Superoxide dismutase RPSOD of deep-sea tubular worm Ridgeia piscesae and preparation method thereof

Abstract

Superoxide dismutase RPSOD of deep-sea tubular worm Ridgeia piscesae and its preparation method, involving the nucleotide sequence of R.piscesae superoxide dismutase gene and the amino acid sequence encoded by it. Based on transcriptome analysis, the nucleotide sequence of R.piscesae superoxide dismutase rpsod was obtained, and the amino acid sequence was deduced. Heterologous recombinant expression of RPSOD was successfully achieved using Escherichia coli BL21 (DE3) as the host. After purification, the function of RPSOD was explored, and the experiment found that it has good tolerance to high temperature, alkalinity and some denaturants (urea, EDTA, PMSF, H2O2), and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to a tube worm, in particular to a nucleotide sequence of a novel superoxide dismutase gene of a tube worm Ridgeiapiscesae from a deep-sea hydrothermal area, an amino acid sequence encoded by the gene and a preparation method thereof. Background Art

[0002] The extreme environment of deep-sea hydrothermal areas can easily cause oxidative stress in the body (1. Mittler R. Oxidative stress, antioxidants and stress tolerance [J]. Trends in Plant Science, 2002, 7 (9): 405-410.). As the dominant species in deep-sea hydrothermal areas, tube worms must have efficient antioxidant mechanisms in their bodies. Superoxide dismutase (SOD) is the first line of defense against oxidative stress. Its function of removing superoxide anion free radicals in organisms has been widely used in anti-aging, anti-inflammatory and tumor inhibition (2. Lin Qingbin, Liao Shengrong, Xiong Yahong, et al. Research and application progress of superoxide dismutase (SOD) [J]. Chemical World, 2006: 378-381.). At present, most of the SOD on the market is extracted from animal blood, which not only has the risks of cross infection and exclusivity, but also is inconvenient to store (3, Xin Bo, Chen Weijun, Xia Qiuyu, et al. Effect of metal ions on the ability of coconut juice to remove superoxide anions [J]. Journal of Tropical Crops, 2009, 30: 1069-1074.). In addition, some SOD is extracted from plants, which has complicated processes and high costs (4, Dong Liang, He Yongzhi, Wang Yuanliang, et al. Research progress on the application of superoxide dismutase (SOD) [J]. China Agricultural Science and Technology Herald, 2013, 15: 53-58.). Therefore, the research and development of new SOD has become a global hotspot.

[0003] With the deepening of research, genetic engineering technology has been widely used in the production of SOD. Today, SOD has made great achievements in many fields such as medical treatment, food, agriculture and daily chemicals (4, Dong Liang, He Yongzhi, Wang Yuanliang, et al. Research progress of superoxide dismutase (SOD) [J]. China Agricultural Science and Technology Herald, 2013, 15: 53-58.). In medical treatment, human therapeutic clinical trials have shown that the supplementation of SOD can prevent or reverse various diseases caused by oxidative stress, including cancer, asthma, diabetes, arthritis, atherosclerosis, ischemia-reperfusion injury, aging, infertility, neurological dysfunction, transplant rejection, autoimmune diseases, rheumatoid arthritis, etc. (5, Mccord JM. Human disease, free radicals, and the oxidant / antioxidant balance [J]. Clinical Biochemistry, 1993, 26 (5): 351.). In food production, SOD fruits, SOD vegetables, SOD active tea and other SOD foods have received strong response since their launch (6, Aoyagi H, Ishii H, Ugwu CU, et al. Effect of heat-generated product from uronic acids on the physiological activities of microbial cells and its application [J]. Bioresource Technology, 2008, 99 (10): 4534-4538.). In daily chemical products such as moisturizing cream, sunscreen, whitening cream, eye cream, nail polish and anti-hair loss spray, the appropriate amount of SOD can effectively eliminate free radical damage and play an anti-aging, anti-inflammatory, anti-viral and anti-radiation role (7, Bafana A, Dutt S, Kumar S, et al. Superoxide dismutase: an industrial perspective [J]. Critical Reviews in Biotechnology, 2011, 31 (1): 65-76.).During the planting process, the increase in SOD levels can activate the metabolic protection mechanism of plants under stress and increase biomass. Taking advantage of this characteristic, researchers are currently trying to use transgenic technology to obtain stress-resistant plants (8, RA, Kim YH, Kim MD, et al. Simultaneous expression of choline oxidase, superoxide dismutase and ascorbate peroxidase in potato plant chloroplasts provides synergistically enhanced protection against various abiotic stresses[J]. Physiologia Plantarum, 2010, 138(4): 520-533.). Summary of the invention

