CN107353345A - MTS SOD2 recombinant proteins and preparation method and application - Google Patents

Abstract

The present invention relates to a MTS-SOD2 recombinant protein and its preparation method and application. The whole gene sequence of human MTS-SOD2 is synthesized by means of genetic engineering and inserted into the prokaryotic expression vector pGEX-4T-1 with GST. The GST‑MTS‑SOD2 fusion protein expression plasmid was successfully constructed. Then, transform the recombinant plasmid pGEX‑4T‑1‑MTS‑SOD2 into Escherichia coli BL21(DE3), induce Escherichia coli to express the fusion protein, and obtain a highly expressed soluble GST‑MTS‑SOD2 fusion protein, which is adsorbed by GST affinity resin After GST‑MTS‑SOD2, the GST tag was cut off by thrombin, and then purified by a heparin affinity column to obtain an electrophoretic pure SOD2 recombinant protein. The protein has SOD activity while carrying the MTS leader peptide. The MTS‑SOD2 recombinant protein can be used to prepare radioprotective drugs.

Description

MTS-SOD2 recombinant protein and its preparation method and application

technical field

The invention relates to a MTS-SOD2 recombinant protein and its preparation method and application, belonging to the field of biotechnology.

Background technique

Radiotherapy is one of the important treatment methods for cancer patients, but radiotherapy will bring various side effects, that is, radiation damage. Radiation damage will seriously affect the quality of life of patients, so radioprotectants should be used in clinical radiotherapy. Amifostine is currently the only radioprotectant approved by the Food and Drug Administration for clinically reducing the toxicity of radiotherapy, but it has shortcomings: 1. Because it is a synthetic compound, it is easy to cause nausea in patients , vomiting, low blood pressure and other symptoms; 2. The effect can only be guaranteed if it is used 20-30 minutes before radiation; 3. The intravenous injection is highly toxic, but it cannot be taken orally; 4. The cost is high and the protective effect on the CNS is small, etc. . Therefore, it is of great significance and necessity to develop new safe and reliable radioprotective drugs for the quality of life of patients and the effect of radiotherapy.

The superoxide dismutase (SOD) family is required to protect oxygen-utilizing cells from the toxicity of reactive oxygen species (ROS) generated during normal metabolism. In addition to protective proteins, these enzymes are also key components of signaling pathways that regulate cell physiology. SOD catalyzes the reaction, followed by removal of hydrogen peroxide with catalase (CAT) and peroxidase. There are 3 known forms of SOD in mammalian cells: the copper- and zinc-containing superoxide dismutase (Cu, Zn-SOD, SOD1) found mainly in the cytoplasm and nucleus, the manganese-containing superoxide dismutase found in the mitochondria Oxide dismutase (Mn-SOD, SOD2) and extracellular superoxide dismutase (EC-SOD, SOD3). Mitochondria-localized SOD2 is considered the most important of the 3 SODs because mitochondria are the main ROS production site. Not only is SOD2 essential for the survival of all aerobic organisms, it has also been shown to be a powerful protective and therapeutic agent against oxidative stress-related diseases such as kidney injury, arthritis, chronic inflammatory pulmonary fibrosis, and Radiation damage caused by ionizing radiation.

SOD2 is synthesized in cells in the form of a precursor with a mitochondrial targeting signal peptide (MTS), enters the mitochondrial matrix through its 2 kDa leader sequence MTS, and the peptide is subsequently cleaved to produce mature and functional proteins that play key roles in mitochondria Enzymatically active protein. A series of studies on the 30 kDa human SOD2 isoform (LSA-type-Mn-SOD) found in human liposarcoma cells LSA showed that the leader peptide MTS of SOD2 also has the ability to mediate protein entry into cells. When using the prokaryotic system to express SOD2 recombinantly, in order to obtain a soluble and enzymatically active protein, the leader peptide sequence of SOD2 will be removed, and the monomeric mature protein will be directly expressed. For example, Wang Feng et al. have used the pET15b plasmid in E. The specific activity of the soluble SOD2 recombinant protein expressed in N-terminal His tag was 1890 U/mg.

