CN110551731B - MsrA gene, protein and gene extraction method of white fin shark - Google Patents
MsrA gene, protein and gene extraction method of white fin shark Download PDFInfo
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Abstract
The invention discloses an MsrA gene of white fin shark, protein and a gene extraction method, wherein the nucleotide sequence of the MsrA gene is shown as SEQ ID No:1 is shown. The invention can provide a targeted MsrA gene sequence without completely sequencing the white fin shark gene, thereby improving the efficiency of non-model biological gene cloning; meanwhile, the white fin shark MsrA protease has high expression quantity and purification degree, and the white fin shark MsrA protease is found to have a remarkable function of repairing methionine oxidation damage in vivo or in vitro in subsequent researches.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an MsrA gene, a protein and a gene extraction method of white fin shark.
Background
The sulfur-containing amino acid methionine in a protein is one of amino acids that can be easily oxidized, and methionine is oxidized to produce two methionine sulfoxides, an S-type and an R-type, which can be specifically reduced to methionine by methionine sulfoxide reductase a (MsrA). MsrA is generally localized in mitochondria and is an important member of the antioxidant system of the organism due to its important resistance to oxidative stress and protein repair. MsrA appeared 30 hundred million years ago and was evolutionarily very primitive. Sharks are primitive marine organisms that are highly conserved evolutionarily, healthy sharks swim for life, presumably with a more powerful antioxidant system.
Disclosure of Invention
The invention aims to provide an MsrA gene, a protein and a gene extraction method of white fin shark, and aims to solve the problem that the MsrA gene sequence, the protein sequence and the protein expression of the white fin shark cannot be obtained because the whole gene sequence of the white fin shark is not determined.
The technical scheme of the invention is as follows:
the MsrA gene of the white fin shark, wherein the MsrA gene has a nucleotide sequence shown as SEQ ID No:1 is shown.
The MsrA gene of the white fin shark, wherein the MsrA cDNA total length of Bai Qisha is 1207bp.
The MsrA gene of white fin shark, wherein 5'UTR of the MsrA gene of Bai Qisha is 20bp, 3' UTR with Ploy A tail is 503bp long, and ORF is 684bp.
The MsrA gene of the white fin shark, wherein the MsrA gene of Bai Qisha encodes 227 amino acids.
The protein for expressing the MsrA gene of the white fin shark is disclosed by the invention, wherein the amino acid sequence of the protein is shown as SEQ ID No:2, respectively.
An MsrA gene extraction method of white fin shark, which comprises the following steps: according to the peptide fragments obtained by mass spectrometry of the total protein of the white fin shark liver, upstream and downstream degenerate primers are designed, cDNA cloning is carried out, both terminal sequences are obtained by a 3'RACE method and a 5' RACE method, and the obtained sequences are spliced to obtain the MsrA gene of the white fin shark.
The MsrA gene extraction method of the white fin shark comprises the following steps:
extracting total RNA from liver tissue of white shark;
carrying out reverse transcription by taking the total RNA as a template to obtain cDNA;
designing an upstream degenerate primer and a downstream degenerate primer, and carrying out PCR amplification by taking the obtained cDNA as a template to obtain an amplification product;
carrying out sequence determination on the obtained amplification product, and carrying out nucleic acid BLAST comparison on the determination result to obtain an MsrA gene fragment of the white fin shark;
both terminal sequences of the MsrA gene were obtained by a 3'RACE and 5' RACE method, and the obtained sequences were ligated to obtain the MsrA gene of white fin shark.
Has the advantages that: the invention can provide a targeted MsrA gene sequence without completely sequencing the white fin shark gene, thereby improving the efficiency of non-model biological gene cloning; meanwhile, the white fin shark MsrA protease has high expression quantity and purification degree, and the white fin shark MsrA protease is found to have a remarkable function of repairing methionine oxidation damage in vivo or in vitro in subsequent researches.
Drawings
FIG. 1 is a graph of total RNA extracted from shark liver tissue of white fin.
FIG. 2 is a PCR electrophoresis chart for identifying bacteria liquid.
FIG. 3 is a diagram of a multiple sequence alignment of MsrA.
