A superoxide dismutase gene of Thalassiosira wissensis and its encoded protein and in vitro expression method

Abstract

The present invention belongs to the field of molecular biology technology, and specifically relates to a superoxide dismutase gene of Thalassiosira wissenii, its encoded protein and an in vitro expression method. Based on the transcriptome sequencing results of Thalassiosira wissenii, the present invention clones a cDNA sequence of a superoxide dismutase gene with a high transcription level from Thalassiosira wissenii, whose open reading frame is 813 bp, encoding a protein sequence containing 270 amino acids, and successfully obtains soluble recombinant protein expression using an in vitro prokaryotic expression system. The SOD protein of Thalassiosira wissenii obtained by the present invention can be soluble in a prokaryotic expression system; and the sequence is found to contain a SodA domain, which is a key part for catalyzing the conversion of reactive oxygen species, and lays a good foundation for the study of antioxidant artificial enzymes.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to a Thalassiosira welshii superoxide dismutase gene, a coded protein and an in vitro expression method thereof. Background Art

[0002] Algae often live in an environment rich in light and oxygen. Under light conditions, the photooxidation reaction of light-harvesting pigments will trigger the production of a large number of free radicals. During photosynthesis, algal cells also release a large number of superoxide anions. At the same time, during metabolism, algal cells can produce a variety of active oxidizing substances, including superoxide anion free radicals, hydroxyl radicals, hydrogen peroxide, etc. These active oxidizing substances may cause irreversible oxidative damage to cell structures. Superoxide dismutase (SOD) is widely present in organisms and belongs to the antioxidant enzyme family. It is currently recognized as an important free radical scavenger internationally. Its main function is to remove highly active superoxide anion free radicals in cells that may cause serious damage to cell structure and function. This process helps to reduce oxidative damage, protect cell integrity, and play an important role in resisting oxidative stress in cells and maintaining the vitality of algal cells.

[0003] As a special algae, the superoxide dismutase produced by Thalassiosira welshii may show unique characteristics in terms of protein primary structure, spatial structure, molecular weight, spectral properties, and sensitivity to different inhibitors. However, there are few studies on superoxide dismutase in Thalassiosira welshii.

[0004] The construction of a prokaryotic expression system will help us gain a deeper understanding of the biological characteristics and functions of superoxide dismutase from Thalassiosira wissensis, while providing new possibilities for its application in related fields. These studies will also help promote the development of environmental protection, medical research, biotechnology and other fields. Summary of the invention

[0005] In view of the above problems, the present invention provides a Thalassiosira wiseri superoxide dismutase gene, its encoded protein and an in vitro expression method.

[0006] The technical solution of the present invention is achieved in this way:

[0007] A superoxide dismutase gene of Thalassiosira welshii has the base sequence in Sequence Listing SEDIQ No.1.

[0008] A protein encoded by a superoxide dismutase gene of Thalassiosira welshii, wherein the protein encoded by the gene has an amino acid sequence in the sequence table SEDIQ No. 2.

[0009] Preferably, the gene-encoded protein is a soluble expressed protein. Through in vitro expression and functional studies, its effectiveness in the antioxidant defense system is evaluated, providing a strong basis for exploring its biological functions and potential applications. The protein encoded by the superoxide dismutase gene of Thalassiosira wissensis of the present invention can be applied to the antioxidant defense system.

[0010] A recombinant expression vector of a superoxide dismutase gene of Thalassiosira welshii, wherein the recombinant expression vector contains a protein encoded by the superoxide dismutase gene of the present invention.

[0011] Preferably, the method for constructing the recombinant expression vector comprises the following steps:

[0012] (1) designing primers to amplify the superoxide dismutase gene of Thalassiosira welshii described in claim 1;

[0013] (2) The E. coli expression vector pET-24a (+) was double-digested with restriction endonucleases BamH I and Xho I, respectively, to obtain a vector pET-24a (+) fragment with sticky ends;

[0014] (3) The superoxide dismutase gene fragment of Thalassiosira wissensis described in step (1) and the vector pET-24a (+) fragment described in step (2) having the same sticky ends are connected by homologous recombination to obtain a recombinant expression vector of the superoxide dismutase gene.

