CN105039363A - Cervus nippon PRDX4 gene, cloning method thereof and encoding protein - Google Patents

Abstract

The invention discloses a cervus nippon PRDX4 gene, a cloning method of the cervus nippon PRDX4 gene and encoding protein, and belongs to the technical field of gene engineering. The nucleotide sequence of the cervus nippon PRDX4 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoding protein is shown as SEQ ID NO. 2. Meanwhile, the invention provides the method for cloning the cervus nippon PRDX4 gene. By means of the cervus nippon PRDX4 gene, the cloning method of the cervus nippon PRDX4 gene and the encoding protein, a cervus nippon PRDX4 cDNA sequence is cloned successfully through a primer design optimization method and an RT-PCR, and bioinformatic analysis is carried out. The primer design optimization method can provide theoretical reference for gene cloning and expression of other non-model organisms or non-whole-genome declared species.

Description

A kind of deer PRDX4 gene and cloning process thereof and proteins encoded

Technical field

The present invention relates to a kind of deer PRDX4 gene and cloning process thereof and proteins encoded, belong to gene engineering technology field.

Background technology

PRDX4 is the important member of PRDXs family, is positioned endoplasmic reticulum.It is the redox reaction participating in cell that the mechanism of action of PRDX4 mainly contains two kinds: one, prevents cell oxidative damage; Two is the peroxidase that can rely on as the class TRX activating NF-κ B and ICAM-1 N-terminal kinase.At present, the PRDX4mRNA sequence of many animals successful clone out, on UNIPROT, the PRDX4 sequence of existing people, dictyostelium discoideum, mouse, rat and ox 5 species is out identified and by checking.The significance of spotted deer PRDX4 albumen in bone knob periosteum Sensitizing area cell is expressed and is illustrated that it may play a significant role in the quick growth and breeding of pilose antler, differentiation migration and maintenance histocyte are stable, but spotted deer genome sequence is not yet measured at present, about the clone of spotted deer PRDX4 gene and functional study are not reported, bring very large difficulty to spotted deer PRDX4cDNA sequence.

Summary of the invention

For solving the problem, the invention provides a kind of deer oxidization-reduction enzyme 4 (Peroxiredoxin4, PRDX4) gene PRDX4, the technical scheme taked is as follows:

One object of the present invention is to provide a kind of deer oxidization-reduction enzyme 4 gene PRDX4, and the nucleotide sequence of this gene is as shown in SEQIDNO.1.Deer oxidization-reduction enzyme 4 gene PRDX4 obtained by analysis, mRNA translated region sequence length is 819bp, include initiator codon ATG and terminator codon TGA, sequence is containing A204, containing C202, containing G212, containing T201, content is respectively 24.9%, 24.7%, 25.9%, 24.5%.

Another object of the present invention is to the albumen providing a kind of described genes encoding, the aminoacid sequence of this albumen is as shown in SEQIDNO.2.By analysis, the deer oxidization-reduction enzyme 4 obtained, its length amino acid sequence is 272 amino acid, hydrophilic protein, having two conserved functional domains, is PRX_Typ2cys (Peroxiredoxinfamily, Typical2-CysPRXsubfamily) and AhpC (Alkylhydroperoxidereductase respectively, AhpC), front 38 amino-acid residue MEAPPPPLPAMTLAPGRSRKLLLLPLLLFLLRAEAVRG may be signal peptide sequences.

Another object of the present invention is to the cloning process providing a kind of described gene, its step is as follows:

1) take the bone knob periosteum Sensitizing area cell of spotted deer and cultivate, obtaining deer horn handle periosteum Sensitizing area cell;

2) extraction step 1) in obtain deer horn handle periosteum Sensitizing area cell in mRNA, according to gained mRNA synthesize cDNA;

3) pcr amplification primer is designed;

4) with step 2) cDNA of gained is template, utilizes step 3) primer that designs carries out pcr amplification, utilize electroresis appraisal and reclaim target amplification product;

5) to step 4) the target amplification product of gained carries out analysis of biological information qualification.

Preferably, step 1) described cultivation, culture condition is: utilize containing 10% foetal calf serum, the DMEM substratum of 1% mycillin, at 37 DEG C, 5.00%CO ₂condition under cultivate 3 days.

