CN110872581A - High-temperature-resistant mutant SOD with PTD (partial transcription degradation) and coding gene and application thereof - Google Patents

High-temperature-resistant mutant SOD with PTD (partial transcription degradation) and coding gene and application thereof Download PDF

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CN110872581A
CN110872581A CN201911218092.XA CN201911218092A CN110872581A CN 110872581 A CN110872581 A CN 110872581A CN 201911218092 A CN201911218092 A CN 201911218092A CN 110872581 A CN110872581 A CN 110872581A
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韩宏岩
许维岸
林安安
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Suzhou Baisi Biotechnology Co ltd
Suzhou Boying Biotechnology Co ltd
Suzhou University
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Abstract

The invention discloses a PTD-SOD-MUTANT protein, which is a fusion protein consisting of a protein transduction structural domain PTD and SOD from thermophilic bacteria HB27, wherein the SOD amino acid sequence has double mutations of I27L and D107A. The PTD-SOD-MUTANT protein has the advantages of good thermal stability, high activity, capability of entering cells through a cell membrane and the like. In vitro and in vivo experiments show that the protein has strong proliferation inhibition effect on mouse B16 melanoma cells and mouse malignant melanoma, and the protein and adriamycin (DOX) are combined to have synergistic effect on tumor cells. The PTD-SOD-MUTANT protein can be used as a pharmaceutical composition, a cosmetic active component, a food additive and the like, and has a great application value and a wide application prospect in the fields of drug research and development, daily necessities, foods and the like.

Description

High-temperature-resistant mutant SOD with PTD (partial transcription degradation) and coding gene and application thereof
Technical Field
The invention relates to an enzyme, a coding gene and application thereof, in particular to a high-temperature resistant mutant SOD with PTD, and a coding gene and application thereof.
Background
Superoxide dismutase (SOD) is an enzyme containing metal ions that was discovered by american scientists Mccord and Fridovich in 1969 from bovine blood red blood cells. The enzyme can catalyze superoxide anion free radical to generate H2O2And O2It is an oxidoreductase that eliminates the excess superoxide anion radical in the organism. SOD can be classified into four types according to their active center metal ions: Cu/Zn-SOD, Mn-SOD, Fe-SOD and Ni-SOD. SOD has certain curative effect on cancer, inflammation, ischemia reperfusion injury, radiation injury and the like, can reduce the toxic and side effect of anticancer drugs on cells and heart, is one of the most effective antioxidases with antitumor activity, and therefore has important application value in the fields of health care products, medicines, cosmetics and the like. However, in practical applications, SOD still has some defects, such as poor thermal stability and easy inactivation at high temperature; the cell membrane has no specific receptor and is difficult to enter cells to play a role; the separation and purification cost of natural SOD is high. Although the basic studies at present have demonstrated the efficacy of SODAnd the mode of action, but because the SOD in the prior art has defects in the aspects of thermal stability, activity maintenance and the like, the application of the SOD in the fields of cosmetics, foods and medicines is greatly limited, and the expected effect cannot be well achieved.
The protein transduction domain PTD in the trans-activation protein (TAT) coded by HIV-1 is a carrier tool capable of bringing substances which cannot enter cells under physiological states to exert biological effects into the cells, and the core sequence of the PTD is YARAAARQARA. The PTD is fused with other proteins for expression, so that biological macromolecules can enter cells in a receptor-independent mode, and the PTD has the advantages of no need of energy in a transfer process, high transfer efficiency, no influence on the biological activity of exogenous proteins and the like.
Disclosure of Invention
The invention discloses a high-temperature resistant mutant SOD with PTD and a coding gene thereof:
1) the gene sequence is SEQ ID NO.1 in the sequence table, wherein the PTD gene sequence is SEQ ID NO.2 in the sequence table;
2) the amino acid sequence is SEQ ID NO.3 in the sequence table, wherein the PTD amino acid sequence is SEQ ID NO.4 in the sequence table.