However, although this method can obtain mature and enzymatically active SOD2, it is difficult for SOD2 without the leader peptide MTS to enter cells and play a role due to the limitation of molecular weight. Therefore, the above-mentioned application of SOD2 is mostly carried out in the form of transgene, which limits the clinical application of SOD2 recombinant protein.

GST is a highly soluble protein, which can be used to increase the solubility of foreign proteins and their expression in E. coli. As one of the commonly used recombination tags, the natural molecular weight of GST is 26 kDa, which is much larger than the His tag, which may affect the structure and function of the foreign protein fused together. The present invention synthesized the full-length human SOD2 gene containing the MTS sequence, cloned it into the pGEX-4T-1 plasmid with the GST tag for the first time, constructed the recombinant plasmid pGEX-4T-1-MTS-SOD2, and expressed it to obtain GST after transformation - MTS-SOD2 fusion protein. The GST-MTS-SOD2 fusion protein was digested to obtain the MTS-SOD2 recombinant protein.

Contents of the invention

The purpose of the present invention is to provide MTS-SOD2 recombinant protein and its preparation method and application, so as to make up for the defect that the existing mature SOD2 cannot transmembrane or the full-length SOD2 has no activity.

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

The sequence of the MTS-SOD2 recombinant protein is shown in SEQ ID NO.1:

MLSRAVCGTSRQLAPALGYLGSRQ KHSLPDLPYDYGALEPHINAQIMQLHHSKHHAAYVNNLNVTEEKYQEALAKGDVTAQTALQPALKFNGGGHINHSIFWTNLSPNGGGEPKGELLEAIKRDFGSFDKFKEKLTAASVGVQGSGWGWLGFNKERGHLQIAACPNQDPLQGTTGLIPLLGIDVWEHAYYLQYKNVRPDYLKAIWNVINWENVTERYMACKK。 (The part in bold is the MTS sequence of the leader peptide).

The preparation method of the GST-MTS-SOD2 fusion protein of the present invention is: using genetic engineering means to synthesize the whole gene sequence of human source MTS-SOD2, and inserting it into the prokaryotic expression vector pGEX-4T-1 with GST, successfully The GST-MTS-SOD2 fusion protein expression plasmid pGEX-4T-1-MTS-SOD2 was constructed. Then, transform the recombinant plasmid pGEX-4T-1-MTS-SOD2 into Escherichia coli BL21(DE3), induce Escherichia coli to express the fusion protein, and obtain a highly expressed soluble GST-MTS-SOD2 fusion protein, which is adsorbed by GST affinity resin After the GST-MTS-SOD2 was cut off the GST tag by thrombin, it was purified by a heparin affinity column to obtain electrophoretic pure MTS-SOD2 recombinant protein.

The MTS-SOD2 recombinant protein is used to prepare radioprotective medicine.

The used protein concentration of the MTS-SOD2 recombinant protein is 2-8 μmol/ml; the sterile filtered MTS-SOD2 recombinant protein is diluted to the used concentration with sterile PBS or normal saline. The method of use is intramuscular injection or intravenous injection 2h-3h before radiation.

Innovation point of the present invention:

(1) In the present invention, the large molecular weight GST tag is used to affect the structure of the fused MTS-SOD2 to change its structure, and finally obtain the MTS-SOD2 recombinant protein with both the leader peptide MTS and good SOD activity.

(2) Under the mediation of MTS, the MTS-SOD2 recombinant protein can enter the cell across the membrane, remove excess free radicals in the cell, and achieve radioprotective effects.

(3) SOD2 has a recognized function of inhibiting cancer cells. The MTS-SOD2 recombinant protein entering cells can protect normal cells from radiation damage and inhibit cancer cells at the same time.

The concrete purposes of medicine of the present invention:

(1) It can be used to prevent or reduce side effects caused by radiotherapy in cancer patients, such as radiation lung injury, radiation hematopoietic system injury and radiation liver injury.