[0015] A recombinant expression strain comprises the recombinant expression vector of the present invention.

[0016] Preferably, the host of the recombinant expression strain is BL21 (DE3) Star competent cells.

[0017] Preferably, the method for preparing the recombinant expression strain comprises the following steps:

[0018] The superoxide dismutase recombinant expression vector is introduced into the host cell BL21 (DE3) Star competent cells to obtain a recombinant expression strain.

[0019] Beneficial Effects

[0020] The beneficial effects of the present invention are summarized as follows:

[0021] The superoxide dismutase (SOD) gene of the present invention is cloned from Thalassiosira wissensis, and the full-length sequence of the open reading frame (ORF) of the gene is obtained by in vitro prokaryotic expression technology, and the characteristics of the encoded protein are predicted and analyzed online; SOD protein is obtained by induced expression, and the solubility of the target protein is detected by SDS-PAGE and Western Blot.

[0022] The protein encoded by the SOD gene of Thalassiosira wissenii obtained by the present invention can be soluble expressed in a prokaryotic expression system; and the sequence is found to contain a SodA domain, which is a key part for catalyzing the conversion of active oxygen, and lays a good foundation for the research on antioxidant artificial enzymes. The protein encoded by the superoxide dismutase gene of Thalassiosira wissenii described in the present invention can be applied to the antioxidant defense system. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The electrophoresis diagram and Western Blot result diagram of the SOD gene cloning of the present invention; wherein, a: SOD gene amplification; b: Western Blot detection result;

[0024] Figure 2 This is a diagram of the Western Blot results of the SOD purified protein provided by the present invention. DETAILED DESCRIPTION

[0025] In order to deepen the understanding of the present invention, the superoxide dismutase gene of Thalassiosira wissensis of the present invention will be further described in detail below with reference to examples and drawings. This example is only used to explain the present invention and does not constitute a limitation on the protection scope of the present invention.

[0026] Based on the transcriptome sequencing results of Thalassiosira wissensis, the present invention cloned a cDNA sequence of a superoxide dismutase (SOD) gene with a high transcription level from Thalassiosira wissensis, whose open reading frame (ORF) is 813 bp and encodes a protein sequence containing 270 amino acids. Soluble recombinant protein expression was successfully obtained using an in vitro prokaryotic expression system.

[0027] Example 1

[0028] The cloning method of the Thalassiosira welshii SOD gene of the present invention mainly comprises the following steps:

[0029] 1. Total RNA extraction and cDNA synthesis of Thalassiosira wiseri;

[0030] 2. Synthesis of SOD gene specific primers and sequencing of PCR amplification products;

[0031] 3. Prediction and analysis of SOD protein structure.

[0032] The specific operations are as follows:

[0033] 1. Extraction of total RNA from Thalassiosira wissenii: Take an appropriate amount of Thalassiosira wissenii sample, grind it in a mortar pre-cooled with liquid nitrogen, repeat 3 times, and extract RNA from the ground powder. Extract total RNA from Thalassiosira wissenii according to the steps of Takara's total RNA extraction kit (RNA Kit). Use Sparkjade's SPARKscript Ⅱ RT Plus Kit to reverse transcribe and synthesize cDNA for gene amplification. For detailed operations, refer to the instructions of each kit.

[0034] 2. Synthesis of SOD gene-specific primers and sequencing of PCR amplification products: Based on the transcriptome data of Thalassiosira wissensis, the specific primers SOD-F and SOD-R for amplifying the ORF of the SOD gene were designed using CE Design software (Table 1). The amplification system was: 0.5 μL Q5 super-fidelity DNA polymerase, 4 μL 2.5mM dNTP, 10 μL cDNA, 2 μL SOD-F, 2 μL SOD-R, 10 μL 5×Q5 Reaction Buffer, 10 μL 5×Q5 High GC Enhancer, 11.5 μL Nuclease-Free Water. The reaction procedure was: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 66℃ annealing for 30 s, 72℃ extension for 30 s, this step was performed for 40 cycles; finally, 72℃ extension for 2 min. The PCR product was detected by DNA electrophoresis, gel recovery, connection and transformation, and then sequenced for verification.