The gene sequence of SEQ ID NO.1 in the sequence table is as follows:
1 TAC GCG CGT GCG GCG GCG CGT CAG GCG CGT
31 GCG ATG CCG TAT CCG TTT AAA CTG CCG GAT
61 CTG GGT TAT CCG TAT GAA GCA CTG GAA CCG
91 CAT ATT GAT GCC AAA ACC ATG GAA CTT CAT
121 CAT CAG AAA CAT CAT GGT GCC TAT GTG ACC
151 AAT CTG AAT GCA GCA CTG GAA AAA TAT CCG
181 TAT CTG CAT GGT GTT GAA GTT GAA GTT CTG
211 CTG CGT CAT CTG GCC GCA CTG CCG CAG GAT
241 ATT CAG ACC GCA GTT CGT AAT AAT GGT GGT
271 GGT CAT CTG AAT CAT AGC CTG TTT TGG CGT
301 CTG CTG ACC CCT GGT GGT GCC AAA GAA CCG
331 GTG GGT GAA CTG AAA AAA GCC ATT GCT GAA
361 CAG TTT GGT GGT TTT CAG GCA CTG AAA GAA
391 AAA CTG ACC CAG GCA GCA ATG GGT CGT TTT
421 GGT AGC GGT TGG GCC TGG CTG GTT AAA GAT
451 CCG TTT GGT AAA CTG CAT GTG CTG AGC ACC
481 CCG AAT CAG GAT AAT CCG GTT ATG GAA GGT
511 TTT ACC CCG ATT GTT GGT ATT GAT GTT TGG
541 GAA CAT GCA TAT TAT CTG AAA TAT CAG AAT
571 CGT CGT GCA GAT TAT CTG CAG GCA ATT TGG
601 AAT GTT CTG AAT TGG GAT GTT GCA GAA GAA
631 TTC TTT AAA AAA GCA TAA。
the SEQ ID NO.2PTD gene sequence in the sequence table is as follows:
TACGCGCGTGCGGCGGCGCGTCAGGCGCGTGCG。
the amino acid sequence of SEQ ID NO.3 in the sequence table is:
YARAAARQARAMPYPFKLPDLGYPYEALEPHIDAKTMELHHQKHHGAYVTNLNAALEKYPYLHGVEVEVLLRHLAALPQDIQTAVRNNGGGHLNHSLFWRLLTPGGAKEPVGELKKAIAEQFGGFQALKEKLTQAAMGRFGSGWAWLVKDPFGKLHVLSTPNQDNPVMEGFTPIVGIDVWEHAYYLKYQNRRADYLQAIWNVLNWDVAEEFFKKA。
the SEQ ID NO.4PTD amino acid sequence in the sequence table is as follows: YARAAARQARA.
The novel high-temperature-resistant PTD-carrying MUTANT SOD (PTD-SOD-MUTANT for short) which easily penetrates through cell membranes is a fusion protein consisting of a protein transduction domain PTD and SOD derived from thermophilic bacteria HB27, wherein the SOD amino acid sequence has double mutations of I27L and D107A, namely mutation of Ile at the 27 th position into Leu and mutation of Asp at the 107 th position into Ala.
Said invention uses pET28b as prokaryotic expression vector by means of gene engineering, and is not limited to said vector, and can also use pGEX-4T-2, pMAL-2c, pQE-9, pBAD-Myc, pBAD-His and pGEX-6T-l, etc., preferably uses pET series. The purification method can adopt affinity chromatography, isoelectric point precipitation, salt dissolution and salting out, dialysis and ultrafiltration, density gradient centrifugation, gel filtration, etc., and preferably adopts nickel column affinity chromatography. To obtain a novel MUTANT SOD (PTD-SOD-MUTANT for short) with PTD, high temperature resistance and easy cell membrane penetration, and a coding gene and application thereof.
The PTD-SOD-MUTANT protein prepared by the invention can be used as a pharmaceutical composition, a cosmetic active component, a food additive and the like.
In the aspect of medicine application, the composition can be used for antitumor medicines, anti-inflammatory medicines, anti-radiation injury medicines and the like, and the medicine dosage form is an oral dosage form or an external dosage form, wherein the oral dosage form is preferably oral liquid, tablets, pills, capsules, granules, oral powder and the like; the external preparation is preferably selected from topical powder, ointment, patch, topical liquid, suppository, spray, aerosol, inhalant, etc. In addition, the PTD-SOD-MUTANT protein provided by the invention can also be chemically modified by adopting trehalose, glycerol, lauroyl chloride, polyethylene glycol (PEG) and the like so as to prolong the retention time of protein drugs in blood.
