9 fusion proteins obtain Correctly expression.

The SOD of GST-SOD1-R9 albumen is 2388 U/mg than work, and GST is 227 U/mg than work.GST-SOD1-X-R9 eggs The ratio of white two kinds of antioxidases is lived and is above GST-SOD1-R9 albumen.

3rd, digestion：

From the figure 3, it may be seen that after GST-SOD1-X-R9 and clostridiopetidase A IV is incubated certain time, protein band concentration is bright at original molecule amount Aobvious to reduce, less than occurring new protein band at original molecule amount, this shows that GST-SOD1-X-R9 can be lost by clostridiopetidase A IV digestions Remove R9 transmembrane peptides.

4th, cross-film is tested：

As shown in Figure 4, compared with the control for being not added with albumen, GST-SOD1-X-R9 fusion proteins have extremely significant cross-film ability（P < 0.01）.

Embodiment two：Protective effect to normal mouse radiation insult

1st, normal raising three days, free diet before mouse radiation.10 groups will be randomly divided into（ n=5 n=5）：Normal group（CON）, Radiocontrast group（XRT）；Amifostine group（AMFT+XRT ）；The experiment of GST-SOD1-R9, GST-SOD1-X-R9 fusion protein Group；

Normal group（CON）：0.5 mL physiological saline of simple intraperitoneal injection, no radiation；

Simple radiation group（XRT）：The preceding mL physiological saline of abdominal cavity note 0.5 is radiated, is irradiated；

Amifostine group（AMFT+XRT）：0.5 mL is injected intraperitoneally by 200 mg/kg dosage body weight ratios in 0.5 h before radiation Amifostine；

Experimental group（GST-SOD1-R9+XRT、GST-SOD1-X-R9+XRT）：2 h inject 0.5 mL and merged accordingly before radiation Albumen, it is 0.5 mg/mL, 0.75 mg/mL, 1.5 mg/mL respectively to be divided into high, normal, basic three dosage.It is denoted as by low middle height GS1R-1, GS1R-2, GS1R-3；GS1XR-1, GS1XR-2, GS1XR-3.

2nd, condition is irradiated

Mouse is fitted into homemade fixing device, receives disposable full-body exposure, every batch of 5 mouse.Radioactive source is X ray, Exposure dose is 6Gy, close rate 2Gy/min.

3rd, Testing index

3.1 mouse weights change

7d is commonly fed after irradiation, the daily identical time weighs and recorded to mouse.By daily body weight all divided by pre-irradiation Body weight, make mouse weight change curve.

3.2 peripheral white blood cells change

After vertebra puts to death mouse, after Hearts take blood vertebra to put to death mouse immediately, Hearts take blood vertebra to put to death small immediately After mouse, Hearts take the μ L of blood 20 immediately, with 380 μ L leucocyte dilution（The mL+1%+1% gentian violets of glacial acetic acid 2.0 The mL of gentian violet 1.0, distilled water are settled to, and distilled water is settled to 100 mL, are mixed）Take drop to enter in tally after dilution, stand 2-3 min, it is placed in ordinary optical microscope after leucocyte sinking and is counted.

3.3 Thymus and Spleen index

The thymus gland and spleen of mouse are won, blots blood with cotton, and weigh.By thymus gland and spleen weight（g）Divided by before putting to death The weight of mouse（g）, multiplied by the Thymus and Spleen index for 100, just obtaining mouse.

Thymus index %=（Mouse weight before thymic weight/execution）×100；

Index and spleen index %=（Mouse weight before spleen weight/execution）×100.

3.4 mouse spleen pathology normal observations

Fritter spleen is fixed in 10% neutral formalin, specimens paraffin embedding slices, the observation of HE stained tissues.

3.5：The detection of internal organs oxidation resistance

Hepatic tissue is made to 10% homogenate, 4000rmp/min centrifugation 20min, supernatant is solution to be measured.Reagent is built up with Nanjing Box determines CAT, MDA, SOD, GST, T-AOC, GSH-Px.

4th, statistical analysis：Examined using the Student's t- in excel 2003.

Experimental result：

1：By Fig. 5（A、B）As can be seen that contrast blank group and radiocontrast group, it is known that under mouse weight occurs seriously after radiation It is sliding.But at the 3rd day of raising, the body weight of all mouse was all slowly being gone up.It is seen that amifostine is given in advance By better than XRT groups according to mouse, daily body weight rise situation.From in figure, middle dose group GST-SOD1-X-R9 effect is imitated Fruit is better than amifostine and GST-SOD1-R9.