[0004] The first object of the present invention is to provide the gene sequence of superoxide dismutase rpsod of the tubular worm R. piscesae;

[0005] The second object of the present invention is to provide the protein sequence of superoxide dismutase RPSOD of the tubular worm R. piscesae;

[0006] The third object of the present invention is to provide a method for preparing superoxide dismutase RPSOD of tubular worm R. piscesae;

[0007] The fourth object of the present invention is to provide the biological activity of superoxide dismutase RPSOD of the tubular worm R. piscesae;

[0008] The superoxide dismutase gene of the tubular worm R. piscesae of the present invention, and the nucleotide sequence encoded by it, are denoted as rpsod.

[0009] The molecular type of the superoxide dismutase gene rpsod of the tubular worm R. piscesae is DNA, and the sequence characteristics are: length of 675 bp, type of nucleic acid, chain type of double chain, and topological structure of linear. The sequence of the superoxide dismutase gene rpsod of R. piscesae is as follows, recorded as SEQ ID No. 1:

[0010]

[0011] The acquisition and preparation of the superoxide dismutase RPSOD of the tubular worm Ridgeia piscesae of the present invention comprises the following steps:

[0012] The total RNA of the tubular worm R.piscesae was extracted and reversed into cDNA. The synthetic sequences SEQ ID No.3 and SEQ ID No.4 were used as primers, and PCR amplification was performed under the conditions of annealing temperature of 52°C and extension time of 42s to obtain the nucleotide sequence of the rpsod gene SEQ ID No.1. The amino acid sequence of the polypeptide SEQ ID No.2 encoded by SEQ ID No.1 was deduced by relevant software. Furthermore, the heterologous recombinant expression of RPSOD was successfully achieved using Escherichia coli BL21 (DE3) as the host. After purification by nickel column, it was proved by biological methods that the polypeptide SEQ ID No.2 encoded by the rpsod gene had superoxide dismutase activity.

[0013] The superoxide dismutase RPSOD of the present invention has the activity of eliminating superoxide anions, is alkali-resistant, high-temperature-resistant, and is insensitive to treatment with 10 mmol / L urea, EDTA, PMSF, and H2O2 denaturants.

[0014] The synthetic sequence SEQ ID No.3: CGC GGATCC ATGCTGTCAGCAACTACAG; (underlined is endonuclease, restriction site BamH I)

[0015] The synthetic sequence SEQ ID No.4: CCG GAATTC CTATTGCCTCGCTTTCT; (underlined is endonuclease, restriction site EcoR I)

[0016] The molecular type of the superoxide dismutase RPSOD of the tubular worm R. piscesae is protein, and the sequence characteristics are: the length is 224aa, the type is amino acid, and the sequence of the superoxide dismutase RPSOD is as follows, recorded as SEQ ID No. 2:

[0017]

[0018] The tubular worm R. piscesae superoxide dismutase RPSOD, the protein has a polypeptide with an amino acid sequence shown in SEQ ID No. 2; or a polypeptide formed by replacing, deleting or adding one or more amino acid residues in the amino acid sequence of SEQ ID No. 2 and having similar functions to the amino acid sequence shown in SEQ ID No. 2.