[0035] 3. SOD protein structure analysis: For the obtained target SOD gene sequence, the ORF finder tool was used to detect its open reading frame, and the nucleic acid sequence was translated into an amino acid sequence. The molecular weight (MW) and isoelectric point (pI) of the protein were measured using the ProtParam online tool, while the signal peptide sequence of the protein was predicted using SignalP 4.1, and whether the protein contained a transmembrane structure was predicted using TMHMM Server v. 2.0. The SOD gene ORF was measured to be 813 bp in length, encoding a 270 amino acid sequence, with a molecular weight of 29.33 kDa and a theoretical isoelectric point of 8.57, indicating that it is an intracellular protein.

[0036] Table 1 Primer names and sequences for PCR amplification of target genes

[0037] Primer name Sequence (5'-3') <![CDATA[T m (℃)]]> pET-24a-SOD-F (BamH I) cagcaaatgggtcgcggatccATGACGACGATTCGTAAACGATG 69.97 pET-24a-SOD-R (Xho I) gtggtggtggtggtgctcgagAGCCTTGGCACTAACAAGCCT 72.40

[0038] SEDIQ No.1 Thalassiosira wiseri superoxide dismutase (SOD) gene base sequence

[0039]

[0040] (a) Sequence characteristics:

[0041] Length: 813

[0042] Type: Base sequence

[0043] Chain type: Single chain

[0044] Topology: Linear

[0045] (b) Molecule type: DNA

[0046] (c) Assumption: No

[0047] (d) Antonym: No

[0048] (e) Original source: Thalassiosira wiseri

[0049] SEDIQ No.2 Amino acid sequence of the protein encoded by the superoxide dismutase (SOD) gene of Thalassiosira wiseri

[0050] MTTIRKRCKKSSTTNTISLPLSYLCLKRTMSAKQIITPALRAAVHVRSSKLNSEVAKSAAAYLSSAPSGGKHVLPDLPYDYAALEPCISAETMEIHHSKHHNTYVTNLNATLEKLDAAVSSGDVSGIISLQSALK FNGGGHVNHTLFWENLTSKGNSEFPTSGPLKEAVDARFGDLEGMKKEVSAMTVAVQGSGWGWLGFDAKTGRIEAATCANQDPLQATTGLVPLLGIDVWEHAYYVDYRNVRPNYVNAIWDVINWKTVEDRLVSAKA

[0051] (a) Sequence characteristics:

[0052] Length: 270

[0053] Type: Amino acid sequence

[0054] Chain type: Single chain

[0055] Topology: Linear

[0056] (b) Molecule type: protein

[0057] (c) Assumption: No

[0058] (d) Antonym: No

[0059] (e) Original source: Thalassiosira wiseri

[0060] Structural features: 71bp-270bp is the SodA domain.

[0061] Example 2

[0062] The method for expressing and detecting the recombinant SOD protein of Thalassiosira welshii of the present invention mainly comprises the following steps:

[0063] 1. Construction and transformation of SOD recombinant plasmid;

[0064] 2. Expression of SOD recombinant protein;

[0065] 3. Solubility test of SOD recombinant protein;

[0066] 4. Purification of SOD recombinant protein;

[0067] 5. Activity Assay of Recombinant Purified SOD Protease

[0068] The specific operations are as follows:

[0069] 1. Construction and transformation of SOD recombinant plasmid: In order to achieve soluble expression of the target protein, the pET series vector pET-24a, which is commonly used for protein expression in E. coli, was selected for the expression of the target protein. The protein expressed by this vector has a His tag at the N-terminus, which can be used for subsequent protein purification. According to the sequence information of the target gene, BamH I and Xho I were selected as double restriction sites, and amplification primers with restriction sites and protective bases were designed, namely SOD-F and SOD-R (Table 1). The target band was obtained by PCR amplification, and the electrophoresis pattern is shown in the figure. Figure 1 a. The expression vector was constructed according to the instructions of ClonExpress® II One Step Cloning Kit, and the recombinant plasmid was transformed into DH5α competent cells. The strains that were positive after sequencing were expanded, the plasmids were extracted, and transformed into BL21(DE3) Star competent cells, and sequenced for verification.