(2) It can be used to protect the instantaneous radiation of nuclear explosions and radiation damage from fission products, prevent radiation damage in the peaceful use of atomic energy in peacetime, enable astronauts to prevent cosmic damage in space, and prevent long-term, low-dose electromagnetic radiation from modern communication tools damage etc.

(3) It can be used to prevent or reduce other oxidative damage caused by free radicals.

The pharmacological action of medicine of the present invention:

The present invention obtains MTS-SOD2 recombinant protein through genetic engineering technology, and the protein not only has the leader peptide MTS but also has good SOD activity. Excess free radicals in the body to achieve radiation protection effect.

Description of drawings

Figure 1 Construction of recombinant plasmid MTS-SOD2. M: DNA marker; 1: pGEX4T-1-MTS-SOD2 recombinant plasmid; 2: identification after pGEX4T-1-MTS-SOD2 double digestion.

Fig. 2 Purification of GTS-MTS-SOD2 and MTS-SOD2 recombinant protein. M: protein marker; 1: The supernatant stock solution after cell crushing; 2: GST-MTS-SOD2 obtained by affinity purification; 3: MTS-SOD2 digested on the GST column; 4: Purified after passing through the heparin column MTS-SOD2.

Figure 3 Stability of MTS-SOD2. (A) Temperature stability of MTS-SOD2; (B) pH stability of MTS-SOD2.

Figure 4 Transmembrane effect of MTS-SOD2. n =3, ** P <0.01 compared with CON group.

Fig. 5 Effect of MTS-SOD2 on body weight of irradiated mice. n=5, compared with normal group ^# P< 0.05, ^## P< 0.01; compared with simple radiation group * P< 0.05, ** P< 0.01.

Fig. 6 Effect of MTS-SOD2 on peripheral blood leukocytes of irradiated mice. n=5, ##P<0.01 compared with CON group, *P<0.05 compared with XRT group.

Fig. 7 Effect of MTS-SOD2 on thymus index of irradiated mice. n=5, ##P<0.01 compared with CON group, *P<0.05 compared with XRT group.

Fig. 8 Effect of MTS-SOD2 on spleen index of irradiated mice. n=5, ##P<0.01 compared with CON group, *P<0.05 compared with XRT group.

Fig. 9A Effect of MTS-SOD2 on the level of antioxidant index MDA in liver tissue of irradiated mice.

Fig. 9B The effect of MTS-SOD2 on the antioxidant index T-AOC in liver tissue of irradiated mice.

Fig. 9C Effect of MTS-SOD2 on the activity of antioxidant index SOD in liver tissue of irradiated mice.

Fig. 9D Effect of MTS-SOD2 on the activity of antioxidant index GST in liver tissue of irradiated mice.

Fig. 9E Effect of MTS-SOD2 on CAT activity of antioxidant index in liver tissue of irradiated mice.

Fig. 9F Effect of MTS-SOD2 on the activity of antioxidant index GSH-PX in liver tissue of irradiated mice.

detailed description

Embodiment 1: Features of MTS-SOD2 Construction

(1) Construction of GST-MTS-SOD2

The human full-length SOD2 gene sequence containing the MTS leader peptide was synthesized by Sangon Bioengineering Shanghai (Co., Ltd.), and EcoRI and XhoI restriction sites were introduced into its upstream and downstream, respectively, and it was combined with pGEX-4T-1 Plasmids (containing the GST tag sequence) were double-digested with EcoRI and XhoI and purified by gel recovery, and then the purified MTS-SOD2 gene fragment and pGEX-4T-1 plasmid fragment were ligated overnight at 4 °C with T4 DNA ligase. Transform Escherichia coli BL21(DE3), pick a single colony for culture, and extract the plasmid for EcoRI and XhoI double enzyme digestion identification, and the positive clones identified by EcoRI and XhoI were identified by DNA sequencing by Sangon Bioengineering Shanghai (Co., Ltd.) Co., Ltd. .

(2) Purification and identification of GTS-MTS-SOD2 and MTS-SOD2

The high-purity GTS-MTS-SOD2 fusion protein was extracted from the broken liquid of the bacterial sludge by ammonium sulfate precipitation and affinity chromatography. Measure the SOD and GST activities of the purified GTS-MTS-SOD2 recombinant protein according to the instructions of the SOD and GST activity detection kits, and combine the protein molecular weight determined by SDS-PAGE electrophoresis to identify whether it is the target protein.