[0019] The method for preparing the superoxide dismutase RPSOD of the tubular worm Ridgeia piscesae of the present invention comprises the following steps:

[0020] 1) The steps of total RNA extraction and reverse transcription synthesis of cDNA from deep-sea tubular worm R. piscesae;

[0021] 2) Steps for PCR amplification and sequence analysis of superoxide dismutase rpsod from the deep-sea tube worm R. piscesae;

[0022] 3) Steps for recombinant expression and purification of superoxide dismutase RPSOD from the deep-sea tubular worm R. Piscesae;

[0023] 4) Steps for activity detection and functional investigation of superoxide dismutase RPSOD in the deep-sea tubular worm R. Piscesae.

[0024] The present invention successfully realizes heterologous recombinant expression of RPSOD by analyzing the superoxide dismutase rpsod gene of deep-sea tubular worm R.piscesae using Escherichia coli BL21 (DE3) as a host. After purification, the enzymatic properties of RPSOD are analyzed, and the results show that the optimum temperature of RPSOD activity is 50°C, and it has excellent stability at 10°C-70°C, and even 41% of residual enzyme activity is still present after incubation at a high temperature of 80°C for 10 hours. The optimum pH of its activity is 9, and its activity is almost lost in an acidic environment. It has strong stability under neutral and alkaline conditions, and still has more than 40% of residual enzyme activity after being treated with a buffer solution of pH 12 for 1 hour. In addition, RPSOD has good tolerance to various common denaturants (urea, EDTA, PMSF, H2O2), and SDS has a certain effect on its activity. In summary, RPSOD is an enzyme that has good tolerance to high temperature, alkalinity, and some denaturants, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The SDS-PAGE electrophoresis of the purification and dialysis of RPSOD after expression. M: protein marker; 1: RPSOD after purification; 2: RPSOD after dialysis.

[0026] Figure 2 The effect of temperature on the activity of recombinant superoxide dismutase RPSOD. The horizontal axis represents different temperatures; the vertical axis represents the relative activity of RPSOD.

[0027] Figure 3 The effect of temperature on the stability of recombinant superoxide dismutase RPSOD. The horizontal axis represents different temperatures; the vertical axis represents the relative activity of RPSOD.

[0028] Figure 4 The effect of pH on the activity of recombinant superoxide dismutase RPSOD. The horizontal axis represents different pH values; the vertical axis represents the relative activity of RPSOD.

[0029] Figure 5 The effect of pH on the stability of recombinant superoxide dismutase RPSOD. The horizontal axis represents different pH values; the vertical axis represents the relative activity of RPSOD.

[0030] Figure 6 The effect of denaturants on recombinant superoxide dismutase RPSOD. The horizontal axis represents different denaturants; the vertical axis represents the relative activity of RPSOD. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as the experimental conditions described in the Molecular Cloning Laboratory Manual (9, Sambrook, Russell (author), Huang Peitang (translator), "Molecular Cloning Laboratory Guide", Science Press, 2002, third edition), or according to the conditions recommended by the reagent or instrument manufacturer.

[0032] To achieve the above object, the present invention adopts the following technical measures, the specific steps of which are:

[0033] 1. Total RNA extraction and reverse transcription of cDNA from deep-sea tubular worm R. piscesae

[0034] The deep-sea tube worm R. piscesae samples were collected from the Juan de Fuca Ridge (47°56'N, 129°05'W, 2181m) and immediately stored in liquid nitrogen. Total RNA of the tube worm was extracted according to the instructions of the TRIzol Reagent kit. 1 μL oligo(dT) was added to the EP tube. 18 Primer (14nmol / L), 2μg total RNA, add DEPC-H2O to 13.5μL and mix well, place in PCR instrument at 70℃ for 10min, and immediately place on ice for 2min. Add 4μL of 5× reverse transcriptase M-MLV buffer, 0.5μL of recombinant ribonuclease inhibitor, 1μL of dNTPs mixture (10mmol / L), and 1μL of reverse transcriptase M-MLV, mix well, and react in PCR instrument at 42℃, 1h; 70℃, 15min, and the result is cDNA.

