CN111235121B

CN111235121B - Construction and application of high-stability superoxide dismutase high-efficiency expression system

Info

Publication number: CN111235121B
Application number: CN202010160119.0A
Authority: CN
Inventors: 夏勇
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-03-10
Filing date: 2020-03-10
Publication date: 2022-03-25
Anticipated expiration: 2040-03-10
Also published as: CN111235121A

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a high-stability superoxide dismutase high-efficiency expression vector, and construction and application of the vector. The protein coded by the open reading frame of the high-efficiency expression vector of the high-stability superoxide dismutase provided by the invention has a sequence shown as SEQ ID NO. 1-7. The nucleotide sequence of the protein sequence shown in SEQ ID NO. 1-7 is SEQ ID NO. 8-17. The method for constructing the vector comprises the following steps: double enzyme digestion, transformation, induction, electrophoresis sample preparation, whole bacteria sample, supernatant sample, immunoblot identification, thermal stability identification and long-term stability identification. The invention has the advantages that the protein expressed by the prepared vector has high stability; the engineering bacteria have high expression yield and easy purification, and can provide different purity levels for downstream.

Description

Construction and application of high-stability superoxide dismutase high-efficiency expression system

Technical Field

The invention belongs to the technical field of enzyme engineering, and particularly relates to a high-stability superoxide dismutase high-efficiency expression vector, and construction and application of the vector.

Background

Free radicals are substances lacking electrons (unsaturated electron pair substances), and after entering a human body, cells, proteins or lipids of the human body are oxidized, so that the human body is injured. There are three main aspects of free radical damage to the human body: firstly, cell membranes are damaged; ② the protease is inactivated; ③ damaging the gene leads to cellular variation or gene mutation. The damage of free radicals to the skin is also not negligible: the skin is exposed to ultraviolet radiation during outdoor exercise to induce the accumulation of free radicals. Melanin is an effective macromolecule for scavenging free radicals, and cells often synthesize a large amount of melanin in order to reduce the harm of free radicals to the cells, which is expressed on the skin as pigmentation. In addition, free radical accumulation can cause oxidation of intracellular proteins and lipids, resulting in pigment-rich lipofuscin, which can produce age spots that are difficult to remove over the course of a day and a month.

Superoxide dismutase is one of the most efficient enzymes for scavenging free radicals, and it catalyzes superoxide radicals to form hydrogen peroxide and oxygen, which are then decomposed into water and oxygen. It can be said that superoxide dismutase is the most effective substance for resisting free radical damage, but sometimes the human body cannot produce enough superoxide dismutase in time, which requires external supplement. However, most of the currently available superoxide dismutase has the defect of unstable activity, namely the superoxide dismutase is stable only in a dry powder state, but the activity of the superoxide dismutase added into an end product is lost quickly, and the activity of the superoxide dismutase in the end product used by a target population is very low or is lost, so that the antioxidant effect of the superoxide dismutase is not ideal.

Therefore, it is necessary to solve the above problems and invent a high-efficiency expression vector of superoxide dismutase with good stability and a construction method of the high-efficiency expression strain.

Disclosure of Invention

In order to solve the above technical problems, the inventors developed superoxide dismutase with good stability by biotechnology and genetic engineering, and established a fermentation and purification process from mature organisms.

Constructing a high-efficiency expression system of the high-stability superoxide dismutase, wherein the protein sequence of the high-stability superoxide dismutase is shown as any one of SEQ ID NO. 1-7;

preferably, the protein sequence of the superoxide dismutase with high stability is shown as SEQ ID NO. 1.

The gene sequence corresponding to the protein sequence of the coded high-stability superoxide dismutase is shown in one of SEQ ID NO. 8-15;

preferably, the gene sequence corresponding to the coded high-stability superoxide dismutase protein sequence SEQ ID NO.1 is SEQ ID NO.8 or SEQ ID NO.9, the gene sequence corresponding to SEQ ID NO.2 is SEQ ID NO.10, the gene sequence corresponding to SEQ ID NO.3 is SEQ ID NO.11, the gene sequence corresponding to SEQ ID NO.4 is SEQ ID NO.12, the gene sequence corresponding to SEQ ID NO.5 is SEQ ID NO.13, the gene sequence corresponding to SEQ ID NO.6 is SEQ ID NO.14, and the gene sequence corresponding to SEQ ID NO.7 is SEQ ID NO. 15;

preferably, the gene sequence corresponding to the protein sequence of the superoxide dismutase with high stability is shown as SEQ ID NO. 8.

The construction method of the high-efficiency expression system of the high-stability superoxide dismutase comprises the following steps:

amplifying a target fragment by using a specific primer, carrying out double enzyme digestion on an amplification gene and a carrier by using a restriction enzyme, recovering an enzyme digestion product by using gel, connecting the target fragment with the carrier by using a ligase, converting the connection product into competent escherichia coli, screening by using a resistant plate, selecting a monoclonal bacterial plaque, carrying out liquid culture and induction, carrying out ultrasonic crushing and centrifugal induction on the bacterial body, carrying out polyacrylamide gel electrophoresis, and verifying the expression quantity and expression solubility of a target protein by using Coomassie brilliant blue staining and an immunoblotting experiment;

(1) template synthesis: synthesizing according to any one of SEQ ID NO. 8-15;

preferably, the synthesis is performed according to SEQ ID NO. 8;

(2) designing a primer: specific primers are designed by taking restriction enzymes NdeI and HindIII as restriction enzyme cutting sites of upstream and downstream restriction enzymes and taking the 5 'end and the 3' end of the SEQ ID NO.8-15 sequence as amplification starting points. Preferably, the primer design sequence is: an upstream primer 5'-GGCATATGATGGGTGTTCATAAATTAG-3'; a downstream primer: 5'-GGCCAAGCTTCTTAATGAAGTCTTTTAAG-3', respectively;

(3) gene amplification: adding the primers and the template into a pfu high-fidelity DNA polymerase amplification system to form a 50 mu l reaction system, and amplifying for 35 cycles in a PCR instrument;

(4) double enzyme digestion: carrying out double enzyme digestion on a target gene and a vector pET30a by adopting NdeI and HindIII, and connecting the products by adopting T4 ligase after the products are recovered by gel to obtain a connecting product;

(5) and (3) transformation: melting competent cells of escherichia coli BL21(DE3), adding the ligation product, performing liquid culture, uniformly mixing the obtained bacterial liquid, and coating the mixture on a kanamycin-resistant plate until monoclonal bacterial plaque is formed;

(6) induction: respectively inoculating the three monoclonals into LB test tubes containing kanamycin, performing shake culture, adding isopropyl-beta-D-thiogalactoside into two samples, and culturing; selecting a sample without adding isopropyl-beta-D-thiogalactoside as a negative reference, and detecting the expression of the protein by adopting polyacrylamide gel electrophoresis and a protein blotting method;

(7) preparing an electrophoresis sample: taking the sediment obtained after the culture medium is centrifuged, suspending the sediment in a lysis buffer solution, and carrying out ultrasonic lysis to obtain a lysate;

(8) taking a whole bacteria sample, namely uniformly mixing part of lysate obtained in the step (4) with a protein loading buffer solution, heating and centrifuging;

(9) taking the residual lysate, centrifuging to obtain supernatant, and mixing the protein sample buffer solution into the supernatant to obtain a supernatant sample; detecting the whole bacteria and cracking the supernatant by polyacrylamide gel electrophoresis and western blotting.

Preferably, in the step (1), T4 ligase is used for ligation for 4-15 h at the temperature of 12-20 ℃ to obtain a ligation product.