FIG. 4 is a SDS-PAGE pattern of total protein.
FIG. 5 is an SDS-PAGE pattern of the purified protein.
Detailed Description
The present invention provides an MsrA gene, a protein and a gene extraction method of white shark, and the present invention is further described in detail below in order to make the objects, technical schemes and effects of the present invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The MsrA gene of the white fin shark, disclosed by the invention, has a nucleotide sequence shown as SEQ ID No:1 is shown.
In the present invention, the total length of the MsrA gene of Bai Qisha is 1207bp, wherein 5'UTR is 20bp, 3' UTR with Ploy A tail is 503bp long, and ORF is 684bp. The MsrA gene of Bai Qisha encodes 227 amino acids.
The invention relates to a protein for expressing the MsrA gene of the white fin shark, wherein the amino acid sequence of the protein is shown as SEQ ID No:2, respectively.
The invention can provide a targeted MsrA gene sequence without completely sequencing the white fin shark gene, thereby improving the efficiency of non-model biological gene cloning; meanwhile, the white fin shark MsrA protease has high expression quantity and purification degree, and the white fin shark MsrA protease is found to have a remarkable function of repairing methionine oxidation damage in vivo or in vitro in subsequent researches.
The invention discloses an MsrA gene extraction method of white fin shark, which comprises the following steps: according to the peptide fragments obtained by mass spectrometry of the total protein of the white fin shark liver, upstream and downstream degenerate primers are designed, cDNA cloning is carried out, both terminal sequences are obtained by a 3'RACE method and a 5' RACE method, and the obtained sequences are spliced to obtain the MsrA gene of the white fin shark.
The invention takes MsrA of white fin shark as a research object to carry out related research. Since the white fin shark genome is not sequenced, degenerate primers are designed to clone cDNA starting from peptide fragment information obtained by mass spectrometry analysis of total protein of the white fin shark liver, and the sequences of the two ends of the cDNA are obtained by a 3'RACE method and a 5' RACE method. The total cDNA length of the white fin shark MsrA is 1207bp, wherein 5'UTR is 20bp, 3' UTR containing Ploy A tail is 503bp, CDS is 684bp, and 227 amino acids are coded. The predicted protein has a molecular weight of 25.74kDa (25.37 kDa for the signal peptide-removed fusion protein expressed herein) and an isoelectric point (pI) of 8.46. Phylogenetic tree analysis shows that white fin shark MsrA has high homology (68% -74%) with mammals, fishes, parts of birds and amphibians, and due to evolutionary conservation, msrA has high homology (62% -67%) with some algae and bacteria. The protein contained 30% alpha helix, 16% beta sheet, and 54% random coil structure at pH 7.5 as determined by circular dichroism.
The invention is further illustrated by the following specific examples.
White fin shark (Bai Qisha, triaenodon obesus) used in experiment was purchased from Shenzhen salt pan region (living body, white fin shark, zhenyanmu), liver tissue was dissected and taken, cut into small pieces and put into EP tube, and after quick freezing with liquid nitrogen, it was stored in refrigerator at-80 deg.C for use.
Total RNA was extracted using TRIZOL reagent (FIG. 1) as follows:
(1) One day ahead, the spoon, tweezers, mortar were placed in an oven at 180 ℃ and baked for 2h, and 0.1% depc water was added;
(2) Precooling a mortar by using liquid nitrogen, clamping about 2g of shark liver tissue blocks by using forceps, putting the shark liver tissue blocks into the mortar, and grinding by using the liquid nitrogen;
(3) Scraping the powder with a spoon to collect the powder, adding 2mL of TRIZOL reagent to completely cover the powder, and moving to an ultra-clean workbench to stand for 5min;
(4) After thawing, moving the homogenate into an RNase-free EP tube in two parts, and centrifuging for 5min at 12500rpm and 4 ℃;
(5) Respectively transferring the supernatant to a new EP tube, adding 200 mu L of chloroform, violently shaking for 15s until the solution is completely emulsified and no phase separation occurs, standing for 5min at room temperature, and centrifuging for 15min at 12500rpm at 4 ℃;
(6) Respectively transferring the supernatant to a new EP tube, adding equal volume of isopropanol, mixing, standing at room temperature for 10min, and centrifuging at 12500rpm at 4 deg.C for 10min;
(7) Discarding the supernatant, adding 1mL of 75% ethanol pre-cooled at-20 deg.C, respectively, and centrifuging at 12500rpm at 4 deg.C for 10min;
(8) Discarding the supernatant and allowing the ethanol to sufficiently volatilize, and adding 0.1% DEPC water 20. Mu.L each to dissolve RNA;
(9) mu.L each of the extracted RNA solutions was subjected to agarose gel (0.8%) electrophoresis to examine the quality of RNA.
