CN110551731A - MsrA gene, protein and gene extraction method of white fin shark - Google Patents

Abstract

the invention discloses an MsrA gene of white shark, protein and a gene extraction method, wherein the MsrA gene has a nucleotide sequence 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

MsrA gene, protein and gene extraction method of white fin shark

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, S-type and 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 can not be obtained because the whole gene sequence of the white fin shark is not determined.

The technical scheme of the invention is as follows:

an MsrA gene of white 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 the white fin shark is 1207 bp.

The MsrA gene of the white fin shark is characterized in that the MsrA gene of the white fin shark has a 5 'UTR of 20bp, a 3' UTR with a Ploy A tail of 503bp in length and an ORF of 684 bp.

The MsrA gene of the white fin shark, wherein the MsrA gene of the white fin shark codes 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: designing upstream and downstream degenerate primers according to a peptide segment obtained by mass spectrometry of the total protein of the white fin shark liver, carrying out cDNA cloning, obtaining two terminal sequences by a 3 'RACE method and a 5' RACE method, and splicing the obtained sequences 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;

obtaining two terminal sequences of the MsrA gene by a 3 'RACE method and a 5' RACE method, and splicing the obtained sequences to obtain the MsrA gene of the 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 shark, provided by the invention, has a nucleotide sequence shown as SEQ ID No: 1 is shown.

In the invention, the total length of the MsrA gene of the white shark is 1207bp, wherein the 5 'UTR is 20bp, the 3' UTR with the Ploy A tail is 503bp long, and the ORF is 684 bp. The MsrA gene of the white fin shark codes for 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: designing upstream and downstream degenerate primers according to a peptide segment obtained by mass spectrometry of the total protein of the white fin shark liver, carrying out cDNA cloning, obtaining two terminal sequences by a 3 'RACE method and a 5' RACE method, and splicing the obtained sequences 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 genome of the white-fin shark has not been sequenced, starting from peptide fragment information obtained by mass spectrometry analysis of total protein of the white-fin shark liver, degenerate primers are designed for cDNA cloning, and the sequences at two ends of the white-fin shark liver are obtained by a 3 'RACE method and a 5' RACE method. The total length of the cDNA spliced by the white fin shark MsrA is 1207bp, wherein the 5 'UTR is 20bp, the 3' UTR containing the Ploy A tail is 503bp, the CDS is 684bp, and 227 amino acids are coded. The predicted protein molecular weight is 25.74kDa (25.37 kDa for the signal peptide-removed fusion protein expressed herein) and the isoelectric point (pI) value is 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 (Triaenodon obesus) for experiment is purchased from Shenzhen salt field (living body, white fin shark, Zhenyanmu), liver tissue is dissected and taken, small pieces are cut and loaded into an EP tube, and the EP tube is placed in a refrigerator at minus 80 ℃ for storage after being frozen by liquid nitrogen.

total RNA was extracted using TRIZOL reagent (FIG. 1) as follows:

(1) Placing the spoon, the tweezers and the mortar in an oven at 180 ℃ for baking for 2 hours one day in advance, and preparing 0.1% DEPC water;

(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 5 min;

(4) After thawing, moving the homogenate into an EP tube of RNase-free in two branches, and centrifuging for 5min at 4 ℃ at 12500 rpm;

(5) Transferring the supernatant to new EP tubes, adding 200 μ L chloroform, shaking vigorously for 15s until the solution is completely emulsified without phase separation, standing at room temperature for 5min, and centrifuging at 12500rpm at 4 deg.C for 15 min;

(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 10 min;

(7) Discarding the supernatant, respectively adding 1mL of 75% ethanol pre-cooled at-20 deg.C, and centrifuging at 12500rpm at 4 deg.C for 10 min;

(8) Discarding the supernatant, and opening to sufficiently volatilize the alcohol, and respectively adding 20 mu L of 0.1% DEPC water to dissolve RNA;

(9) mu.L of each of the extracted RNA solutions was subjected to agarose gel (0.8%) electrophoresis to examine the quality of RNA.