Preferably, in the step (2), BL21(DE3) competent cells are taken out of the ultra-low temperature refrigerator, and the temperature of the ultra-low temperature refrigerator is-80 to-70 ℃;

preferably, in the step (2), 30-100 mu l of BL21(DE3) competent cells are taken out of the ultra-low temperature refrigerator, placed on ice for melting, then 3-15 mu l of connecting products are added, placed on ice for 25-35 min, thermally shocked in water bath at 42-45 ℃ for 45-90 s, placed on ice for 2-10 min, added with 400-1000 mu l of room-temperature LB liquid culture medium, and shaken in a constant-temperature shaking table at 30-37 ℃ for 45-60 min;

preferably, in the step (2), the obtained bacterial liquid is uniformly mixed and then coated on a kanamycin-resistant flat plate, the flat plate is inverted, and the bacterial liquid is cultured for 16-24 hours at the temperature of 30-37 ℃ until monoclonal bacterial plaque is formed;

preferably, the kanamycin concentration in (2) is 45 to 55. mu.g/ml.

(3) Selecting three monoclonals, respectively inoculating the monoclonals into 1-8 ml LB test tubes containing 45-55 mu g/ml kanamycin, carrying out shake culture in a shaking table at the temperature of 30-40 ℃ at the rotating speed of 200r/min, adding 0.1-1.0 mM isopropyl-beta-D-thiogalactoside into two test tubes when OD600 reaches 0.6-0.9, respectively, culturing one test tube at the temperature of 14-20 ℃ for 12-20 h, culturing the other test tube at the temperature of 30-40 ℃ for 3-8 h, and detecting the expression of the protein by using a polyacrylamide gel electrophoresis and a western blot method by using the test tube without the addition of the isopropyl-beta-D-thiogalactoside as a negative reference.

(4) Taking the sediment of the culture medium after centrifugation, and suspending the sediment in a lysis buffer solution for 4-6 min by ultrasonic lysis; the volume ratio of the medium to the lysis buffer was: 5: 1-2: 1;

preferably, in (4), 3ml of the sediment after the culture medium is centrifuged is taken and resuspended in 1ml of lysis buffer, and the sediment is subjected to ultrasonic lysis for 5 min;

preferably, (4) during ultrasonic cracking, stopping ultrasonic treatment for 2.5-3.5 s every 1.5-2.5 s, and carrying out ultrasonic treatment alternately and circularly for 4-6 min;

preferably, in (4), the lysis buffer is 50mM Tris-HCl,150mM NaCl, 5% glycerol, pH 8.0.

(5) 1/4 of the lysate obtained in the step (4) is taken, evenly mixed with a protein loading buffer solution, heated and centrifuged; the volume ratio of lysate to protein loading buffer was 4: 1;

preferably, (5) the loading buffer is a protein loading buffer with 5 times concentration;

preferably, in the step (5), the mixture is heated to 95-100 ℃ and kept for 8-12 min;

preferably, in the step (5), centrifuging for 5-15 min at the rotating speed of 12000-15000 r/min;

(6) mixing a protein loading buffer solution to the supernatant as a supernatant sample, wherein the volume ratio of the protein loading buffer solution to the supernatant is 1: 3.5-1: 4.5; detecting the whole bacteria and cracking supernatant by polyacrylamide gel electrophoresis and western blotting;

preferably, in the step (6), centrifuging for 10-15 min at the rotating speed of 13000-15000 r/min, and taking the supernatant;

preferably, (6) the volume ratio of the protein loading buffer to the supernatant is 1: 4;

preferably, in (6), the protein loading buffer is a 5-fold concentration of the protein loading buffer.

Preferably, the above construction method comprises the steps of:

(1) primer design and gene amplification and double enzyme digestion: designing specific primers by taking restriction enzymes NdeI and HindIII as restriction enzyme cutting sites of upstream and downstream restriction enzymes and taking the 5 'end and the 3' end of the SEQ ID NO.8 sequence as amplification starting points;

carrying out double enzyme digestion on the target gene and a vector pET30a by NdeI and HindIII, recovering the product through gel, and connecting for 4-15 h at 12-20 ℃ by adopting T4 ligase to obtain a connected product;

(2) and (3) transformation: taking 30-100 mu l of BL21(DE3) competent cells out of an ultralow-temperature refrigerator at the temperature of-80 to-70 ℃, putting the cells on ice for melting, adding 3-15 mu l of connecting products, placing the cells on ice for 25-35 min, thermally exciting the cells in water bath at the temperature of 42-45 ℃ for 80-100 s, then placing the cells on ice for 2-6 min, adding 400-1000 mu l of room-temperature LB liquid culture medium, and placing the cells in a shaking table for shaking culture at the temperature of 30-37 ℃ and at the rotating speed of 150-250 r/min for 45-70 min; uniformly mixing the obtained bacterial liquid, coating the mixture on a kanamycin-resistant LB flat plate with the concentration of 45-55 mu g/ml, inverting the flat plate, and culturing at 33-37 ℃ until monoclonal bacterial plaque is formed;

(3) induction: selecting three monoclonals, respectively inoculating the monoclonals into 1-5 ml LB test tubes containing 45-55 mug/ml kanamycin, performing shake culture at the temperature of 30-37 ℃ and the rotating speed of 200r/min in a shaking table, adding 0.1-1.0 mM isopropyl-beta-D-thiogalactoside into two test tubes when OD600 reaches 0.6-0.9, respectively, culturing one test tube at the temperature of 14-20 ℃ for 12-20 h, culturing the other test tube at the temperature of 30-37 ℃ for 1-8 h, taking the test tube without the addition of the isopropyl-beta-D-thiogalactoside as a negative reference, and detecting the expression of the protein by adopting polyacrylamide gel electrophoresis and a protein blotting method;

(4) preparing an electrophoresis sample: taking the sediment obtained after the culture medium is centrifuged, re-suspending the sediment in a lysis buffer solution, and carrying out ultrasonic lysis for 4-6 min to obtain a lysate;

the volume ratio of the medium to the lysis buffer was: 5: 1-2: 1; lysis buffer was 50mM Tris-HCl,150mM NaCl, 5% glycerol, pH 8.0;

during ultrasonic cracking, stopping the ultrasonic treatment for 2.5-3.5 s every 1.5-2.5 s, and carrying out the ultrasonic treatment alternately and circularly for 4-6 min;

(5) taking 1/3 of the lysate obtained in the step (4), uniformly mixing the lysate with 5X protein loading buffer solution, heating to 95-100 ℃, keeping for 8-12 min, and centrifuging for 5-10 min at the rotating speed of 12000-15000 r/min; the volume ratio of the protein loading buffer to the supernatant was 1: 4;

(6) taking the supernatant sample, namely centrifuging the residual lysate for 8-12 min at the rotating speed of 12000-15000 r/min, and taking the supernatant; mixing a 5X protein loading buffer solution into the supernatant to be used as a supernatant sample; the volume ratio of the protein loading buffer to the supernatant was 1: 4; detecting the whole bacteria and cracking the supernatant by polyacrylamide gel electrophoresis and western blotting.

The high-efficiency expression vector of the high-stability superoxide dismutase provided by the invention is applied to the preparation of skin care products, food processing and medicine production, and is also within the protection range of the invention.

The invention has the beneficial effects that:

(1) the engineering bacteria has high expression yield, and is realized by optimizing codons, so that the finally obtained superoxide dismutase has the characteristics, as shown in the attached figures 1-5;

(2) the stability is high, the activity is not easy to lose, for example, the thermal stability and the normal temperature stability can be well maintained, as shown in the attached figures 6-7;

(3) easy purification, and can provide different purity levels for downstream, because the invention improves the thermal stability of superoxide dismutase, and the invention designs and improves the fusion expression label.