Preferably, step 3) described amplimer, its nucleotide sequence is as shown in SEQIDNO.3-SEQIDNO.4.

Preferably, step 4) described pcr amplification, amplification condition is: 95 DEG C of denaturation 5min, 94 DEG C of 30s, 67.3 DEG C of 30s, 72 DEG C of 150s, 30 circulations, 72 DEG C of 1min after loop ends.

Preferably, step 5) described Analysis and Identification, being the band whether utilize nucleic acid electrophoresis to judge to detect be 800-900bp containing size in swimming lane, then by being analyzed with known species sequence, determining fragment for the purpose of object fragment is whether.

The concrete steps of described method are as follows:

1) take the bone knob periosteum Sensitizing area cell of spotted deer, and utilize containing 10% foetal calf serum, the DMEM substratum of 1% mycillin, at 37 DEG C, 5.00%CO ₂condition under cultivate 3 days, obtain deer horn handle periosteum Sensitizing area cell;

2) extraction step 1) in obtain deer horn handle periosteum Sensitizing area cell in mRNA, according to gained mRNA synthesize cDNA;

3) pcr amplification step 2 is designed) primer of described cDNA, obtain the primer as SEQIDNO.3-SEQIDNO.4;

4) with step 2) cDNA of gained is template, utilizes step 3) primer that obtains carries out pcr amplification, amplification condition is: 95 DEG C of denaturation 5min, 94 DEG C of 30s, 67.3 DEG C of 30s, 72 DEG C of 150s, 30 circulations, 72 DEG C of 1min after loop ends, obtain amplified production;

5) nucleic acid electrophoresis determining step 4 is utilized) by contrast marker, the DNA band whether containing a 800-900bp in gained amplified production, then by being analyzed with known species sequence, determines fragment for the purpose of object fragment is whether.

The beneficial effect that the present invention obtains:

The existence of PRDX4 albumen can ensure the normal growth propagation of biological cells and tissues; The peroxidase that the class TRX activating NF-κ B and ICAM-1 N-terminal kinase relies on; PRDX4 can protect beta Cell of islet from damage, anti-ageing.After cloning this gene, the protein expression vector that can carry out next step builds, research PRDX4 protein expression and regenerate the interaction of associated protein with other pilose antlers, to disclosing its effect in pilose antler regenerative process.

The present invention is optimized by primer design method and RT-PCR successful clone goes out spotted deer PRDX4cDNA sequence, and carries out bioinformatic analysis.Design of primers optimization method in the present invention provides theoretical reference can to biological or without full-length genome row species the gene diffusion of other non-mode.

Accompanying drawing explanation

Fig. 1 is the nucleic acid electrophoresis figure of deer oxidization-reduction enzyme 4 gene PRDX4.

Embodiment

Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not by the restriction of embodiment.

Material therefor, reagent, instruments and methods in following examples, without specified otherwise, be the conventional material in this area, reagent, instruments and methods, all obtain by commercial channel.

Spotted deer (Cervusnippon) bone knob periosteum takes from Gao Yun's experiment deer field.

The extraction (SunH etc., 2012) of embodiment 1 deer horn handle periosteum cell and the extraction of mRNA

After the spotted deer bone knob periosteum obtained from Scientia Agricultura Sinica special product institute experiment deer field is fetched, according to the people such as SunH (SunH, YangF, ChuW, ZhaoH, McMahonC, LiC.Lentiviral-mediatedRNAiknockdownofCbfa1geneinhibitse ndochondralossificationofantlerstemcellsinmicromasscultu re.PLoSOne.2012,7 (10): e47367) method processes gained periosteum, and obtains the bone knob periosteum Sensitizing area cell of spotted deer.

Pilose antler bone knob periosteum Sensitizing area cell is placed on 10% foetal calf serum containing 20ml, the 75cm of the DMEM substratum of 1% mycillin ²in culturing bottle, 37 DEG C, 5.00%CO ₂condition under cultivate 2 days; Discard old nutrient solution, under changing the same new nutrient solution equal conditions of 20ml, continue cultivation 1 day, obtain deer horn handle periosteum Sensitizing area cell.