[0035] 2. PCR Amplification and Sequence Analysis of Superoxide Dismutase (rpsod) from the Deep-sea Tubular Worm R. piscesae

[0036] Primer 5.0 software was used to design rpsod primers. The forward primer (the underlined part is the BamHI restriction site) is: 5′-CGC GGATCCATGCTGTCAGCAACTACAG-3′, reverse primer (the underlined part is the EcoRI restriction site): 5′-CCG GAATTC CTATTGCCTCGCTTTCT-3′. The PCR reaction system was: 4 μL of dNTP mixture (2.5 mmol / L), 0.5 μL of PrimerSTAR HS DNA polymerase, 5× PrimerSTAR buffer (containing Mg 2+ ) 10μL, 1μL of forward and reverse primers (10μmol / L), 1μL of cDNA template, add water to 50μL, mix well and place in PCR instrument for reaction. PCR amplification program is as follows: 98℃ pre-denaturation for 5min; 98℃ denaturation for 1min, 52℃ annealing for 30s, 72℃ extension for 42s, 35 cycles; finally 72℃ extension for 10min.

[0037] 3. Recombinant expression and purification of superoxide dismutase RPSOD from deep-sea tubular worm R. piscesae

[0038] The PCR amplification product was subjected to 1% agarose gel electrophoresis, and the target band was cut out after observation by a gel imager. DNA was recovered from the gel using a gel recovery kit. The gel recovery product and the pET-His vector plasmid were double-digested with BamHI and EcoRI respectively. After the double-digestion products were cut and recovered, the recovered pET-His vector after digestion was connected to the target fragment at 16°C for 8h to construct the recombinant plasmid pET-His-RPSOD. The recombinant plasmid was transformed into E. coli Top10 competent cells, evenly spread on LB solid medium (1% NaCl, 1% tryptone, 0.5% yeast extract) containing ampicillin sodium (100mg / mL), and cultured at 37°C for 12h. Single colonies were picked for colony PCR, positive clones were screened, and a small amount of recombinant plasmid was extracted using a plasmid extraction kit. The recombinant plasmid was then double-digested for verification and sent to Hubei Qingke Biotechnology Co., Ltd. for sequencing and analysis.

[0039] The recombinant plasmid pET-His-RPSOD was transformed into E.coli BL21 (DE3) competent cells, and positive clones were screened by colony PCR. A single colony was inoculated into 5 mL LB liquid culture medium containing ampicillin sodium (100 mg / mL), and cultured at 220 r / min for 9 h in a 37°C constant temperature shaker to prepare seed liquid. The seed liquid was inoculated into 250 mL LB liquid culture medium containing ampicillin sodium (100 mg / mL) at a ratio of 1:100, and cultured at 220 r / min in a 37°C shaker until OD 600=0.6~0.8. Take 1mL of bacterial solution as negative control, add IPTG (final concentration is 0.125mmol / L) and continue to induce culture at 150r / min in a 16℃ shaker for 12h. Centrifuge the induced bacterial solution at 8000r / min at 4℃ for 30min to collect the bacteria. Resuspend the bacteria with 20mmol / L imidazole solution (0.5M NaCl, 20mmol / L Na3PO4, 20mmol / L imidazole, pH=7.4), and ultrasonically crush on ice for 40min according to the program of power 200W, crush for 5s, and stop for 20s. Centrifuge at 13000r / min at 4℃ for 30min to separate the supernatant and inclusion bodies.