Drawings

FIG. 1 is a SDS-PAGE analysis of the expression of high-stability superoxide dismutase protein in BL21(DE 3);

lane M protein Marker, Lane C1 BSA (1. mu.g), Lane C2 BSA (2. mu.g), Lane N uninduced whole cells, Lane 1:15 ℃ induced whole cells for 16h, Lane 2:37 ℃ induced whole cells for 4h, Lane N1 uninduced cell lysis supernatant, Lane 3:15 ℃ induced cell lysis supernatant for 16h, Lane 4:37 ℃ induced cell lysis supernatant for 4 h;

FIG. 2 is a Western blot immunoblot analysis of the expression of high-stability superoxide dismutase protein in BL21(DE 3). Lane M protein Marker, Lane N uninduced whole cells, Lane 1:15 ℃ induced whole cells for 16h, Lane 2:37 ℃ induced whole cells for 4h, Lane 3:15 ℃ induced cell lysis supernatant for 16h, Lane 4:37 ℃ induced cell lysis supernatant for 4 h; the antibody used for immunoblotting was the anti-His antibody (GenScript, cat. No. a 00186);

FIG. 3 is the original image of the vitality determination of the present invention and the similar products;

FIG. 4 is a bar graph comparing the activity measurements of the present invention and similar products;

FIG. 5 is a graph comparing total activities of superoxide dismutase obtained by inoculating 100ml LB fermentation liquid with the same bacterial amount and inducing for the same time;

FIG. 6 is an original graph showing the comparison of activities of the present invention and similar products after heat treatment in a 70 ℃ water bath for 1 hour, respectively;

FIG. 7 is a bar graph of the activity determination of superoxide dismutase obtained after the heat treatment of the present invention and the like in a water bath at 70 ℃ for 1 hour;

FIG. 8 is a graph comparing the stability of the present invention in solution at room temperature with similar products;

FIG. 9 is a photograph showing the state of the solution of the product of the present invention at 5mg/ml, 0.5mg/ml and 0.1 mg/ml;

FIG. 10 is a graph showing the effect of the present invention on dermatitis.

Detailed Description

In order that those skilled in the art will better understand the present invention, the inventors will further describe and illustrate the present invention by the following specific examples, but do not limit the present invention.

Example 1

The construction of the high-efficiency expression system of the high-stability superoxide dismutase is characterized in that the protein sequence of the high-stability superoxide dismutase is shown as any one of SEQ ID NO. 1-7.

The gene sequence corresponding to the protein sequence of the superoxide dismutase with high stability is shown in one of SEQ ID NO. 8-15.

Specifically, the gene sequence corresponding to the coded high-stability superoxide dismutase protein sequence SEQ ID NO.1 is SEQ ID NO.8 or SEQ ID NO.9, the gene sequence corresponding to SEQ ID NO.2 is SEQ ID NO.10, the gene sequence corresponding to SEQ ID NO.3 is SEQ ID NO.11, the gene sequence corresponding to SEQ ID NO.4 is SEQ ID NO.12, the gene sequence corresponding to SEQ ID NO.5 is SEQ ID NO.13, the gene sequence corresponding to SEQ ID NO.6 is SEQ ID NO.14, and the gene sequence corresponding to SEQ ID NO.7 is SEQ ID NO. 15;

in SEQ ID No. 9-15, R is A or G, Y is C or T, H is A, C or T, N is A, C, G or T; in the sequence table listed in the invention, only one of SEQ ID NO. 9-15 is given; there are numerous possible variations on the sequence listing below that are within the scope of the present invention.

The sequence table in SEQ ID NO. 9-15 of the invention is as follows:

SEQ ID No.9

wherein, R is A or G, Y is C or T, H is A, C or T, N is A, C, G or T

SEQ ID No.10

Wherein, R is A or G, Y is C or T, H is A, C or T, N is A, C, G or T

SEQ ID No.11

Wherein, R is A or G, Y is C or T, H is A, C or T, N is A, C, G or T

SEQ ID No.12

Wherein, R is A or G, Y is C or T, H is A, C or T, N is A, C, G or T

SEQ ID No.13

Wherein, R is A or G, Y is C or T, H is A, C or T, N is A, C, G or T

SEQ ID No.14

Wherein, R is A or G, Y is C or T, H is A, C or T, N is A, C, G or T

SEQ ID No.15

Wherein, R is A or G, Y is C or T, H is A, C or T, N is A, C, G or T

The expression vector constructed by the invention preferably has a high-stability superoxide dismutase protein sequence shown as SEQ ID NO. 1;

the gene sequence corresponding to the protein sequence of the coded high-stability superoxide dismutase is preferably shown as SEQ ID NO. 8;

the nucleotide sequence of the open reading frame of the expression vector constructed by the invention is any one of SEQ ID NO.8-15, and the specific SEQ ID NO.8-15 is shown in the attached sequence table of the invention.

Example 2

(1) template synthesis: synthesizing according to any one of SEQ ID NO. 8-15;

designing a primer: restriction enzymes NdeI and HindIII are used as restriction enzyme cutting sites of the upstream and downstream primers, and the 5 'end and the 3' end of the SEQ ID NO.8-15 sequence are used as amplification starting points to design specific primers. Preferably, the primer design sequence is: an upstream primer 5'-GGCATATGATGGGTGTTCATAAATTAG-3'; a downstream primer: 5'-GGCCAAGCTTCTTAATGAAGTCTTTTAAG-3', respectively;

gene amplification: adding the primers and the template into a pfu high-fidelity DNA polymerase amplification system to form a 50 mu l reaction system, and amplifying for 35 cycles in a PCR instrument;

double enzyme digestion: carrying out double enzyme digestion on a target gene and a vector pET30a by NdeI and HindIII, recovering a product through gel, and connecting overnight at 16 ℃ for about 12 hours by adopting T4 ligase to obtain a connection product; the target gene is synthesized by biological companies;

(2) and (3) transformation: taking 50 μ l of BL21(DE3) competent cells out of an ultra-low temperature refrigerator, placing on ice for melting, adding 5 μ l of ligation product, placing on ice for 30min, thermally shocking in water bath at 42 ℃ for 90s, placing on ice for 3min, adding 600 μ l of room temperature LB liquid culture medium, placing in a shaking table, and shaking-culturing at 37 ℃ and at the rotating speed of 200r/min for 60 min; uniformly mixing the obtained bacterial liquid, coating the mixture on a kanamycin-resistant flat plate with the concentration of 50 mug/ml, inverting the flat plate, and culturing at 37 ℃ for 16-24 hours until monoclonal bacterial plaque is formed;

(3) induction: selecting three monoclonals, respectively inoculating the monoclonals into 5ml LB test tubes containing 45-55 mu g/ml kanamycin, performing shake culture at 37 ℃ in a shaking table at a rotating speed of 200r/min, when OD600 reaches about 0.7, respectively adding 0.5mM isopropyl-beta-D-thiogalactoside into two test tubes, respectively culturing one test tube at 15 ℃ for 16h, culturing the other test tube at 37 ℃ for 4h, using the test tube without the addition of the isopropyl-beta-D-thiogalactoside as a negative reference, and detecting the expression of protein by adopting polyacrylamide gel electrophoresis and a western blotting method;

(4) preparing an electrophoresis sample: taking the sediment obtained after the culture medium is centrifuged, suspending the sediment in a lysis buffer solution, and carrying out ultrasonic lysis for 5min to obtain a lysate;

the volume ratio of medium to lysis buffer was 3: 1; lysis buffer was 50mM Tris-HCl,150mM NaCl, 5% glycerol, pH 8.0;

during ultrasonic cracking, stopping the ultrasonic treatment for 3 seconds every 2 seconds, and carrying out the ultrasonic treatment for 5min in total in an alternating and circulating manner;

(5) and (3) taking 1/3 of the lysate obtained in the step (4), and uniformly mixing the lysate with a protein loading buffer solution with the concentration of 5 times, wherein the volume ratio of the protein loading buffer solution to the lysate is 1: 4, heating to 100 ℃, keeping for 10min, and centrifuging for 5min at the rotating speed of 15000 r/min;

(6) taking the supernatant sample, centrifuging the rest lysate at 15000r/min for 10min, and taking the supernatant; mixing a protein loading buffer solution with 5 times concentration into the supernatant to be used as a supernatant sample; the volume ratio of the protein loading buffer to the supernatant was 1: 4; detecting the whole bacteria and cracking the supernatant by polyacrylamide gel electrophoresis and western blotting.