Then according to pimeScript TM RT-PCR Kit instructions for RT-PCR reaction, white fin shark liver total RNA. Obtaining the peptide segment information of the total protein of the white fin shark liver by mass spectrometry, further analyzing to obtain peptide segment sequences ELLKVFWESHDPTQGMR, EVCTGMTGHAEVVR, LQEALASK and LQEASK, simultaneously designing upstream and downstream primers according to the nucleic acid sequences of MsrA in four species of Anoplopoma fimbria (BT 082637), nothobrachius furzeri (EU 400617), homo sapiens (BC 054033) and Xenopus laevis (BC 053804) in NCBI database, degenerating the nucleic acid sequences corresponding to MsrA in the peptide segments, amplifying the midstream sequence of the white fin shark MsrA gene, connecting the segment sequence to a T-vector and transferring the segment into Escherichia coli TOP10 strain, culturing a single clone colony, picking the colony, performing suspension culture, performing PCR (figure 2), extracting the single clone bacterial liquid plasmid corresponding to the positive strip, performing sequence determination on the GenBank database, and determining the nucleic acid sequence of the white fin shark gene as a segment. And obtaining MsrA 3 'and 5' terminal sequences by RACE technology, and finally splicing into a Bai Qisha MsrA full-length sequence.
Bai Qisha MsrA has a cDNA full length of 1207bp, 5'UTR (untranslated region) of 20bp, 3' UTR with Ploy A tail of 503bp in length, and ORF of 684bp, and encodes 227 amino acids in total. The predicted protein molecular weight is 25.74kDa, and the isoelectric point (pI) value is 8.46.
The white fin shark (Trigonodon obesus) MsrA related sequence information was submitted to NCBI GenBank (Accession number:). Through protein sequence BLAST analysis, the white fin shark MsrA has homology of 70-74% with MsrA sequences of mammals such as jungle fowl, cattle, rabbits, human beings and the like, has homology of 68-72% with fishes and amphibians such as African odontophorus carpio, zebra fish, african magaina and the like, and also has homology of 62-67% with MsrA sequences of some algae and bacteria due to evolutionary conservation (Table 1).
The protein sequence of white fin shark MsrA was subjected to multiple sequence alignment with other species such as bovine (Bos taurus), nematode (Caenorhabditis elegans), yeast (Saccharomyces cerevisiae), escherichia coli (Escherichia coli), pseudomonas (Pseudomonas) and cyanobacteria (oscillatoria cyanobacteria), wherein white fin shark MsrA had 35% sequence identity with nematode MsrA, 37% with yeast and 56% with Escherichia coli (fig. 3).
Like MsrA of other species, msrA of white fin shark contains three conserved cysteines, cys-66 at the N-terminus, and Cys-212 and Cys-221 at the C-terminus, in addition to which the protein structure contains 3 cysteines, which totals more cysteine than other species, and whether this correlates with its antioxidant capacity requires further experimental verification. Subsequently, a pet28a-MsrA expression vector is constructed and successfully transferred into a BL21 escherichia coli strain, 1mM IPTG with the final concentration is added to induce Bai Qisha MsrA fusion protein to be expressed in a large quantity (figure 4), and the protein is extracted and purified (figure 5) and is consistent with the predicted molecular weight.
TABLE 1 homology alignment of white fin shark MsrA with other species
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (7)
1. The MsrA gene of white shark, which is characterized in that the MsrA gene has a nucleotide sequence shown as SEQ ID No:1 is shown.