In cosmetic applications, it can be used as an active ingredient to be added to cosmetics in the form of soaps, facial cleansers, shower gels, skin lotions, skin creams, essences, skin oils, eye creams, masks, aerosols or sprays, and the like. Can be used in spot-removing and whitening cosmetics, anti-aging cosmetics, antioxidant cosmetics or their combination.
The invention discloses a high temperature resistant mutant SOD with PTD and a coding gene thereof, which utilizes a bioinformatics method to carry out molecular dynamics simulation by taking the crystal structure of the SOD coded by thermophilic bacteria HB27 as a template, and finds that the substrate binding affinity of the SOD coded by thermophilic bacteria HB27 can be greatly improved under the premise of not influencing the SOD binding specificity by carrying out I27L and D107A double mutation on the SOD amino acid sequence, thereby leading to the improvement of the SOD activity and thermal stability. In addition, the mutant protein has an optimized protein structure and a new biological function, and has great superiority compared with the natural protein.
Drawings
FIG. 1 is an SDS-PAGE identification of purified PTD-SOD-MUTANT proteins;
FIG. 2 is a graph showing the results of inhibition of proliferation of mouse B16 melanoma cells in vitro by PTD-SOD-MUTANT protein and SOD protein after 48 hours of culture;
FIG. 3 is a graph showing the results of in vivo treatment of malignant melanoma in mice with PTD-SOD-MUTANT protein and SOD protein,
a is the relative tumor volume growth curve within 10 days of administration, b is the tumor body weight 10 days after administration, c is the tumor photograph 10 days after administration;
FIG. 4 is a graph showing the results of in vitro proliferation inhibition of mouse B16 melanoma cells by DOX, PTD-SOD-MUTANT protein and a combination thereof after 48 hours of culture;
FIG. 5 is a morphogram of mouse B16 melanoma cells treated with DOX, PTD-SOD-MUTANT protein and a combination of both, after 48 hours of culture.
Detailed Description
The present invention will be described in further detail below with reference to specific embodiments and with reference to the accompanying drawings. The following examples are intended to illustrate, but not limit, the invention.
Example 1
Expression and purification of PTD-SOD-MUTANT protein
The PTD-SOD-MUTANT gene sequence is synthesized by Shanghai (stock) of bioengineering, wherein a prokaryotic expression vector is pet28b, recombinant plasmids are transformed into competent escherichia coli BL21(DE3) by a heat shock method, isopropyl- β -D-thiogalactopyranoside (IPTG) is added for shaking table shaking culture to induce the expression of PTD-SOD-MUTANT protein, the shaking table rotating speed is 220rpm/min (the rotating speed range is 200 rpm/min and is 250rpm/min, preferably 220rpm/min), the induction temperature is 37 ℃ (the induction temperature range is 30-38 ℃, preferably 37 ℃), the final concentration of IPTG is 1mM (the final concentration range of IPTG is 0.1-1.5mM and is preferably 1mM), the induction time is 7 hours (the induction time range is 3-8 hours and is preferably 7 hours), after the induced expression is treated by an ultrasonic cracking method, the bacterial affinity chromatographic column is utilized, under natural conditions, the Buffer with the imidazole concentration of 25mM and the Washing concentration of 250mM and the MUD-MUTANT protein is purified by the Ni-NTA affinity chromatographic column under natural conditions, the SOD molecular weight of the SOD-TANT protein is about 26.
Example 2
PTD-SOD-MUTANT protein has in vitro proliferation inhibiting effect on mouse B16 melanoma cells
The MTT method was used to examine the effect of PTD-SOD-MUTANT protein and SOD protein (control) on the proliferation of mouse B16 melanoma cells. MTT principle: succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into difficultly soluble bluish purple crystals and deposit in cells, while dead cells do not have the function. Dimethyl Maple (DMSO) can dissolve purple crystals in cells, and the light absorption value is measured at the wavelength of 570nm by using an enzyme-labeling instrument, so that the number of living cells can be indirectly reflected.
The specific operation is as follows: taking mouse B16 melanoma cells in logarithmic growth phase, inoculating the cells into a 96-well plate, wherein the number of the cells in each well is 1 multiplied by 103(ii) a The two proteins are respectively set into treatment groups according to three gradients of 0.2, 0.4 and 0.8mg/mL, each group has 3 auxiliary holes, and a 1 multiplied PBS treatment group is used as a control; after 48 hours of culture, 20 mu L of MTT with the concentration of 5g/L is added for further culture for 4 hours; removing culture medium, adding DMSO 150 μ L, placing on horizontal shaker, acting at room temperature for 10min, measuring absorbance at 570nm with microplate reader, and drawing histogram according to the measured value.