As can be seen from the attached figure 1, the target strip has obvious high-efficiency expression after the induction of the cells than before the induction, and the superoxide dismutase generated by the expression system constructed by the invention has good solubility from the distribution of the target protein in the supernatant.

As shown in the attached figure 2, Western blot analysis of the expression of the high-stability superoxide dismutase protein in BL21(DE3) shows that the superoxide dismutase constructed by the method has a correct structure.

Example 3

While the inventor of the present invention has searched and studied the present invention, the comparison is also made with respect to other types of products, specifically as follows:

after the invention and three similar products sold in the market are prepared into a solution with the same protein concentration (0.1mg/ml), the activity is detected by a superoxide dismutase detection kit (NBT method). As shown in fig. 3; in FIG. 3, the substrate shows purple color after undergoing autoxidation, and superoxide dismutase can prevent the autoxidation of the substrate, so that lighter color represents higher enzyme activity, and darker color represents lower enzyme activity.

After the invention and three similar products sold in the market are prepared into a solution with the same protein concentration (0.1mg/ml), the activity is detected by a superoxide dismutase detection kit (NBT method), and as shown in figure 4, the activity difference between the invention and a comparative product sold in the market is shown by a bar chart.

Through the attached figures 3 and 4, the vitality of the invention is far higher than that of three similar products sold on the market.

Example 4

The inventor also finds out that the gene sequences of the target protein, namely a comparison sequence 1 and a comparison sequence 2, can not be efficiently expressed when researching the invention, and the gene sequences are shown as SEQ ID No. 16-17; as for the enzyme activities of the

comparison sequences

1 and 2, as shown in figure 5, it can be seen from the histogram of the enzyme activity of the superoxide dismutase in figure 5 that after the fermentation liquor with the same volume is inoculated with the same bacterial amount and is induced for the same time, the total activity of the superoxide dismutase product is more than 9 times of the total activity of the enzyme in the comparison sequence 1 and more than 6 times of the total activity of the enzyme in the comparison sequence 2. Therefore, the sequence provided by the invention has high activity of superoxide dismutase.

Example 5

The method and the similar products sold in the market are respectively subjected to heat treatment for 1 hour in a water bath at 70 ℃, and then the activity of superoxide dismutase before and after the heat treatment is measured.

As shown in fig. 6 and 7, it can be seen from fig. 6 and 7 that the product of the present invention has excellent thermal stability and exhibits excellent activity even after 1 hour of hot water bath. This also shows that the present invention solves the problem mentioned in the background of the invention that the activity of superoxide dismutase in the end product is very low or has lost its activity.

Example 6

The invention and the similar products sold in the market are prepared into solution by the same buffer solution, then sterilized by a sterile 0.22 mu M microporous filter membrane in a sterile operating platform, then subpackaged in a sterile small centrifuge tube, sealed and stored, then placed at room temperature, and activity determination is carried out at a set time point.

The invention and the similar products sold in the market are prepared into solution by the same buffer solution, then sterilized by a sterile 0.22 mu M microporous filter membrane in a sterile operating platform, then subpackaged in a sterile small centrifuge tube, sealed and stored, then placed at normal temperature, and activity determination is carried out at a set time point. The curve formed by the circle marks in the figure represents the change record of the enzyme activity along with the time under the room temperature condition; the curve formed by the square marks represents the record of the change of the enzyme activity of the similar products sold in the market with time under the room temperature condition, as shown in the attached figure 8.

The curve formed by the circular marks represents the change record of the enzyme activity along with the time under the room temperature condition; the curve formed by the square marks represents the record of the change of the enzyme activity of the similar products sold in the market with time under the room temperature condition. The result shows that the product of the invention also shows stronger enzyme activity after 50 weeks, and the enzyme activity of the similar products sold in the market is almost lost.

Example 7

The graph of the superoxide dismutase enzyme solution with different concentrations is shown in figure 9, and the protein concentration of the superoxide dismutase in the three tubes is 5mg/ml, 0.5mg/ml and 0.1mg/ml respectively according to the graph of the solution state of the superoxide dismutase enzyme solution with different concentrations.

Application example 1

The effect of treating dermatitis with the aqueous solution of the present invention at 200U/ml was reduced in redness after 1 hour of application, and the redness inflammation portion was significantly reduced and the bulge portion caused by dermatitis was significantly flattened after another application and overnight (12 hours). The subject was a 37-year-old male, and the test site was the skin on the back of the neck.

Sequence listing

<110> courage in summer

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 213

<212> PRT

<213> superoxide dismutase (superoxide dismutase)

<400> 1

Met Gly Val His Lys Leu Glu Pro Lys Asp His Leu Lys Pro Gln Asn

1 5 10 15

Leu Glu Gly Ile Ser Asn Glu Gln Ile Glu Pro His Phe Glu Ala His

20 25 30

Tyr Lys Gly Tyr Val Ala Lys Tyr Asn Glu Ile Gln Glu Lys Leu Ala

35 40 45

Asp Gln Asn Phe Ala Asp Arg Ser Lys Ala Asn Gln Asn Tyr Ser Glu

50 55 60

Tyr Arg Glu Leu Lys Val Glu Glu Thr Phe Asn Tyr Met Gly Val Val

65 70 75 80

Leu His Glu Leu Tyr Phe Gly Met Leu Thr Pro Gly Gly Lys Gly Glu

85 90 95

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

100 105 110

Asp Ala Cys Thr Asn Glu Leu Lys Ala Ala Ala Met Ala Phe Arg Gly

115 120 125

Trp Ala Ile Leu Gly Leu Asp Ile Phe Ser Gly Arg Leu Val Val Asn

130 135 140

Gly Leu Asp Ala His Asn Val Tyr Asn Leu Thr Gly Leu Ile Pro Leu

145 150 155 160

Ile Val Ile Asp Thr Tyr Glu His Ala Tyr Tyr Val Asp Tyr Lys Asn

165 170 175

Lys Arg Pro Pro Tyr Ile Asp Ala Phe Phe Lys Asn Ile Asn Trp Asp

180 185 190

Val Val Asn Glu Arg Phe Glu Lys Ala Met Lys Ala Tyr Glu Ala Leu

195 200 205

Lys Asp Phe Ile Lys

210

<210> 2

<211> 211

<212> PRT

<213> superoxide dismutase (superoxide dismutase)

<400> 2

Met Gly Val His Lys Leu Thr Pro Arg Asp His Leu Lys Pro Gln Asn

1 5 10 15

Leu Asp Gly Ile Ser Asn Glu Gln Ile Glu Pro His Phe Glu Ala His

20 25 30

Tyr Lys Gly Tyr Val Thr Lys Tyr Asn Glu Ile Gln Glu Lys Leu Ala

35 40 45

Asp Gln Ser Phe Ser Asp Arg Gly Lys Ala Asn Gln Asn Tyr Ser Glu

50 55 60

Tyr Arg Glu Leu Lys Val Glu Glu Thr Phe Asn Tyr Met Gly Ile Val

65 70 75 80

Leu His Glu Leu Tyr Phe Gly His Leu Gly Ala Lys Gly Glu Pro Ser

85 90 95

Glu Ala Phe Lys Lys Lys Val Glu Glu Asp Leu Gly Gly Trp Asp Thr

100 105 110

Cys Thr Asn Glu Leu Lys Ala Ala Ala Val Ala Phe Arg Gly Trp Ala

115 120 125

Val Leu Gly Leu Asp Leu Phe Ser Gly Arg Leu Val Val Asn Gly Leu

130 135 140

Asp Ala His Asn Val Tyr Asn Leu His Gly Phe Ile Pro Ile Ile Val

145 150 155 160

Val Asp Thr Tyr Glu His Ala Tyr Tyr Val Asp Tyr Lys Asn Lys Arg

165 170 175

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

180 185 190

Asn Ala Arg Phe Glu Lys Ala Met Lys Ala Tyr Glu Ala Met Lys Asp

195 200 205

Tyr Ile Gly

210

<210> 3

<211> 213

<212> PRT

<213> superoxide dismutase (superoxide dismutase)