GST-MTS-SOD2 was adsorbed by GST affinity resin, and then the GST tag was cut off by thrombin and purified by heparin affinity column to obtain electrophoretic pure MTS-SOD2 recombinant protein. According to the instructions of the SOD activity detection kit, the SOD activity of the purified MTS-SOD2 recombinant protein was measured respectively, combined with the protein molecular weight determined by SDS-PAGE electrophoresis, to identify whether it was the target protein.

(3) Stability of MTS-SOD2

1: Thermal stability of MTS-SOD2

Take 300 µL of MTS-SOD2 recombinant protein, place them in water baths at 25, 37, 50, 60, and 80 °C for 30 minutes, take them out, and wait until they return to room temperature, then take samples to measure the residual enzyme activity and analyze the thermal stability of the fusion protein .

2: pH stability of MTS-SOD2

Take 300 µL of MTS-SOD2 recombinant protein, adjust it to pH 4, 5, 6, 7, 8 with extensive buffer solution, place it at 4 ℃ for 30 minutes, take it out, wait for it to return to room temperature, take samples to measure the residual enzyme activity, and analyze the fusion protein. pH stability.

(4) Transmembrane effect of MTS-SOD2

The MTS-SOD2 recombinant protein was labeled with FITC, respectively.

L-02 cells in the logarithmic growth phase were prepared into a single cell suspension with cell culture medium containing 15% calf serum, inoculated into a 24-well plate with 40,000 cells per well, and cultured for 24 h. Aspirate the culture solution, add PBS to wash twice, add 400 µL 42 µg/mL FITC-protein to each well, and incubate at 37 °C for 3 h. Aspirate off the protein solution, wash twice with PBS, add 250 µL cell lysate (0.5% Triton-X-100 + 0.1% SDS), vortex for 2 min, place in 4°C refrigerator for 15 min, then centrifuge at 1,200 r/min for 5 min , 200 µL of the supernatant was detected with a fluorescent microplate reader, and the protein content of the supernatant was determined with a BCA kit. The transmembrane efficiency of the two fusion proteins was compared by the fluorescence intensity corresponding to each µg of cell protein.

Experimental results

1: Construction of MTS-SOD2

The synthetic human SOD2 whole gene fragment and pGEX-4T-1 plasmid containing the MTS leader peptide were double-digested with EcoRI and XhoI endonucleases, and the digested MTS-SOD2 gene fragment and pGEX-4T were purified by gel recovery. -1 Plasmid fragment, insert the MTS-SOD2 gene into the multiple cloning site of the pGEX-4T-1 plasmid through T4 DNA ligase. After picking a single colony and culturing, the plasmid was extracted for EcoRI and XhoI digestion and identification. The results are shown in Figure 1. After the recombinant was identified by enzyme digestion, two target bands were obtained, about 4,946 bp and 669 bp, which were consistent with the expected molecular weights of pGEX-4T-1 plasmid and MTS-SOD2 respectively. The correct plasmid identified by enzyme digestion was identified by DNA sequencing, and the sequencing result was consistent with the expected sequence, which proved that the MTS-SOD2 gene had been successfully inserted into the pGEX-4T-1 vector, and the pGEX-4T-1-MTS-SOD2 recombinant plasmid was successfully constructed.

2. Purification and identification of MTS-SOD2

The theoretical molecular weights of GST-MTS-SOD2 fusion protein and MTS-SOD2 recombinant protein are about 51 kDa and 25 kDa respectively, and the actual molecular weights of GST-MTS-SOD2 fusion protein and MTS-SOD2 recombinant protein in Figure 2 are about 46 kDa and 25 kDa respectively . Since the GST-MTS-SOD2 fusion protein is a unique protein fused by two antioxidant enzymes, SOD2 and GST, and has both SOD and GST activities, we further used SOD and GST detection kits to measure the enzyme activities of the protein. . The results showed that the SOD activity of the purified GST-MTS-SOD2 fusion protein was 1788 U/mg, and the GST activity was 150 U/mg. The SOD activity of the purified MTS-SOD2 recombinant protein after digesting the GST-MTS-SOD2 fusion protein was 2000 U/mg. Based on the results of molecular weight and activity analysis, it can be confirmed that the GST-MTS-SOD2 protein is correctly expressed, and the MTS-SOD2 recombinant protein obtained by enzymatic digestion is the expected protein.