The experimental results are shown in fig. 2, the PTD-SOD-MUTANT protein has stronger cell inhibition effect than SOD protein (control) under the same concentration, and the PTD-SOD-MUTANT protein has significant difference under each concentration, when the concentration is 0.8mg/mL, the inhibition degree of the PTD-SOD-MUTANT protein on mouse B16 melanoma cells is twice as high as that of the SOD protein. The results show that, under in vitro conditions, the PTD-SOD-MUTANT protein is easier to penetrate cell membranes to enter cells to play a role than the SOD protein, and the activity of the PTD-SOD-MUTANT protein is higher than that of the SOD protein.
Example 3
In vivo therapeutic effect of PTD-SOD-MUTANT protein on malignant melanoma of mice
Mice in logarithmic growth phase, B16 melanoma cells, were aspirated, trypsinized and counted. Centrifuging at 1000rpm for 5 min, preparing cell suspension with DMEM culture solution, and adjusting cell concentration to 1 × 106one/mL. Get jian (medicine for health)Kangchun breed of 9 nude mice of 4-6 weeks old were injected with 0.1mL of prepared tumor cell suspension in the left axilla, and repeated once on day 2. The weight of the nude mice is measured every 2 days, the basic vital signs are observed, and the success of the molding is marked when tumors grow out under the armpits of the nude mice. The tumor diameter was measured with a vernier caliper every 2 days, and when the tumor diameter was about 1cm, the animals were randomly divided into 3 groups, and were intraperitoneally injected with 0.1mL of PBS, PTD-SOD-MUTANT protein (concentration 0.8mg/mL), and SOD protein (concentration 0.8mg/mL), 1 time every 2 days, and continuously injected for 10 days. The survival of the nude mice was observed every day, the longest and shortest path of tumor of the nude mice were measured with a vernier caliper every 2 days and the body weight of the nude mice was weighed, starting on the day of treatment. Using the formula V as a x b2The tumor volume V was calculated at 0.52 (V: tumor volume; a: longest diameter of tumor; b: shortest diameter of tumor). By the formula V ═ Vt-V0)/V0×100％(Vt: tumor volume, V, measured at various time points0: initial tumor volume at the start of dosing) the rate of change of tumor volume was calculated. Nude mice were sacrificed 10 days after administration by depithing, and subcutaneous tumors (sparing necrotic tissue as much as possible) were removed and weighed. The experimental results are shown in fig. 3, and it can be seen from fig. 3a and 3b that compared with the PBS control group, both SOD protein and PTD-SOD-MUTANT protein have a certain degree of inhibition effect on tumor growth, and at the same concentration, the inhibition effect of PTD-SOD-MUTANT protein is significantly stronger than that of SOD protein, and the difference has statistical significance. After the end of the administration, the mice were sacrificed and the tumors were taken out for photography, and it can be seen from fig. 3c that the tumors of the mice of the PTD-SOD-MUTANT protein group were significantly smaller than those of the control group and SOD protein group, which is consistent with the results of the relative tumor volume growth curve. The result shows that under the in vivo condition, the PTD-SOD-MUTANT protein is easier to penetrate through cell membranes and enter cells to play a role than the SOD protein, and has better anti-tumor effect.
Example 4
In-vitro proliferation inhibition effect of PTD-SOD-MUTANT protein and DOX composition on mouse B16 melanoma cells
Doxorubicin (DOX) is a common antitumor antibiotic, can inhibit the synthesis of RNA and DNA, the antitumor spectrum is wider, inhibit many tumors, belong to cycle nonspecific medicament, have killing effects on tumor cells of various growth cycles, but because DOX specificity is not strong, will kill normal cells while killing tumor cells, so there are many side effects, such as bone marrow hematopoietic function decline, hepatic function damage, heart failure, potential cytotoxicity, etc. The previous experiments have proved that the PTD-SOD-MUTANT protein is easier to enter tumor cells to play a role compared with SOD protein, and the purpose of the experiment is to explore whether the combination of the PTD-SOD-MUTANT protein and DOX has stronger cell specificity compared with single DOX administration, thereby having stronger tumor inhibition effect.