<400> 3

Met Ala Val His Lys Leu Gln Pro Lys Asp His Leu Lys Pro Gln Asn

1 5 10 15

Leu Glu Gly Ile Ser Asn Glu Gln Ile Glu Pro His Phe Glu Ala His

20 25 30

Tyr Lys Gly Tyr Val Thr Lys Tyr Asn Glu Ile Gln Glu Lys Leu Ala

35 40 45

Asp Gln Asn Phe Ala Asp Arg Ser Lys Ala Asn Gln Asn Tyr Ser Glu

50 55 60

Tyr Arg Glu Leu Lys Val Glu Glu Thr Phe Asn Tyr Met Gly Val Val

65 70 75 80

Leu His Glu Leu Tyr Phe Gly Met Leu Thr Ser Gly Gly Lys Gly Glu

85 90 95

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

100 105 110

Asp Ala Cys Thr Asn Glu Leu Lys Ala Ala Ala Ile Ala Phe Arg Gly

115 120 125

Trp Ala Ile Leu Gly Leu Asp Ile Phe Ser Gly Arg Leu Val Val Asn

130 135 140

Gly Leu Asp Ala His Asn Val Tyr Asn Leu Thr Gly Leu Ile Pro Ile

145 150 155 160

Ile Val Ile Asp Thr Tyr Glu His Ala Tyr Tyr Val Asp Tyr Lys Asn

165 170 175

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

180 185 190

Val Val Asn Glu Arg Phe Glu Lys Ala Met Lys Ala Tyr Glu Ala Leu

195 200 205

Lys Asp Phe Ile Lys

210

<210> 4

<211> 211

<212> PRT

<213> superoxide dismutase (superoxide dismutase)

<400> 4

Met Ala Val His Lys Leu Gln Pro Lys Asp His Leu Lys Pro Ser Asn

1 5 10 15

Leu Lys Gly Ile Ser Asn Glu Gln Ile Glu Pro His Phe Glu Ala His

20 25 30

Tyr Lys Gly Tyr Val Thr Lys Tyr Asn Glu Ile Gln Glu Lys Leu Ala

35 40 45

Asp Leu Asn Phe Ser Asp Arg Ser Lys Ala Asn Gln Asn Tyr Ser Glu

50 55 60

Tyr Arg Glu Leu Lys Val Glu Glu Ser Phe Asn Tyr Met Gly Val Val

65 70 75 80

Leu His Glu Leu Tyr Phe Gly His Leu Gly Ala Lys Gly Gln Pro Ser

85 90 95

Glu Ala Phe Lys Lys Lys Val Glu Glu Asp Phe Gly Ser Trp Asp Ala

100 105 110

Cys Val Gln Glu Leu Lys Ala Thr Gly Ile Ala Phe Arg Gly Trp Ala

115 120 125

Val Leu Gly Leu Asp Ile Phe Ser Gly Arg Leu Val Val Asn Gly Leu

130 135 140

Asp Ala His Asn Val Tyr Asn Phe Thr Gly Leu Ile Pro Leu Ile Val

145 150 155 160

Leu Asp Thr Tyr Glu His Ala Tyr Tyr Val Asp Tyr Lys Asn Lys Arg

165 170 175

Pro Pro Tyr Ile Asp Ala Phe Leu Glu Asn Ile Asn Trp Asp Val Val

180 185 190

Asn Glu Arg Phe Glu Lys Ala Met Lys Ala Tyr Glu Thr Leu Lys Asp

195 200 205

Phe Val Lys

210

<210> 5

<211> 211

<212> PRT

<213> superoxide dismutase (superoxide dismutase)

<400> 5

Met Gly Val His Lys Leu Gln Ala Arg Asp His Leu Lys Pro Gln Asn

1 5 10 15

Leu Asp Gly Ile Ser Asn Glu Gln Ile Glu Pro His Phe Glu Ala His

20 25 30

Tyr Lys Gly Tyr Val Gly Lys Tyr Asn Glu Ile Gln Glu Lys Leu Ala

35 40 45

Asp Gln Asn Phe Ser Asp Arg Ser Lys Ala Asn Gln Asn Tyr Ser Glu

50 55 60

Tyr Arg Glu Leu Lys Val Glu Glu Thr Phe Asn Tyr Met Gly Ile Val

65 70 75 80

Leu His Glu Leu Tyr Phe Gly His Leu Gly Ala Lys Gly Glu Pro Ser

85 90 95

Glu Ala Phe Lys Lys Lys Val Glu Glu Asp Leu Gly Gly Trp Asp Ala

100 105 110

Cys Thr Asn Glu Leu Lys Ala Ala Ala Val Ala Phe Arg Gly Trp Ala

115 120 125

Val Leu Gly Leu Asp Leu Phe Ser Gly Arg Leu Val Val Asn Gly Leu

130 135 140

Asp Ala His Asn Val Tyr Asn Phe His Gly Phe Ile Pro Ile Ile Val

145 150 155 160

Ile Asp Thr Tyr Glu His Ala Tyr Tyr Val Asp Tyr Lys Asn Lys Arg

165 170 175

Pro Pro Tyr Val Asp Ala Val Leu Lys Asn Leu Asn Trp Glu Val Val

180 185 190

Asn Ala Arg Phe Glu Lys Ala Met Lys Ala Cys Glu Ala Leu Gly Asp

195 200 205

Tyr Ile Gly

210

<210> 6

<211> 211

<212> PRT

<213> superoxide dismutase (superoxide dismutase)

<400> 6

Met Pro Val His Lys Leu Glu Pro Lys Asn His Leu Lys Pro Ser Asn

1 5 10 15

Leu Asn Gly Ile Ser Asn Glu Gln Ile Glu Pro His Phe Glu Ala His

20 25 30

Tyr Lys Gly Tyr Val Thr Lys Tyr Asn Glu Ile Gln Glu Lys Leu Ala

35 40 45

Asp Leu Asn Phe Ser Asp Arg Ser Lys Ala Asn Gln Asn Tyr Ser Glu

50 55 60

Tyr Arg Glu Leu Lys Val Glu Glu Thr Phe Asn Tyr Met Gly Val Val

65 70 75 80

Leu His Glu Leu Tyr Phe Gly His Leu Gly Ala Lys Gly Glu Pro Ser

85 90 95

Glu Ala Phe Lys Lys Lys Val Glu Glu Asp Phe Gly Ser Trp Asp Ala

100 105 110

Cys Ile Gln Glu Ile Lys Ala Ala Gly Met Ala Phe Arg Gly Trp Ala

115 120 125

Ile Leu Gly Leu Asp Ile Phe Ser Gly Arg Leu Val Val Asn Gly Leu

130 135 140

Asp Ala His Asn Val Tyr Asn Tyr Thr Gly Leu Ile Pro Leu Ile Val

145 150 155 160

Leu Asp Thr Tyr Glu His Ala Tyr Tyr Val Asp Gln Lys Asn Lys Arg

165 170 175

Pro Pro Tyr Ile Asp Ala Phe Leu Gln Asn Leu Asn Trp Glu Val Ile

180 185 190

Asn Glu Arg Phe Glu Lys Ala Met Lys Ala Tyr Glu Thr Leu Lys Asp

195 200 205

Phe Val Lys

210

<210> 7

<211> 211

<212> PRT

<213> superoxide dismutase (superoxide dismutase)