3. Stability of MTS-SOD2

It can be seen from Figure 3-A that the activity of the MTS-SOD2 recombinant protein is more sensitive to temperature. When the heat treatment temperature is less than 40°C (under physiological conditions), its activity does not change much, but when the heat treatment temperature exceeds 40°C, the activity changes with With the increase of temperature, the enzyme activity of MTS-SOD2 recombinant protein began to decrease rapidly. After 30 minutes in 60 ℃ water bath, the activity was only about 9%, and after 80 ℃ water bath for 30 minutes, the activity was close to 0.

It can be seen from Figure 3-B that pH also has a great influence on the enzyme activity of MTS-SOD2 recombinant protein, and its SOD activity decreases with the decrease of pH in the range of pH ≤ 7, and in the range of pH 7-8 ( Under physiological conditions) the activity remains basically unchanged, and when the pH exceeds 8, the activity decreases with the increase of pH.

It can be seen from the above that MTS-SOD2 has good stability under physiological conditions and is suitable for clinical application.

4. Transmembrane test:

It can be seen from Figure 4 that compared with the normal control, the MTS-SOD2 recombinant protein has a significant transmembrane ability (P < 0.05).

Example 2: Protective Effect on Mice Radiation Injury

1. The mice were fed normally for three days before irradiation, and they were free to eat and drink. Will be randomly divided into 6 groups (n=5): normal group (CON), radiation control group (XRT); amifostine group (AMFT+XRT); MTS-SOD2 group (MTS-SOD2+XRT).

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

Radiation-only group (XRT): Before radiation, 0.5 mL of normal saline was injected into the abdominal cavity for irradiation;

Amifostine group (AMFT+XRT): 0.5 mL of amifostine was injected intraperitoneally at a dose of 200 mg/kg body weight 0.5 h before radiation;

Experimental group (MTS-SOD2+XRT): 0.5 mL of the corresponding fusion protein was injected 2 hours before radiation, divided into three doses: 2 μM, 4 μM, and 8 μM. According to the low, medium and high records, do MS2-1+XRT, MS2-2+XRT, MS2-3+XRT.

2. Irradiation conditions

Mice were housed in homemade fixtures and received one-time whole-body irradiation, 5 mice per batch. The radiation source is X-ray, the irradiation dose is 6Gy, and the dose rate is 2Gy/min.

3. Detection indicators

3.1 Changes in body weight of mice

The mice were fed for 7 days after irradiation, and the mice were weighed and recorded at the same time every day. Divide the daily body weight by the body weight before irradiation to create a mouse body weight change curve.

3.2 Changes in the number of white blood cells in peripheral blood

Immediately after the mouse was sacrificed by the spine, the heart was dissected to take blood. Gentian violet 1.0 mL, distilled water to 100 mL, mix well) after dilution, take the liquid and drop it into the counting plate, let it stand for 2-3 min, and put the leukocytes into an ordinary optical microscope for counting after sinking.

3.3 Thymus index and spleen index

The thymus and spleen of the mice were removed, the blood was blotted dry with cotton, and weighed. Divide the thymus and spleen weight (g) by the weight of the mouse before sacrifice (g), and multiply by 100 to obtain the thymus index and spleen index of the mouse.

Thymus index%=(thymus weight/mouse body weight before sacrifice)×100

Spleen index%=(spleen weight/mouse body weight before sacrifice)×100

3.4 Routine observation of mouse spleen pathology

A small piece of spleen was fixed in 10% neutral formaldehyde, embedded in paraffin, sectioned, and histologically observed after HE staining.