[0040] The collected supernatant solution was combined with the His-tagged protein purification medium at 4°C for 3h, and washed with 20mmol / L, 50mmol / L, 100mmol / L, 200mmol / L, 300mmol / L, and 400mmol / L imidazole solutions (0.5mol / L NaCl, 20mmol / L Na3PO4, 20-400mmol / L imidazole, pH=7.4) to remove impurities, and finally eluted with 500mmol / L imidazole solution (0.5mol / L NaCl, 20mmol / L Na3PO4, 500mmol / L imidazole, pH=7.4). The collected target protein was placed in a dialysis bag and dialyzed stepwise into 0mM imidazole Na3PO4 buffer at 4°C. The protein samples prepared from the above parts were subjected to 10% SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining to observe the expression and purification of the recombinant protein ( Figure 1 ).

[0041] 1. Detection of superoxide dismutase (RPSOD) activity in deep-sea tubular worm R. piscesae

[0042] 4.1 Measurement of RPSOD activity by WST-1 method

[0043] The substrate stock solution and buffer solution in the total superoxide dismutase (SOD) assay kit (WST-1 method) of Nanjing Jiancheng Company were prepared into substrate application solution (prepared and used immediately) at a volume ratio of 1:200, and the enzyme stock solution and enzyme diluent were prepared into enzyme working solution (prepared and used immediately) at a volume ratio of 1:10. Add each reagent to the enzyme label strip according to Table 1:

[0044] Table 1 WST method detection system configuration table

[0045]

[0046] After mixing, incubate at 37°C for 20 min, and calculate the SOD activity according to the following formula after reading the results with a 450 nm microplate reader (the amount of enzyme corresponding to a 50% SOD inhibition rate is one enzyme activity unit (U)):

[0047]

[0048] 4.2 Detection of RPSOD activity by NBT method

[0049] Take three transparent glass test tubes and mark them as RPSOD experimental group, RPSOD buffer control group, and RPSOD buffer light-shielded group. Prepare 2.7mL of mixed solution for each group, add L-methionine with a final concentration of 13mmol / L, 10μmol / L EDTA, 75μmol / L NBT, and 2μmol / L riboflavin, and fill the rest of the system with the corresponding buffer. Add 300μL of each of the RPSOD experimental group, RPSOD buffer control group, and RPSOD light-shielded group to the prepared mixed solution, irradiate the RPSOD experimental group and the RPSOD control group with a strong light of 4000lx for 30min, and strictly protect the RPSOD buffer light-shielded group from light. Pipette 200μL of each of the reaction solutions into a 96-well plate, detect at the absorption peak of 560nm with an enzyme reader, and calculate the SOD activity according to the formula (where 50% inhibition of NBT photoreduction is one enzyme activity unit (U)):

[0050]

[0051] 4.3 Effect of temperature on RPSOD activity

[0052] The entire reaction system was placed at 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 1h at the same time. The activity of RPSOD under different temperature treatments was detected by WST-1 method, and the relative enzyme activity was calculated and a line graph was drawn. The experiment was repeated three times. The results showed that 50℃ was the optimal temperature for RPSOD to react ( Figure 2 ).

[0053] 4.4 Analysis of RPSOD thermal stability

[0054] The RPSOD protein was treated at 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for a certain period of time. The WST-1 method was used to detect the activity of RPSOD after treatment at different temperatures, and the relative enzyme activity was calculated to draw a line graph. The experiment was repeated three times. The results showed that when RPSOD was incubated at 10℃-70℃, whether it was incubated for 3h or 6h, its relative enzyme activity was above 95%, and its stability was excellent. After incubation at 80℃ for 3h, the relative enzyme activity of RPSOD was still above 90%, and its remaining enzyme activity dropped to about 60% after 6h of treatment ( Figure 3 ). This indicates that RPSOD has good stability under high temperature conditions.