<400> 7

Met Pro Val His Lys Leu Gln Pro Arg Asp His Leu Lys Pro Ser Asn

1 5 10 15

Leu Asn Gly Ile Ser Asn Glu Gln Ile Glu Pro His Phe Glu Ala His

20 25 30

Tyr Lys Gly Tyr Val Ala Lys Tyr Asn Glu Ile Gln Glu Lys Leu Ala

35 40 45

Asp Leu Ser Phe Ser Asp Arg Ser Lys Ala Asn Gln Asn Tyr Ser Glu

50 55 60

Tyr Arg Glu Leu Lys Val Glu Glu Thr Phe Asn Tyr Met Gly Val Val

65 70 75 80

Leu His Glu Leu Tyr Phe Gly His Leu Gly Pro Lys Gly Gln Pro Ser

85 90 95

Asp Ala Leu Lys Lys Lys Val Glu Glu Asp Phe Gly Ser Trp Asp Ala

100 105 110

Cys Ile Gln Glu Ile Lys Ala Ala Gly Ile Ala Phe Arg Gly Trp Ala

115 120 125

Ile Leu Gly Leu Asp Ile Phe Ser Gly Arg Leu Val Val Asn Gly Leu

130 135 140

Asp Ala His Asn Val Tyr Asn Tyr Thr Gly Leu Ile Pro Leu Ile Val

145 150 155 160

Leu Asp Thr Tyr Glu His Ala Tyr Tyr Val Asp Gln Lys Asn Lys Arg

165 170 175

Pro Pro Tyr Ile Glu Ala Phe Leu Gln Ser Leu Asn Trp Asp Val Ile

180 185 190

Asn Glu Arg Phe Glu Lys Ala Met Lys Ala Tyr Glu Ala Leu Lys Asp

195 200 205

Phe Ile Lys

210

<210> 8

<211> 639

<212> DNA/RNA

<213> superoxide dismutase (superoxide dismutase)

<400> 8

atgggtgttc ataaattaga acctaaggat cacttgaaac cgcaaaatct tgagggcatt 60

tctaacgaac agatcgagcc acattttgaa gctcactata agggatacgt cgccaaatat 120

aatgagatac aagaaaagct cgcagaccag aacttcgcgg atcgttccaa agctaatcaa 180

aactactcag agtatcgcga acttaaggta gaggaaactt ttaattacat gggggtggtt 240

ctgcatgagt tatatttcgg tatgttgacc ccgggcggaa aaggggaacc ttcggaggcc 300

cttaagaaaa agattgaaga ggacatcggt ggcctcgatg catgtacaaa cgaactaaaa 360

gcggctgcca tggcatttcg tggatgggcg atactggggt tagacatttt cagtggtcgc 420

ttggtcgtaa atggccttga tgctcacaac gtgtacaatc tcacgggact tatcccgctg 480

atagttattg acacttatga gcatgcctac tatgtcgatt acaagaacaa acgtccaccg 540

tatatcgacg catttttcaa gaatataaac tgggatgtag tgaatgaacg ctttgagaaa 600

gcgatgaagg cttacgaagc cttaaaagac ttcattaag 639

<210> 9

<211> 639

<212> DNA/RNA

<213> superoxide dismutase (superoxide dismutase)

<400> 9

atgggggtgc acaaactgga accgaaagac cacctgaaac cgcaaaacct ggaagggatt 60

tcgaacgaac aaattgaacc gcacttcgaa gcgcactaca aagggtacgt ggcgaaatac 120

aacgaaattc aagaaaaact ggcggaccaa aacttcgcgg accggtcgaa agcgaaccaa 180

aactactcgg aataccggga actgaaagtg gaagaaacgt tcaactacat gggggtggtg 240

ctgcacgaac tgtacttcgg gatgctgacg ccggggggga aaggggaacc gtcggaagcg 300

ctgaaaaaaa aaattgaaga agacattggg gggctggacg cgtgcacgaa cgaactgaaa 360

gcggcggcga tggcgttccg ggggtgggcg attctggggc tggacatttt ctcggggcgg 420

ctggtggtga acgggctgga cgcgcacaac gtgtacaacc tgacggggct gattccgctg 480

attgtgattg acacgtacga acacgcgtac tacgtggact acaaaaacaa acggccgccg 540

tacattgacg cgttcttcaa aaacattaac tgggacgtgg tgaacgaacg gttcgaaaaa 600

gcgatgaaag cgtacgaagc gctgaaagac ttcattaag 639

<210> 10

<211> 633

<212> DNA/RNA

<213> superoxide dismutase (superoxide dismutase)

<400> 10

atgggggtgc acaaactgac gccgcgggac cacctgaaac cgcaaaacct ggacgggatt 60

tcgaacgaac aaattgaacc gcacttcgaa gcgcactaca aagggtacgt gacgaaatac 120

aacgaaattc aagaaaaact ggcggaccaa tcgttctcgg accgggggaa agcgaaccaa 180

aactactcgg aataccggga actgaaagtg gaagaaacgt tcaactacat ggggattgtg 240

ctgcacgaac tgtacttcgg gcacctgggg gcgaaagggg aaccgtcgga agcgttcaaa 300

aaaaaagtgg aagaagacct gggggggtgg gacacgtgca cgaacgaact gaaagcggcg 360

gcggtggcgt tccgggggtg ggcggtgctg gggctggacc tgttctcggg gcggctggtg 420

gtgaacgggc tggacgcgca caacgtgtac aacctgcacg ggttcattcc gattattgtg 480

gtggacacgt acgaacacgc gtactacgtg gactacaaaa acaaacggcc gccgtacgtg 540

gacgcggtgc tgaaaaacct gaactgggac gtggtgaacg cgcggttcga aaaagcgatg 600

aaagcgtacg aagcgatgaa agactacatt ggt 633

<210> 11

<211> 639

<212> DNA/RNA

<213> superoxide dismutase (superoxide dismutase)

<400> 11

atggcggtgc acaaactgca accgaaagac cacctgaaac cgcaaaacct ggaagggatt 60

tcgaacgaac aaattgaacc gcacttcgaa gcgcactaca aagggtacgt gacgaaatac 120

aacgaaattc aagaaaaact ggcggaccaa aacttcgcgg accggtcgaa agcgaaccaa 180

aactactcgg aataccggga actgaaagtg gaagaaacgt tcaactacat gggggtggtg 240

ctgcacgaac tgtacttcgg gatgctgacg tcggggggga aaggggaacc gtcggaagcg 300

ctgaaaaaaa aaattgaaga agacctgggg gggttcgacg cgtgcacgaa cgaactgaaa 360

gcggcggcga ttgcgttccg ggggtgggcg attctggggc tggacatttt ctcggggcgg 420

ctggtggtga acgggctgga cgcgcacaac gtgtacaacc tgacggggct gattccgatt 480

attgtgattg acacgtacga acacgcgtac tacgtggact acaaaaacaa acggccgccg 540

tacgtggacg cggtgctgaa aaacattaac tgggacgtgg tgaacgaacg gttcgaaaaa 600

gcgatgaaag cgtacgaagc gctgaaagac ttcattaag 639

<210> 12

<211> 633

<212> DNA/RNA

<213> superoxide dismutase (superoxide dismutase)

<400> 12

atggcggtgc acaaactgca accgaaagac cacctgaaac cgtcgaacct gaaagggatt 60

tcgaacgaac aaattgaacc gcacttcgaa gcgcactaca aagggtacgt gacgaaatac 120

aacgaaattc aagaaaaact ggcggacctg aacttctcgg accggtcgaa agcgaaccaa 180

aactactcgg aataccggga actgaaagtg gaagaatcgt tcaactacat gggggtggtg 240

ctgcacgaac tgtacttcgg gcacctgggg gcgaaagggc aaccgtcgga agcgttcaaa 300

aaaaaagtgg aagaagactt cgggtcgtgg gacgcgtgcg tgcaagaact gaaagcgacg 360

gggattgcgt tccgggggtg ggcggtgctg gggctggaca ttttctcggg gcggctggtg 420

gtgaacgggc tggacgcgca caacgtgtac aacttcacgg ggctgattcc gctgattgtg 480

ctggacacgt acgaacacgc gtactacgtg gactacaaaa acaaacggcc gccgtacatt 540

gacgcgttcc tggaaaacat taactgggac gtggtgaacg aacggttcga aaaagcgatg 600

aaagcgtacg aaacgctgaa agacttcgtg aag 633

<210> 13

<211> 633

<212> DNA/RNA

<213> superoxide dismutase (superoxide dismutase)