The specific operation is as follows: taking mouse B16 melanoma cells in logarithmic growth phase, inoculating the cells into a 96-well plate, wherein the number of the cells in each well is 1 multiplied by 103(ii) a Setting 3 treatment groups, each group having 3 auxiliary wells, respectively treating with 0.8mg/mL PTD-SOD-MUTANT protein, 0.8mg/mL LDOX, 0.4mg/mL PTD-SOD-MUTANT protein +0.4mg/mL DOX, and treating with 1xPBS as a control group; after 48 hours of culture, 20 mu L of MTT with the concentration of 5g/L is added for further culture for 4 hours; removing culture medium, adding DMSO 150 μ L, placing on horizontal shaker, acting at room temperature for 10min, measuring absorbance at 570nm with microplate reader, and drawing histogram according to the value.
The results of the experiment are shown in fig. 4 and 5. As can be seen from FIG. 4, both the PTD-SOD-MUTANT protein and DOX inhibited the growth of melanoma cells of mouse B16, the inhibition rate of DOX was about 10%, the inhibition rate of PTD-SOD-MUTANT protein was about 20%, and the inhibition effect of PTD-SOD-MUTANT protein was stronger than that of DOX. However, when the two are combined to act together in the form of a composition, the inhibition effect is remarkably increased, and the inhibition rate is over 50 percent, which is greatly stronger than the effect when the two are independently acted. Therefore, it is speculated that DOX may interact with PTD-SOD-MUTANT protein, penetrate through cell membrane to enter tumor cells through PTD protein transduction, and simultaneously have synergistic effect with the PTD-SOD-MUTANT protein. From FIG. 5, it can be seen that DOX, PTD-SOD-MUTANT protein and the cells treated by the combination of both generate different degrees of cell necrosis, which is expressed by cell group death, cell membrane lysis, and whole cell lysis, wherein the number of cell necrosis processed by the combination of both is the largest, the PTD-SOD-MUTANT protein is the next to that of cell necrosis, and DOX is the smallest, which is consistent with the MTT experimental result, and it is proved that PTD-SOD-MUTANT protein and DOX have synergistic effect on the inhibition of tumor cell growth, and the combination of both can be used as anticancer drugs, and has wide application prospect in the field of tumor treatment.
Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Sequence listing
<110> Suzhou university
<120> high-temperature-resistant PTD-carrying mutant SOD, and coding gene and application thereof
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>648
<212>DNA
<213> Artificial sequence ()
<400>1
tacgcgcgtg cggcggcgcg tcaggcgcgt gcgatgccgt atccgtttaa actgccggat 60
ctgggttatc cgtatgaagc actggaaccg catattgatg ccaaaaccat ggaacttcat 120
catcagaaac atcatggtgc ctatgtgacc aatctgaatg cagcactgga aaaatatccg 180
tatctgcatg gtgttgaagt tgaagttctg ctgcgtcatc tggccgcact gccgcaggat 240
attcagaccg cagttcgtaa taatggtggt ggtcatctga atcatagcct gttttggcgt 300
ctgctgaccc ctggtggtgc caaagaaccg gtgggtgaac tgaaaaaagc cattgctgaa 360
cagtttggtg gttttcaggc actgaaagaa aaactgaccc aggcagcaat gggtcgtttt 420
ggtagcggtt gggcctggct ggttaaagat ccgtttggta aactgcatgt gctgagcacc 480
ccgaatcagg ataatccggt tatggaaggt tttaccccga ttgttggtat tgatgtttgg540
gaacatgcat attatctgaa atatcagaat cgtcgtgcag attatctgca ggcaatttgg 600
aatgttctga attgggatgt tgcagaagaa ttctttaaaa aagcataa 648
<210>2
<211>33
<212>DNA
<213> Artificial sequence ()
<400>2
tacgcgcgtg cggcggcgcg tcaggcgcgt gcg 33
<210>3
<211>215
<212>PRT
<213> Artificial sequence ()
<400>3
Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala Met Pro Tyr Pro Phe
1 5 10 15
Lys Leu Pro Asp Leu Gly Tyr Pro Tyr Glu Ala Leu Glu Pro His Ile
20 25 30