2. The MsrA gene of white fin shark according to claim 1, wherein the MsrA cDNA of Bai Qisha has a total length of 1207bp.
3. The MsrA gene from white fin shark according to claim 1, wherein the MsrA gene of Bai Qisha is 20bp in 5'UTR, 503bp in 3' UTR with Ploy A tail, 684bp in ORF.
4. The MsrA gene from white fin shark of claim 1, wherein the MsrA gene of Bai Qisha encodes 227 amino acids.
5. A protein for expressing the MsrA gene of the white fin shark of claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID No:2, respectively.
6. The MsrA gene extraction method of white fin shark as claimed in claim 1, comprising the steps of: according to the peptide fragments obtained by mass spectrometry of the total protein of the white fin shark liver, upstream and downstream degenerate primers are designed, cDNA cloning is carried out, both terminal sequences are obtained by a 3'RACE method and a 5' RACE method, and the obtained sequences are spliced to obtain the MsrA gene of the white fin shark.
7. The MsrA gene extraction method of white fin shark as claimed in claim 6, comprising the steps of:
extracting total RNA from liver tissue of white shark;
carrying out reverse transcription by taking the total RNA as a template to obtain cDNA;
designing an upstream degenerate primer and a downstream degenerate primer, and carrying out PCR amplification by taking the obtained cDNA as a template to obtain an amplification product;
carrying out sequence determination on the obtained amplification product, and carrying out nucleic acid BLAST comparison on the determination result to obtain an MsrA gene fragment of the white fin shark;
both terminal sequences of the MsrA gene were obtained by a 3'RACE and 5' RACE method, and the obtained sequences were ligated to obtain the MsrA gene of white fin shark.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5798243A (en) * | 1996-05-01 | 1998-08-25 | The Rockefeller University | Bacterial peptide methionine sulfoxide reductase, and adhesion-associated protein, and antibiotic therapies based thereon |
| CN1246534A (en) * | 1998-08-31 | 2000-03-08 | 上海新黄浦复旦基因工程有限公司 | Coding sequence of human methionine sulfoxide reductase, its encoded polypeptide and its preparing process |
| WO2003074674A2 (en) * | 2002-03-01 | 2003-09-12 | Exelixis, Inc. | MSRAs AS MODIFIERS OF THE p53 PATHWAY AND METHODS OF USE |
| CN1563386A (en) * | 2004-03-18 | 2005-01-12 | 上海交通大学 | Reducing enzyme protein coded sequence of sulfoxide methionine of cotton |
| WO2011150007A2 (en) * | 2010-05-26 | 2011-12-01 | Opko Curna Llc | Treatment of methionine sulfoxide reductase a (msra) related diseases by inhibition of natural antisense transcript to msra |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5798243A (en) * | 1996-05-01 | 1998-08-25 | The Rockefeller University | Bacterial peptide methionine sulfoxide reductase, and adhesion-associated protein, and antibiotic therapies based thereon |
| CN1246534A (en) * | 1998-08-31 | 2000-03-08 | 上海新黄浦复旦基因工程有限公司 | Coding sequence of human methionine sulfoxide reductase, its encoded polypeptide and its preparing process |
| WO2003074674A2 (en) * | 2002-03-01 | 2003-09-12 | Exelixis, Inc. | MSRAs AS MODIFIERS OF THE p53 PATHWAY AND METHODS OF USE |
| CN1563386A (en) * | 2004-03-18 | 2005-01-12 | 上海交通大学 | Reducing enzyme protein coded sequence of sulfoxide methionine of cotton |
| WO2011150007A2 (en) * | 2010-05-26 | 2011-12-01 | Opko Curna Llc | Treatment of methionine sulfoxide reductase a (msra) related diseases by inhibition of natural antisense transcript to msra |
Non-Patent Citations (1)
| Title |
|---|
| Drosophila methionine sulfoxide reductase A (MSRA) lacks methionine oxidase activity;Tarafdar等;《Free Radic Biol Med》;20181204;第131卷;第154-161页 * |
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