Then, performing RT-PCR reaction according to pimeScript ^TM RT-PCR Kit instructions to obtain white fin shark liver total RNA, obtaining white fin shark liver total protein peptide fragment information by mass spectrometry, further analyzing to obtain peptide fragment sequences ELLKVFWESHDPTQGMR, EVCTGMTGHAEVVR, LQEALASK and LQEALSK, simultaneously designing upstream and downstream primers according to the nucleic acid sequences corresponding to MsrA in four species of anoploma fimbria (BT082637), Nothophanchius furzar (EU400617), Homo sapiens (BC 053) and Xenopus lais (BC053804) in NCBI database, amplifying the midstream sequence of the MsrA gene of the white fin shark, connecting the fragment sequences to a T-vector and transferring into Escherichia coli strain 10, culturing a single clone colony, performing PCR after degenerating the colony culture, extracting the corresponding single clone of the obtained positive band, determining the sequence of the single clone plasmid, determining the sequence of the sequence in the GenBank, determining the sequence of the MsrA plasmid, and obtaining a fragment of MsrA sequence of the MsrA gene by splicing technology, and obtaining the MsrA DNA sequence of MsrA library.

The total length of cDNA of the white shark MsrA is 1207bp, wherein the 5 'UTR (untranslated region) is 20bp, the 3' UTR with Ploy A tail is 503bp in length, the ORF is 684bp, and 227 amino acids are coded in total. The predicted protein molecular weight is 25.74kDa, and the isoelectric point (pI) value is 8.46.

White fin shark (Triaerodon 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, a final concentration of 1mMIPTG is added to induce the massive expression of the white finfish MsrA fusion protein (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.

Sequence listing

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acaggagcag atggctgcag ctctgaaatc caaagaggcc tatcaggagg aattgaacag gaatgggtat 480

ggtcaaatta caacagagat ccgtgagggt caagtattct actatgctga agattaccat caacaatact 540

tggataagaa cccaggcggt tattgtggat tgggtggcac tggggtatct tgcccaattg atattcagaa 600

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tctaaaaaag tggaaaccta tcccaatata ttgaaaatag ttgaataaaa ctctgcattt gattctggaa 780

ccctgttaag cattaagttg cagcaaaaga tgtaataatt tttaaatgtt tgctcttaca acattctcat 840

ctaaaatatg tgtaaatgtc aattgttgag aataaggcac aaaaaatata ttttcatatg tgtatatgtt 900

tctatatata tacatatata tttactgact gttttgttcc catagtcagt gccattggta caaagtgaat 960

tgcattattc tgaaaaagca ggaaaataaa tgaaagccat tacaatttta aaaaaaaaaa aaaaaaaaaa 1020

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Met Arg Thr Trp Gln Arg Phe Leu Leu Gly Arg Ile Gly Leu Ala Met

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Ala Phe Arg Val Gln Met Leu Ser Lys Glu Gln Ala Cys Pro Gly Arg

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Thr Glu Thr Met Lys Val Ser Ala Arg His His Val Asn Gly Asn Arg

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Thr Val Gly Pro Phe Pro Glu Gly Leu Gln Met Val Met Phe Gly Met

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

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Tyr Ser Thr Gln Val Gly Tyr Ala Gly Gly Phe Thr Pro Asn Pro Thr

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Tyr Lys Glu Val Cys Ser Gly Met Ser Gly His Ala Glu Val Val Arg

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Val Val Tyr His Pro Asp Lys Ile Cys Phe Glu Lys Leu Leu Lys Val

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Met Ala Ala Ala Leu Lys Ser Lys Glu Ala Tyr Gln Glu Glu Leu Asn

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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 a white fin shark as claimed in claim 1, wherein the MsrA cDNA of the white fin shark has a total length of 1207 bp.

3. the MsrA gene from white fin shark of claim 1, wherein the MsrA gene from white fin shark has a 5 'UTR of 20bp, a 3' UTR with a Ploy A tail of 503bp long and an ORF of 684 bp.

4. The MsrA gene from a white fin shark of claim 1, wherein the MsrA gene from a white fin shark 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. An MsrA gene extraction method of white fin shark is characterized by comprising the following steps: designing upstream and downstream degenerate primers according to a peptide segment obtained by mass spectrometry of the total protein of the white fin shark liver, carrying out cDNA cloning, obtaining two terminal sequences by a 3 'RACE method and a 5' RACE method, and splicing the obtained sequences 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;

Obtaining two terminal sequences of the MsrA gene by a 3 'RACE method and a 5' RACE method, and splicing the obtained sequences to obtain the MsrA gene of the white fin shark.