2：As shown in Figure 6, compared with normal group, there is extremely significant decline in peripheral white blood cells after mouse irradiation（P ＜ 0.01）, only the 14.8% of normal group.Compared with radiocontrast group, the radiation murine interleukin for giving amifostine in advance counts It is now significant to rise（P ＜ 0.05）, about improve 62.9%.Compared with radiocontrast group, two kinds of fusions of various dose are given in advance Albumen, there is the protection of certain protective effect, GS1R-2, GS1R-3 and GS1XR-3 to mouse to the hemopoietic system of mouse Effect reaches the level of signifiance（P ＜ 0.05）, make 103.7%, 107.4% and has been respectively increased by the number of white blood cells according to mouse 77.8%.The protective effect of two kinds of fusion proteins is all better than amifostine.

3：As shown in Figure 7, compared with normal group, its thymus index significantly reduces radiocontrast group（P ＜ 0.01）, it is only The 54.6% of normal group.The pre- radiation mouse for giving amifostine can make to be improved by the thymus index according to mouse compared with radiation group 8.4%.Pre-irradiation injects the mouse of free radical scavenger, and thymus index is also improved, and from Fig. 9, we draw：GS1R-1 and GS1R-3 makes 41.5% and 52.8% is respectively increased by the thymus index according to mouse；GS1XR-3, GS1XR-2, GS1XR-1 make by according to small The thymus index of mouse has been respectively increased 38.7%, 58.6% and 8.6%, and wherein GS1R-3 and GS1XR-1 reach the level of signifiance（P ＜ 0.05）.Middle dose group GST-SOD1-X-R9 action effect is better than amifostine and GST-SOD1-R9.

4：As seen from Figure 8, the index and spleen index of radiocontrast group mouse is substantially less than normal group, only normal group 25.9%.Compared with radiocontrast, giving amifostine can improve by the index and spleen index according to mouse, but not have remarkable result, pre-irradiation Freer base scavenger, can make to be improved by the index and spleen index according to mouse, and GS1R-3 makes index and spleen index significantly improve 17.3%； GS1XR-2, GS1XR-3 make the index and spleen index of mouse that 21.2% and 17.3% be respectively increased.Middle dose group GST-SOD1-X-R9's Action effect is better than amifostine and GST-SOD1-R9.

5：The pathological section result of spleen tissue is as shown in figure 9, red pulp in the spleen tissue of normal mouse（Dark portion in figure Point）And white pulp（Light-colored part in figure）Clear-cut, after mouse irradiation, spleen is impaired serious, and white pulp amount greatly reduces.Before radiation Amifostine and fusion protein are given, can effectively maintain the amount of white pulp.GST-SOD1-R9 protection effect can be found from figure Not as GST-SOD1-X-R9 and amifostine, GST-SOD1-X-R9 effect is again better than amifostine.It was found from E figures, middle dose Amount group GST-SOD1-X-R9 action effect is better than amifostine and GST-SOD1-R9.

6：By Figure 10 analysis fusioning proteins to by the protection effect according to mouse liver organization.Mouse by after Systemic radiation, its Mouse liver antioxidant system, MDA is horizontal significantly to be risen, in addition to GST vigor, SOD vigor, GSH-PX vigor, CAT vigor and T- AOC vigor is remarkably decreased.Half an hour gives amifostine before radiation, improves with can reducing MDA levels and the conspicuousness of liver SOD, GSH-PX and T-AOC vigor.2 h give fusion protein and can made by the horizontal reductions of MDA according to mouse liver organization before radiation, SOD vigor, GSH-PX vigor, T-AOC vigor and CAT vigor significantly rise.GS1R-3 is suitable with amifostine effect, GS1XR-2, GS1XR-3 are to better than GS1R and amifostine by the radioactive protection effect according to mouse.Wherein again with middle dose group GST- SOD1-X-R9 action effect is optimal.

Embodiment 3：The influence of tumour after being radiated to tumor-bearing mice

（1）Knurl is subcutaneously injected into：

Cell culture：By Hep G2 cells use containing 10% NBCS, 1% dual anti-DMEM culture mediums, be placed in saturated humidity, 5%CO₂, cultivate under the conditions of 37 DEG C, when cell length to 70-80% degrees of fusion, pancreatin had digestive transfer culture culture；

Cell subcutaneous implantation：When culture cell reaches certain amount, after cleaning 2 times with serum-free cell culture medium, pancreatin disappears Change and collect cell, cell count is carried out with cell counting count board.1000rpm, 5min collect cell, use serum-free cell culture medium The cell of collection is diluted to 2 × 10⁷Individual/ml, by 1:1 ratio adds after matrigel mixes and cell suspension quickly is pressed into 100 μ L/ only carries out nude mice（20g or so）Forelimb oxter is subcutaneously injected, and must mix cell suspension before per injection.All realities Test laminar flow of the mouse under the conditions of constant temperature (25 ± 1) DEG C, constant humidity (50% ± 10%), no-special pathogen (SPF) Raised in frame, free diet.