3.5: Detection of organ antioxidant capacity

The liver tissue was made into a 10% homogenate, centrifuged at 4000rmp/min for 20min, and the supernatant was the solution to be tested. CAT, MDA, SOD, GST, T-AOC, GSH-Px were measured with Nanjing Jiancheng kit.

4. Statistical analysis: Student's t-test in excel 2003 was used.

Experimental results:

1: Combined with Figure 5, 2 days after radiation, the body weight of the radiation group alone decreased by 16%, which was extremely significant (P<0.01) lower than that of the normal group, and the body weight growth rate of the amifostine group was significantly higher than that of the radiation group alone Increased by 6.5% (P<0.05). In the protein group, the weight gain of the MTS-SOD2 low-dose group (MS2-1+XRT) was better than that of amifostine, which was significantly increased by about 11% compared with the simple radiation group. The effect of MTS-SOD2 medium dose group (MS2-2+XRT) and high dose group (MS2-3+XRT) was comparable to that of amifostine.

On the 4th day after radiation, the body weight of the simple radiation group was significantly lower than that of the normal group (P<0.01). The body weight of the amifostine group has begun to recover, while that of the radiation group has not yet recovered. The body weight of the MTS-SOD2 low-dose group (MS2-1+XRT) increased by 5 %. The growth rate of MTS-SOD2 high-dose group (MS2-3+XRT) was slightly higher than that of amifostine. The other groups were similar to the previous two days. MTS-SOD2 medium-dose group (MS2-2+XRT) had a similar effect to amifostine.

On the 7th day after radiation, the body weight growth rate of the amifostine group was better than that of the radiation group alone. It can also be seen from the figure that the MTS-SOD2 (MS2-1+XRT) given to the low-dose group had the best effect, with a significant increase of 9.5 % (P<0.05), the effect of MTS-SOD2 high-dose group (MS2-3+XRT) was comparable to that of amifostine.

2: As shown in Figure 6, compared with the normal group, the peripheral blood leukocytes of the mice after irradiation decreased significantly (P<0.01), which was only 14.8% of the normal group. Compared with the radiation control group, the number of leukocytes in the radiation mice pre-administered with amifostine was significantly increased (P<0.05), which increased by about 62.9%. Compared with the radiation control group, the number of peripheral blood leukocytes increased after pre-administration of different doses of recombinant protein MTS-SOD2. The protective effect of (MS2-3+XRT) on mice reached a very significant level (P<0.01), an increase of 81.5%, and its protective effect exceeded that of the amifostine group.

3: As can be seen from Figure 7, compared with the normal group, the thymus index in the radiation-only group was significantly (P<0.01) lower, only 57.4% of the original. The thymus index of the irradiated mice preadministered with amifostine had no significant change compared with the irradiated group. However, in the pre-administration of MTS-SOD2 recombinant protein, the MS2-1 group could significantly (P<0.05) protect the thymus of the mice, and the thymus index increased by about 40%.

4: According to Figure 8, it can be observed that the normal spleen organ is crescent-shaped, with uniform texture and no folds. After irradiation, mice with the same body weight had smaller and more irregular spleen organs than before. In the MTS-SOD2 recombinant protein administration, many nodules appeared, but it was not observed in the radiation alone group. It can be seen from the figure that the spleen index of mice in the radiation control group was significantly (P<0.01) lower than that of the normal group, only 27.3% of the normal group. Compared with the radiation control, the administration of amifostine can slightly increase the spleen index of the irradiated mice, but there is no significant effect. In the fusion protein MS2-2, compared with the simple radiation group, the spleen index was significantly (P<0.05) increased by 60%.

5: Analyzing the protective effect of MTS-SOD2 on the liver tissue of irradiated mice from Figure 9, it can be obtained that the MDA level in the antioxidant index of the liver in the radiation group was significantly increased by 2 times (P<0.05), and the activity of GSH-Px was significantly decreased by 27.9% (P<0.05), while the activities of SOD and T-AOC decreased significantly by 34.9% and 55% (P<0.01), respectively.