Asp Ala Lys Thr Met Glu Leu His His Gln Lys His His Gly Ala Tyr
35 40 45
Val Thr Asn Leu Asn Ala Ala Leu Glu Lys Tyr Pro Tyr Leu His Gly
50 55 60
Val Glu Val Glu Val Leu Leu Arg His Leu Ala Ala Leu Pro Gln Asp
65 70 75 80
Ile Gln Thr Ala Val Arg Asn Asn GlyGly Gly His Leu Asn His Ser
85 90 95
Leu Phe Trp Arg Leu Leu Thr Pro Gly Gly Ala Lys Glu Pro Val Gly
100 105 110
Glu Leu Lys Lys Ala Ile Ala Glu Gln Phe Gly Gly Phe Gln Ala Leu
115 120 125
Lys Glu Lys Leu Thr Gln Ala Ala Met Gly Arg Phe Gly Ser Gly Trp
130 135 140
Ala Trp Leu Val Lys Asp Pro Phe Gly Lys Leu His Val Leu Ser Thr
145 150 155 160
Pro Asn Gln Asp Asn Pro Val Met Glu Gly Phe Thr Pro Ile Val Gly
165 170 175
Ile Asp Val Trp Glu His Ala Tyr Tyr Leu Lys Tyr Gln Asn Arg Arg
180 185 190
Ala Asp Tyr Leu Gln Ala Ile Trp Asn Val Leu Asn Trp Asp Val Ala
195 200 205
Glu Glu Phe Phe Lys Lys Ala
210 215
<210>4
<211>11
<212>PRT
<213> Artificial sequence ()
<400>4
Tyr Ala ArgAla Ala Ala Arg Gln Ala Arg Ala
1 5 10

Claims (10)

1. A high temperature resistant mutant SOD with PTD and a coding gene thereof:
1) the gene sequence is SEQ ID NO.1 in the sequence table, wherein the PTD gene sequence is SEQ ID NO.2 in the sequence table;
2) the amino acid sequence is SEQ ID NO.3 in the sequence table, wherein the PTD amino acid sequence is SEQ ID NO.4 in the sequence table.
2. The mutant SOD with PTD and its coding gene with high temperature resistance as claimed in claim 1, wherein: the fusion protein consists of a protein transduction structural domain PTD and SOD derived from thermophilic bacteria HB27, wherein the SOD amino acid sequence has double mutations of I27L and D107A.
3. The mutant SOD with PTD and its coding gene with high temperature resistance as claimed in claim 1, wherein: any one of pet28b, pGEX-4T-2, pMAL-2c, pQE-9, pBAD-Myc, pBAD-His and pGEX-6T-l is taken as a prokaryotic expression vector.
4. A high-temp resistant MUTANT SOD with PTD and its coding gene expressing and purifying process includes such steps as thermally shocking to transform the recombinant plasmid to competent colibacillus BL21, adding isopropyl- β -D-thiogalactopyranoside, shaking culture to induce the expression of PTD-SOD-MUTANT protein, ultrasonic cracking to treat the thallus, purifying, sequentially adding shift Buffer with imidazole concentration of 25mM and experiment Buffer with imidazole concentration of 250mM, and purifying.
5. The expression and purification method of the high temperature resistant mutant SOD with PTD and its coding gene as claimed in claim 4, wherein: the rotating speed of the shaking table is 200-250 rpm/min.
6. The expression and purification method of the high temperature resistant mutant SOD with PTD and its coding gene as claimed in claim 4, wherein: the induction temperature is 30-38 ℃.
7. The expression and purification method of the high temperature resistant mutant SOD with PTD and its coding gene as claimed in claim 4, wherein: the induction time is 3-8 h.
8. The expression and purification method of the high temperature resistant mutant SOD with PTD and its coding gene as claimed in claim 4, wherein: in the expression of PTD-SOD-MUTANT protein, the final concentration range of IPTG is 0.1-1.5 mM.
9. The expression and purification method of the high temperature resistant mutant SOD with PTD and its coding gene as claimed in claim 1, wherein: the purification method adopts any one of affinity chromatography, isoelectric point precipitation, salt dissolution and salting out, dialysis and ultrafiltration, density gradient centrifugation and gel filtration.
10. A high temperature resistant mutant SOD with PTD and its coding gene are applied in inhibiting melanoma cell.
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