（2）Packet：10 d or so, select tumor size and grow to 100 mm³~200 mm³Nude mice be used for test.With Machine is grouped, every group 7：

Experimental group（GST-SOD1-R9+XRT、GST-SOD1-X-R9+XRT）：Effect in being tested according to normal mouse radioactive protection Best dose point, 2 h inject 0.5 mL high dose GST-SOD1-R9+XRT high doses respectively before radiation（GS1R-3,1.5 mg/mL）With middle dosage GST-SOD1-X-R9+XRT（GS1XR-2,0.75mg/mL）

（3）Radiation：Mouse is fitted into homemade fixing device, receives disposable local irradiation（Tumor locus）, 7 every batch Mouse.Radioactive source is X ray, exposure dose 15Gy, close rate 2Gy/min.

（4）Raising：Continue raising 21 days after radiation.

（5）Anaesthetize and put to death nude mice, peeling operation tumor resection tissue is weighed.

Experimental result：

As shown in Figure 11, compared with blank group tumor-bearing mice, the tumor-bearing mice tumour knurl weight of simple radiation group is remarkably decreased, radiation The knurl weight of the preceding tumor-bearing mice for having injected amifostine in advance is 2 times or so of simple radiation group, has injected height before radiation in advance The knurl weight of dosage GST-SOD1-R9 tumor-bearing mice is close with preform injection amifostine group, injection in advance before only radiating The knurl weight of middle dosage GST-SOD1-X-R9 tumor-bearing mice is close to simple radiation group.It these results suggest that, amifostine and GST- SOD1-R9 has certain protective effect to tumour, and GST-SOD1-X-R9 is because cannot be introduced into tumour, to tumour unprotect Effect.

The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, it should all belong to the covering scope of the present invention.

SEQUENCE LISTING

<110>University of Fuzhou

<120>GST-SOD1-X-R9 fusion proteins and preparation method and application

<130> 3

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 408

<212> PRT

<213>GST-SOD1-X-R9 fusion proteins

<400> 1

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg

210 215 220

Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp Met

225 230 235 240

Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly

245 250 255

Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp

260 265 270

Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His

275 280 285

Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe

290 295 300

Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His

305 310 315 320

Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp

325 330 335

Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile

340 345 350

Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys

355 360 365

Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu

370 375 380

Ala Cys Gly Val Ile Gly Ile Ala Gln Pro Leu Gly Leu Ala Gly Arg

385 390 395 400

Arg Arg Arg Arg Arg Arg Arg Arg

405

<210> 2

<211> 6

<212> PRT

<213>Connect 1-9Nac MBP

<400> 2

Pro Leu Gly Leu Ala Gly

1 5

<210> 3

<211> 9

<212> PRT

<213>Cell-penetrating peptide R9

<400> 3

Arg Arg Arg Arg Arg Arg Arg Arg Arg

1 5

Claims

1.GST-SOD1-X-R9 fusion proteins, it is characterised in that：The fusion protein includes human copper zinc superoxide dismutase SOD1, can by matrix metalloproteinase MMP-2 and MMP-9 are identified and are degraded connection 1-9Nac MBP, with nine arginic cell-penetrating peptides R9, glutathione s-transferase GST.

2. GST-SOD1-X-R9 fusion proteins according to claim 1, it is characterised in that：Connect the amino acid sequence of 1-9Nac MBP For PLGLAG；Cell-penetrating peptide R9 amino acid sequence is RRRRRRRRR.

3. the preparation method of GST-SOD1-X-R9 fusion proteins as claimed in claim 1, it is characterised in that：Using gene weight Group technology, according to the gene order of human copper zinc superoxide dismutase in Genbank, fusion can be by matrix metalloproteinase MMP- Connection 1-9Nac MBP that 2 and MMP-9 is identified and degraded and with nine arginic cell-penetrating peptide R9, and by the SOD1-X-R9 bases of synthesis Because inserting the pGEX4T-1 plasmids containing GST sequences, pGEX4T-1-SOD1-X-R9 expression plasmids are obtained, then in Escherichia coli BL21（DE3）In obtain the solvable GST-SOD1-X-R9 of high expression quantity；By ammonium sulfate precipitation and GST affinity chromatographys from thalline The GST-SOD1-X-R9 fusion proteins of high-purity are extracted in broken liquid.

4. GST-SOD1-X-R9 fusion proteins as claimed in claim 1 are preparing the application in preventing and treating radiation insult medicine.

5. application according to claim 1, it is characterised in that：GST-SOD1-X-R9 fusion proteins use protein concentration For 0.5-1.5 mg/ml；By the GST-SOD1-X-R9 fusion proteins being sterile filtered with sterile PBS or normal saline dilution To concentration.

6. GST-SOD1-X-R9 fusion proteins as claimed in claim 1 are preparing preventing and treating oxidative damage as caused by free radical Application in medicine.