[0055] 4.5 Effect of pH on RPSOD activity

[0056] RPSOD protein was added to NBT systems containing different buffer solutions with pH values ​​of 2-8 (citric acid-Na2HPO4), 9-10 (glycine-NaOH), 7.1-9 (Tris-HCl), 9.3-11 (Na2CO3-NaHCO3), and 11-12 (Na2HPO4-NaOH). The activity of RPSOD was detected by NBT method, and the relative enzyme activity was calculated and a line graph was drawn. The experiment was repeated three times. The experimental results showed that the optimal pH for RPSOD reaction was 9 ( Figure 4 ).

[0057] 4.6 Analysis of pH stability of RPSOD

[0058] RPSOD was treated with different buffer solutions with pH values ​​of 2-8 (citric acid-Na2HPO4), 9-10 (glycine-NaOH), 7.1-9 (Tris-HCl), 9.3-11 (Na2CO3-NaHCO3), and 11-12 (Na2HPO4-NaOH) for 1 hour. The activity of RPSOD after treatment with different pH values ​​was determined by the WST-1 method, and the relative enzyme activity was calculated and a line graph was drawn. The experiment was repeated three times. The results showed that when pH ≤ 3, the residual enzyme activity of RPSOD was 0% after treatment with the corresponding buffer solution for 1 hour. After treatment with a buffer solution at pH 4 for 1 hour, the residual enzyme activity was only 17%. Subsequently, after treatment with a buffer solution at pH 4-8 for 1 hour, the stability of RPSOD mainly showed an upward trend; after treatment with a buffer solution at pH 9-12 for 1 hour, its stability gradually weakened: after treatment with a buffer solution at pH 11 for 1 hour, the residual activity of RPSOD was still more than 40%, but after treatment with a buffer solution at pH 12 for 1 hour, the residual activity of RPSOD was less than 20% ( Figure 5 ). It can be seen that RPSOD has good stability in neutral and weak alkaline environments.

[0059] 4.7 Effects of denaturants on RPSOD activity

[0060] The RPSOD protein was treated with 10mmol / L urea, EDTA, SDS, PMSF, and H2O2 for 1h. The activity of RPSOD was detected by WST-1 method, and the relative enzyme activity was calculated and a bar graph was drawn. The experiment was repeated three times. The experimental results showed that urea, EDTA, and H2O2 had almost no effect on the activity of RPSOD. The activity of RPSOD after PMSF treatment changed within 20%. After SDS treatment for 1h, RPSOD was relatively affected, but there was still 65% of the remaining activity ( Figure 6 ).

Claims

1. Tube Worms Ridgeia piscesae Superoxide dismutase gene rpsod , characterized in that The tube worm Ridgeia piscesae Superoxide dismutase gene rpsod The molecular type is DNA, the sequence characteristics are: length is 675 bp, the type is nucleic acid, the chain is double-stranded, the topology is linear, the tubular worm Ridgeia piscesae Superoxide dismutase gene rpsod The nucleotide sequence is shown in SEQ ID No.

1.

2. Tube Worms Ridgeia piscesae Superoxide dismutase RPSOD, characterized by The molecular type of the superoxide dismutase RPSOD is protein, and the sequence characteristics are: length is 224aa, and the type is amino acid. The amino acid sequence of the superoxide dismutase RPSOD is shown in SEQ ID No.

2.

3. The tubular worm according to claim 2 Ridgeia piscesae The preparation method of superoxide dismutase RPSOD is characterized in that The following steps are involved: 1) Deep-sea tube worms Ridgeia piscesae The steps of total RNA extraction and reverse transcription to synthesize cDNA; 2) Deep-sea tube worms Ridgeia piscesae Steps for PCR amplification and sequence analysis of superoxide dismutase RPSOD; 3) Deep-sea tube worms Ridgeia piscesae The steps of recombinant expression and purification of superoxide dismutase RPSOD; and 4) Deep-sea tube worms Ridgeia piscesae Steps for activity detection and functional exploration of superoxide dismutase RPSOD.

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