The amifostine group had a significant (P<0.05) protective effect on the anti-oxidation index of the mouse liver, and compared with the simple radiation group, the MOD level decreased by 32.6%. SOD activity increased by 22.2%, GSH-Px activity increased by 13.69%, and T-AOC activity increased by 32.2%. The indicators in the low-dose group and high-dose group pre-administered with MTS-SOD2 were better than that of amifostine.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

<110> Fuzhou University

<120> MTS-SOD2 recombinant protein and its preparation method and application

<130> 3

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 222

<212> PRT

<213> MTS-SOD2 recombinant protein

<400> 1

Met Leu Ser Arg Ala Val Cys Gly Thr Ser Arg Gln Leu Ala Pro Ala

1 5 10 15

Leu Gly Tyr Leu Gly Ser Arg Gln Lys His Ser Leu Pro Asp Leu Pro

20 25 30

Tyr Asp Tyr Gly Ala Leu Glu Pro His Ile Asn Ala Gln Ile Met Gln

35 40 45

Leu His His Ser Lys His His Ala Ala Tyr Val Asn Asn Leu Asn Val

50 55 60

Thr Glu Glu Lys Tyr Gln Glu Ala Leu Ala Lys Gly Asp Val Thr Ala

65 70 75 80

Gln Thr Ala Leu Gln Pro Ala Leu Lys Phe Asn Gly Gly Gly His Ile

85 90 95

Asn His Ser Ile Phe Trp Thr Asn Leu Ser Pro Asn Gly Gly Gly Glu

100 105 110

Pro Lys Gly Glu Leu Leu Glu Ala Ile Lys Arg Asp Phe Gly Ser Phe

115 120 125

Asp Lys Phe Lys Glu Lys Leu Thr Ala Ala Ser Val Gly Val Gln Gly

130 135 140

Ser Gly Trp Gly Trp Leu Gly Phe Asn Lys Glu Arg Gly His Leu Gln

145 150 155 160

Ile Ala Ala Cys Pro Asn Gln Asp Pro Leu Gln Gly Thr Thr Gly Leu

165 170 175

Ile Pro Leu Leu Gly Ile Asp Val Trp Glu His Ala Tyr Tyr Leu Gln

180 185 190

Tyr Lys Asn Val Arg Pro Asp Tyr Leu Lys Ala Ile Trp Asn Val Ile

195 200 205

Asn Trp Glu Asn Val Thr Glu Arg Tyr Met Ala Cys Lys Lys

210 215 220

Claims (5)

1.MTS-SOD2 recombinant proteins, it is characterised in that：The sequence of the MTS-SOD2 recombinant proteins such as SEQ ID NO.1 institutes Show.

2. the preparation method of MTS-SOD2 recombinant proteins as claimed in claim 1, it is characterised in that：Utilize genetic engineering skill Art, synthesis people source Mn-SOD complete genome sequences, and be inserted into the prokaryotic expression carrier pGEX-4T-1 with GST, construct GST-MTS-SOD2 fusion protein expression plasmids pGEX-4T-1-MTS-SOD2；Then, by recombinant plasmid pGEX-4T-1-MTS- SOD2 conversion e. coli bl21s (DE3）, Bacillus coli expression fusion protein is induced, can obtain the soluble g ST- of high expression quantity MTS-SOD2 fusion proteins, GST labels are cut first with fibrin ferment after GST affine resins adsorb GST-MTS-SOD2, then passed through Heparin affinity column purifies to have obtained electrophoretically pure MTS-SOD2 recombinant proteins.

3. MTS-SOD2 recombinant proteins according to claim 1 are preparing the application in preventing and treating radiation insult medicine.

4. MTS-SOD2 recombinant proteins according to claim 3 are preparing the application in preventing and treating radiation insult medicine, it is special Sign is：MTS-SOD2 recombinant proteins are 8 μm of ol/ml of 2- using protein concentration；By the MTS-SOD2 being sterile filtered weights Histone is with sterile PBS or normal saline dilution to concentration；The administering mode of the material is injection or oral.

5. MTS-SOD2 recombinant proteins according to claim 1 are preparing preventing and treating oxidative damage medicine as caused by free radical In application.