[0070] 2. Expression of SOD recombinant protein: Take an appropriate amount of positive bacterial solution and shake it overnight at 37°C; divide it into 200 mL LB medium (containing kanamycin) and adjust the OD 600 When the pH was about 0.8, add 0.1 mM IPTG and shake the culture at 16°C and 120 rpm for 18 h.

[0071] 3. Recombinant protein solubility test: centrifuge the bacterial solution at 10000 rpm and 4℃ for 10 min, collect the bacterial precipitate, resuspend in Buffer A (20 mM sodium dihydrogen phosphate, 20 mM sodium dihydrogen phosphate, 20 mM imidazole, 500 mM NaCl, 20 mL glycerol, 1 LddH2O, pH7.5), and ultrasonically disrupt the bacterial solution in an ice bath for 5 s, with an interval of 10 s, until the bacterial solution is clear; centrifuge the disrupted solution at 4℃ and 12000 rpm for 45 min, and filter the supernatant obtained by centrifugation with a 0.45 μm filter membrane. Take 15 μL of protein supernatant and 5 μL of protein loading buffer and mix them in a centrifuge tube, denature at 95℃ for 10 min in a PCR instrument, and react SDS-PAGE at 150V for 50 min. According to the instructions of Anti His-Tag Mouse Monoclonal Antibody and Goat Anti-Mouse IgG, HRP Conjugated, the primary antibody and secondary antibody were diluted at a ratio of 1:500 and 1:2000 (v / v), respectively. The protein was transferred to the membrane and incubated using the iBlot transfer instrument and the iBind protein blotting instrument. After color development using the high-sensitive ECL chemiluminescent reagent, the membrane was observed using the BIO-RAD gel imaging system. Obvious bands appeared at the predicted position, indicating that the recombinant protein obtained was a soluble protein. The results are shown in Figure 1 As shown in b.

[0072] 4. Purification of SOD recombinant protein: Collect the cells induced for 24 hours by centrifugation, add 20 mL sodium phosphate buffer to suspend the cells, divide into 3 tubes for disruption, and the disruption conditions are: disruption for 5 seconds, interval for 10 seconds, power of 30%, and time for 25 minutes. After disruption, centrifuge and take the supernatant. AKTA was used to purify the protein. Before using the instrument, the pump system was flushed with ultrapure water, and then the chromatography column was installed at a flow rate of 1 mL / min; 50 mL of ultrapure water was used to wash the chromatography column, and the flow rate was set to 5 mL / min; 75 mL of Binding Buffer was used to equilibrate the chromatography column, and the flow rate was set to 5 mL / min; the sample was filtered through a 0.45 μm filter membrane before loading, and the sample was diluted to 150 mL with Binding Buffer; the diluted sample was loaded at a flow rate of 1.2 mL / min; after loading, the impurities were washed with 50 mL of Binding Buffer; then 10% Elution Buffer was used for elution, the flow rate was set to 3 mL / min, and the time was set to 10 min, and then 15% Elution Buffer was used for elution, and the flow rate was set to 3 mL / min until the peak appeared, and the target protein was collected by volume, and 1.5 mL was collected in each centrifuge tube. After protein collection, use 50 mL of Elution Buffer to wash away all proteins; use ultrapure water and ethanol to wash the chromatography column; then remove the chromatography column at a flow rate of 1 mL / min and store it at 4°C; finally, use ultrapure water and ethanol to wash the pump system.

[0073] 5. Western blot detection of recombinant purified SOD protein: The method is the same as above, and a clear band appears at the predicted position. The results are as follows Figure 2 shown.

[0074] 6. Determination of the activity of the recombinant protease after SOD purification: The purified protein obtained above was tested for enzyme activity using the total superoxide dismutase assay kit (WST-1 method) of Nanjing Jiancheng Bioengineering Institute. For detailed operations, refer to the kit manual. The purified SOD recombinant protein was tested for enzyme activity.

[0075] From the above, it can be seen that the Thalassiosira welshii SOD protein obtained in the present invention has soluble expression in the prokaryotic expression system.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A superoxide dismutase gene of Thalassiosira welshii, characterized in that: Its base sequence is shown in the sequence table As shown in SEQ ID No.