<400> 13

atgggggtgc acaaactgca agcgcgggac cacctgaaac cgcaaaacct ggacgggatt 60

tcgaacgaac aaattgaacc gcacttcgaa gcgcactaca aagggtacgt ggggaaatac 120

aacgaaattc aagaaaaact ggcggaccaa aacttctcgg accggtcgaa agcgaaccaa 180

aactactcgg aataccggga actgaaagtg gaagaaacgt tcaactacat ggggattgtg 240

ctgcacgaac tgtacttcgg gcacctgggg gcgaaagggg aaccgtcgga agcgttcaaa 300

aaaaaagtgg aagaagacct gggggggtgg gacgcgtgca cgaacgaact gaaagcggcg 360

gcggtggcgt tccgggggtg ggcggtgctg gggctggacc tgttctcggg gcggctggtg 420

gtgaacgggc tggacgcgca caacgtgtac aacttccacg ggttcattcc gattattgtg 480

attgacacgt acgaacacgc gtactacgtg gactacaaaa acaaacggcc gccgtacgtg 540

gacgcggtgc tgaaaaacct gaactgggaa gtggtgaacg cgcggttcga aaaagcgatg 600

aaagcgtgcg aagcgctggg ggactacatt ggt 633

<210> 14

<211> 633

<212> DNA/RNA

<213> superoxide dismutase (superoxide dismutase)

<400> 14

atgccggtgc acaaactgga accgaaaaac cacctgaaac cgtcgaacct gaacgggatt 60

tcgaacgaac aaattgaacc gcacttcgaa gcgcactaca aagggtacgt gacgaaatac 120

aacgaaattc aagaaaaact ggcggacctg aacttctcgg accggtcgaa agcgaaccaa 180

aactactcgg aataccggga actgaaagtg gaagaaacgt tcaactacat gggggtggtg 240

ctgcacgaac tgtacttcgg gcacctgggg gcgaaagggg aaccgtcgga agcgttcaaa 300

aaaaaagtgg aagaagactt cgggtcgtgg gacgcgtgca ttcaagaaat taaagcggcg 360

gggatggcgt tccgggggtg ggcgattctg gggctggaca ttttctcggg gcggctggtg 420

gtgaacgggc tggacgcgca caacgtgtac aactacacgg ggctgattcc gctgattgtg 480

ctggacacgt acgaacacgc gtactacgtg gaccaaaaaa acaaacggcc gccgtacatt 540

gacgcgttcc tgcaaaacct gaactgggaa gtgattaacg aacggttcga aaaagcgatg 600

aaagcgtacg aaacgctgaa agacttcgtg aag 633

<210> 15

<211> 633

<212> DNA/RNA

<213> superoxide dismutase (superoxide dismutase)

<400> 15

atgccggtgc acaaactgca accgcgggac cacctgaaac cgtcgaacct gaacgggatt 60

tcgaacgaac aaattgaacc gcacttcgaa gcgcactaca aagggtacgt ggcgaaatac 120

aacgaaattc aagaaaaact ggcggacctg tcgttctcgg accggtcgaa agcgaaccaa 180

aactactcgg aataccggga actgaaagtg gaagaaacgt tcaactacat gggggtggtg 240

ctgcacgaac tgtacttcgg gcacctgggg ccgaaagggc aaccgtcgga cgcgctgaaa 300

aaaaaagtgg aagaagactt cgggtcgtgg gacgcgtgca ttcaagaaat taaagcggcg 360

gggattgcgt tccgggggtg ggcgattctg gggctggaca ttttctcggg gcggctggtg 420

gtgaacgggc tggacgcgca caacgtgtac aactacacgg ggctgattcc gctgattgtg 480

ctggacacgt acgaacacgc gtactacgtg gaccaaaaaa acaaacggcc gccgtacatt 540

gaagcgttcc tgcaatcgct gaactgggac gtgattaacg aacggttcga aaaagcgatg 600

aaagcgtacg aagcgctgaa agacttcatt aag 633

<210> 16

<211> 639

<212> DNA/RNA

<213> superoxide dismutase (superoxide dismutase)

<400> 16

atgggtgttc ataaactaga acccaaggat cacctaaaac cccaaaatct agagggcata 60

tcgaacgaac agatagagcc acattttgaa gctcactata agggatacgt cgccaaatat 120

aatgagatac aagaaaagct agcagaccag aacttcgcgg atcgatcgaa agctaatcaa 180

aactactcgg agtatcgaga actaaaggta gaggaaactt ttaattacat gggggtggtt 240

ctacatgagt tatatttcgg tatgttgacc ccgggcggaa aaggggaacc ttcggaggcc 300

ctaaagaaaa agatagaaga ggacataggt ggcctcgatg catgtacaaa cgaactaaaa 360

gcggctgcca tggcatttcg aggatgggcg atactagggt tagacatatt ctcgggtcga 420

ttggtcgtaa atggcctaga tgctcacaac gtgtacaatc taacgggact aataccccta 480

atagttattg acacttatga gcatgcctac tatgtcgatt acaagaacaa acgacctccc 540

tatatagacg catttttcaa gaatataaac tgggatgtag tgaatgaaag gtttgagaaa 600

gcgatgaagg cttacgaagc cctaaaagac ttcattaag 639

<210> 17

<211> 639

<212> DNA/RNA

<213> superoxide dismutase (superoxide dismutase)

<400> 17

atgggtgttc ataaattaga acccaaggat cacttgaaac cccaaaatct tgagggcatt 60

tctaacgaac agatcgagcc acattttgaa gctcactata agggatacgt cgccaaatat 120

aatgagatac aagaaaagct cgcagaccag aacttcgcgg atcgatccaa agctaatcaa 180

aactactcag agtatcgaga actaaaggta gaggaaactt ttaattacat gggggtggtt 240

ctgcatgagt tatatttcgg tatgttgacc ccgggcggaa aaggggaacc ttcggaggcc 300

cttaagaaaa agattgaaga ggacatcggt ggcctcgatg catgtacaaa cgaactaaaa 360

gcggctgcca tggcatttcg aggatgggcg atactggggt tagacatttt cagtggtcga 420

ttggtcgtaa atggccttga tgctcacaac gtgtacaatc tcacgggact aatccccctg 480

atagttattg acacttatga gcatgcctac tatgtcgatt acaagaacaa acgacctccc 540

tatatcgacg catttttcaa gaatataaac tgggatgtag tgaatgaaag gtttgagaaa 600

gcgatgaagg cttacgaagc cttaaaagac ttcattaag 639

Claims

1. The method for constructing the high-stability superoxide dismutase is characterized in that a gene sequence for coding the high-stability superoxide dismutase is shown as SEQ ID No. 8;

the method comprises the following steps:

(1) primer design and gene amplification: designing specific primers by taking restriction enzymes NdeI and HindIII as restriction enzyme cutting sites of upstream and downstream restriction enzymes and taking the 5 'end and the 3' end of the SEQ ID NO.8 sequence as amplification starting points;

(2) double enzyme digestion: carrying out double enzyme digestion on a target gene and a vector pET30a by NdeI and HindIII, recovering the product through gel, and then connecting by adopting T4 ligase, wherein the T4 ligase is connected for 4-15 hours at the temperature of 12-20 ℃ to obtain a connected product;

(3) and (3) transformation: taking out 30-100 mu l of escherichia coli BL21 competent cells from an ultralow-temperature refrigerator, placing the escherichia coli BL21 competent cells on ice for melting, adding 3-15 mu l of connecting products, placing the escherichia coli BL21 competent cells on ice for 25-35 min, thermally shocking the escherichia coli BL21 competent cells in water bath for 45-90 s at 42-45 ℃, placing the escherichia coli BL21 competent cells on ice for 2-10 min, adding 400-1000 mu l of LB liquid medium at room temperature, and shaking the escherichia coli BL21 competent cells in a constant-temperature shaking table at 30-37 ℃ for 45-60 min; liquid culture, namely uniformly mixing the obtained bacterial liquid, coating the mixture on a kanamycin-resistant flat plate, inverting the flat plate, and culturing at the temperature of 30-37 ℃ for 16-24 hours until monoclonal bacterial plaque is formed;

(4) induction: selecting three monoclonals, respectively inoculating the monoclonals into 1-8 ml test tubes containing LB culture medium containing 45-55 mu g/ml kanamycin, carrying out shake culture at the temperature of 30-40 ℃ and the rotation speed of 200r/min in a shaking table, adding 0.1-1.0 mM isopropyl-beta-D-thiogalactoside into two test tubes when OD600 reaches 0.6-0.9, respectively, culturing one test tube at the temperature of 14-20 ℃ for 12-20 h, culturing the other test tube at the temperature of 30-40 ℃ for 3-8 h, and detecting the expression of protein by using polyacrylamide gel electrophoresis and western blotting with the test tube without adding isopropyl-beta-D-thiogalactoside as a negative reference;

(5) preparing an electrophoresis sample: taking the sediment obtained after the culture medium is centrifuged, suspending the sediment in a lysis buffer solution, and carrying out ultrasonic lysis to obtain a lysate;

(6) taking part of lysate obtained in the step (5), uniformly mixing the lysate with the protein loading buffer solution, heating and centrifuging;

(7) taking the residual lysate, centrifuging to obtain supernatant, and mixing the protein sample buffer solution into the supernatant to obtain a supernatant sample; detecting the whole bacteria and cracking the supernatant by polyacrylamide gel electrophoresis and western blotting.

2. The method for constructing superoxide dismutase with high stability as claimed in claim 1, wherein in (3), BL21 competent cells are taken out from the ultra-low temperature refrigerator, and the temperature of the ultra-low temperature refrigerator is-80 to-70 ℃.

3. The method for constructing the superoxide dismutase with high stability as claimed in claim 1, wherein in (5), the sediment after the culture medium centrifugation is taken and is suspended in the lysis buffer, and the ultrasonic lysis time is 4-6 min; the volume ratio of the medium to the lysis buffer was: 5: 1-2: 1.

4. the method of claim 1, wherein in step (5), 3ml of the culture medium is centrifuged, and the pellet is resuspended in 1ml of lysis buffer and sonicated for 5 min.

5. The method for constructing superoxide dismutase with high stability as claimed in claim 1, wherein (5) ultrasonic cleavage is performed for 1.5-2.5 s and then stopped for 2.5-3.5 s, and the ultrasonic treatment is performed in an alternating cycle for 4-6 min.

6. The method for constructing superoxide dismutase with high stability as claimed in claim 1, wherein in (5), the lysis buffer is 50mM Tris-HCl,150mM NaCl, 5% glycerol, pH 8.0.

7. The method for constructing a high-stability superoxide dismutase as claimed in claim 1, wherein in (6), 1/4 of the lysate obtained in (5) is taken, mixed with the protein loading buffer solution uniformly, heated and centrifuged; the volume ratio of lysate to protein loading buffer was 4: 1.

8. the method for constructing superoxide dismutase with high stability as claimed in claim 7, wherein in (6), the protein loading buffer has a concentration 5 times that of the protein loading buffer.

9. The method for constructing a high-stability superoxide dismutase as claimed in claim 7, wherein in (6), the mixture is heated to 95 to 100 ℃ for 8 to 12 min.

10. The method for constructing a high-stability superoxide dismutase as claimed in claim 7, wherein in (6), the centrifugation is performed at 12000-15000 r/min for 5-15 min.

11. The method for constructing superoxide dismutase with high stability as claimed in claim 1, wherein in (7), a protein loading buffer is mixed with the supernatant as a supernatant sample, wherein the volume ratio of the protein loading buffer to the supernatant is 1: 3.5-1: 4.5; detecting the whole bacteria and cracking the supernatant by polyacrylamide gel electrophoresis and western blotting.

12. The method for constructing a high-stability superoxide dismutase as claimed in claim 1, wherein in (7), the mixture is centrifuged at 13000 to 15000r/min for 10 to 15min, and the supernatant is collected.

13. The method for constructing a high-stability superoxide dismutase as claimed in claim 1, wherein in (7), the volume ratio of the protein loading buffer to the supernatant is 1: 4.

14. the method for constructing superoxide dismutase with high stability as claimed in claim 1, wherein in (7), the protein loading buffer has a concentration 5 times that of the protein loading buffer.

15. The method for constructing the high-stability superoxide dismutase as claimed in claim 1, comprising the steps of:

(2) double enzyme digestion: carrying out double enzyme digestion on the target gene and a vector pET30a by NdeI and HindIII, recovering the product through gel, and connecting for 4-15 h at 12-20 ℃ by adopting T4 ligase to obtain a connected product;

(3) and (3) transformation: taking out 30-100 mu l of BL21 competent cells from an ultralow-temperature refrigerator at the temperature of-80 to-70 ℃, putting the cells on ice for melting, adding 3-15 mu l of connecting products, putting the cells on ice for 25-35 min, thermally exciting the cells in a water bath at the temperature of 42-45 ℃ for 45-90 s, putting the cells on ice for 2-6 min, adding 400-1000 mu l of LB liquid medium at room temperature, and putting the cells in a shaking table for shaking culture at the temperature of 30-37 ℃ and the rotating speed of 150-250 r/min for 45-60 min; uniformly mixing the obtained bacterial liquid, coating the mixture on a kanamycin-resistant LB flat plate with the concentration of 45-55 mu g/ml, inverting the flat plate, and culturing at 33-37 ℃ until monoclonal bacterial plaque is formed;

(4) induction: selecting three monoclonals, respectively inoculating the monoclonals into 1-5 ml LB test tubes containing 45-55 mug/ml kanamycin, performing shake culture at the temperature of 30-37 ℃ and the rotating speed of 200r/min in a shaking table, adding 0.1-1.0 mM isopropyl-beta-D-thiogalactoside into two test tubes when OD600 reaches 0.6-0.9, respectively, culturing one test tube at the temperature of 14-20 ℃ for 12-20 h, culturing the other test tube at the temperature of 30-37 ℃ for 3-8 h, taking the test tube without the addition of the isopropyl-beta-D-thiogalactoside as a negative reference, and detecting the expression of the protein by adopting polyacrylamide gel electrophoresis and a protein blotting method;

(5) preparing an electrophoresis sample: taking the sediment obtained after the culture medium is centrifuged, re-suspending the sediment in a lysis buffer solution, and carrying out ultrasonic lysis for 4-6 min to obtain a lysate;

the volume ratio of the medium to the lysis buffer was: 5: 1-2: 1; lysis buffer 50mM Tris-HCl,150mM NaCl, 5% glycerol, pH 8.0;

(6) taking 1/3 of the lysate obtained in the step (5), uniformly mixing the lysate with 5X protein loading buffer solution, heating to 95-100 ℃, keeping the temperature for 8-12 min, and centrifuging for 5-10 min at the rotating speed of 12000-15000 r/min; the volume ratio of the protein loading buffer to the supernatant was 1: 4;

(7) taking the supernatant sample, namely centrifuging the residual lysate for 8-12 min at the rotating speed of 12000-15000 r/min, and taking the supernatant; mixing 5 times of protein loading buffer solution into the supernatant to be used as a supernatant sample; the volume ratio of the protein loading buffer to the supernatant was 1: 4; detecting the whole bacteria and cracking the supernatant by polyacrylamide gel electrophoresis and western blotting.