1.

2. The method for cloning the superoxide dismutase gene of Thalassiosira welshii according to claim 1, characterized in that: The following steps are involved: Total RNA extraction and cDNA synthesis of Thalassiosira wiseri: Take an appropriate amount of Thalassiosira wissenii sample, grind it in a mortar pre-cooled with liquid nitrogen, repeat several times, and extract RNA from the ground powder; extract total RNA from Thalassiosira wissenii according to the steps of the total RNA extraction kit; use the kit to reverse transcribe and synthesize cDNA for gene amplification; SOD gene specific primer synthesis and PCR amplification: Based on the transcriptome data of Thalassiosira welshii, specific primers SOD-F and SOD-R were designed to amplify the ORF of SOD gene. The sequence of the primer SOD-F is: 5'-cagcaaatgggtcgcggatccATGACGACGATTCGTAAACGATG-3'; The sequence of the primer SOD-R is: 5'- gtggtggtggtggtgctcgagAGCCTTGGCACTAACAAGCCT -3'; The amplification system was: 0.5 μL Q5 ultra-fidelity DNA polymerase, 4 μL 2.5 mM dNTP, 10 μL cDNA, 2 μL SOD-F, 2 μL SOD-R, 10 μL 5´Q5 Reaction Buffer, 10 μL 5´Q5 High GC Enhancer, 11.5 μL Nuclease-Free Water; The reaction procedure was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 66°C for 30 s, and extension at 72°C for 30 s, for 40 cycles; and finally extension at 72°C for 2 min. The PCR products were detected by DNA electrophoresis, recovered by gel, connected and transformed, and then verified by sequencing.

3. The protein encoded by the superoxide dismutase gene of Thalassiosira welshii according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID No.2 in the sequence listing.

4. A recombinant expression vector of the superoxide dismutase gene of Thalassiosira welshii, characterized in that: The recombinant expression vector contains the superoxide dismutase gene according to claim 1.

5. The recombinant expression vector of the superoxide dismutase gene of Thalassiosira welshii according to claim 4, characterized in that: The method for constructing the recombinant expression vector comprises the following steps: (1) designing primers to amplify the superoxide dismutase gene of Thalassiosira welshii described in claim 1; (2) The E. coli expression vector pET-24a (+) was cleaved with restriction endonucleases BamH and XOt Double enzyme digestion to obtain the vector pET-24a (+) fragment with sticky ends; (3) The superoxide dismutase gene fragment of Thalassiosira wissensis described in step (1) and the vector pET-24a (+) fragment described in step (2) having the same sticky ends are connected by homologous recombination to obtain a recombinant expression vector of the superoxide dismutase gene.

6. A recombinant expression strain, characterized in that: Comprising the recombinant expression vector described in any one of claims 4-5.

7. The recombinant expression strain according to claim 6, characterized in that: The host of the recombinant expression strain is BL21 (DE3) Star competent cells, and the preparation method of the recombinant expression strain comprises the following steps: The superoxide dismutase recombinant expression vector according to any one of claims 4 to 5 is introduced into a host cell BL21 (DE3) Star competent cell to obtain a recombinant expression strain.

8. The in vitro expression method of the recombinant protein encoded by the superoxide dismutase gene of Thalassiosira welshii according to claim 1, characterized in that: The following steps are involved: (1) Construction and transformation of SOD recombinant plasmid: The vector pET-24a was selected for the expression of the target protein. According to the superoxide dismutase gene sequence information of Thalassiosira wissensis, BamH and XOt As double restriction sites, amplification primers with restriction sites and protective bases were designed, SOD-F and SOD-R; PCR amplification to obtain the target band; construct expression vector, and transform the recombinant plasmid into DH5a competent cells; expand the culture of the strains that are positive after sequencing, extract the plasmid, transform into BL21(DE3) Star competent cells, and sequence verification; (2) Expression of SOD recombinant protein: Take an appropriate amount of positive bacterial solution and shake overnight; then transfer to LB medium, add IPTG, and shake at 16°C and 120 rpm to induce expression.

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