Cultured spotted deer bone knob periosteum Sensitizing area cell in Tissue Culture Flask, discard nutrient solution, collected by trypsinisation cell, 1* cell washing solution (the raw work in Shanghai) washes twice, for the extraction (PureLink of spotted deer bone knob periosteum Sensitizing area cell total rna ^tMrNAMiniKit, LifeTechnologies), then synthesize cDNA first chain (PrimeScript ^tMfirstStandcDNASynthesisKit, Takara).

Embodiment 2 design of primers

According to ox PRDX4mRNA primers, devise 11 groups of primers, as shown in table 1.These 11 groups of primers are utilized to increase.

Table 111 group primer sequence

First group to the 9th group primer is the primer by Primer software design, utilizes these nine groups of primers to increase, does not all obtain target product.Tenth group of primer is directly intercept two sections of sequences as primer from the two ends of ox PRDX4mRNA sequence, utilizes this primer sets to carry out increasing and also there is no target product.Tenth group of primer near deer PRDX4mRNA initiator codon and terminator codon complementary sequence, carries out upstream and downstream primer choose two sections of sequences, after comprehensive evaluation is carried out to the CG content of primer, annealing temperature, dimer and complementarity etc., then primer two ends add a small amount of base balance CG content after the primer that goes out of final engineer.The tenth group of primer achievement is utilized to clone target product.

The process of clone is: RT-PCR clones spotted deer cDNA fragment, 50 μ LPCR reaction systems: 2*PremixTaq25 μ L, each 2 μ L, the ddH2O20 μ L of 1 μ LcDNA, primers F orward11 and Reverse11 (10mmol/L).PCR reaction conditions: 95 DEG C of denaturation 5min, 94 DEG C of 30s, 67.3 DEG C of 30s, 72 DEG C of 150s, 30 circulations, 72 DEG C of 1min after loop ends.PCR primer agarose gel electrophoresis detected result.Object fragment carries out gel recovery, and glue recovery product is served Hai Shenggong biotechnology Services Co., Ltd and carried out sequencing.

Embodiment 3 bioinformatic analysis

Sequencing result shows that this test clones the mRNA sequence of deer PRDX4 first.With deer horn handle periosteum Sensitizing area cell cDNA for template, with sequence shown in SEQIDNO.3-SEQIDNO.4 for primer carries out pcr amplification, amplification utilizes nucleic acid electrophoresis to detect, and result as shown in Figure 1.As can be known from Fig. 1, nov nucleic acid swimming lane has a bright single slice and size is about 800-900bp, the PRDX4 clip size that pcr amplification goes out and other species length basically identical, the clone mRNA sequence length of real income is 819bp.Found by bioinformatic analysis, some physico-chemical properties of deer PRDX4 albumen, biological procedures and the species such as ox, people basic simlarity.The mRNA cloned and ox PRDX4mRNA sequence (AccessionNumber:BC109824.1) and people PRDX4mRNA sequence (AccessionNumber:BC016770.1) similarity are respectively 98%, 90%.The similarity of coded protein amino acid sequence and ox, people is for being respectively 97.1%, 91.2%.

Contrasted by the aminoacid sequence of the oxidization-reduction enzyme 4 (PRDX4) of different plant species, result shows, and the PRDX4 amino acid sequence similarity of spotted deer and ox is the highest, and people takes second place, and Mouse and rat is minimum.

The PRDX4 Amino acid sequences alignment result of deer PRDX4 aminoacid sequence and people, ox, Mouse and rat, between different plant species, the aminoacid sequence system of PRDXs coding grows tree.

PRDX4 aminoacid sequence phylogenetic tree display people and P of Rats RDX4 derive from same branch, and ox and mouse PRDX4 derive from same branch, and the common branch of spotted deer PRDX4 and ox and mouse PRDX4 derives from same branch.

Deer PRDX4 albumen is the same with other species belongs to secreted protein.Prediction deer PRDX4 albumen has two conserved functional domains, is PRX_Typ2cys (Peroxiredoxinfamily, Typical2-CysPRXsubfamily) and AhpC (Alkylhydroperoxidereductase, AhpC) respectively.Wherein PRX_Typ2cys belongs to typical 2-CysPRX subtribe in peroxiredoxin (PRX) family, belongs to sulfydryl specific anti-oxidative family (Thiol-specificantioxidant (TSA).And AhpC is also the important component part of TSA protein family.And also comprise this two conserved functional domains at known ox PRDX4 albumen and people PRDX4 albumen.

Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention; any person skilled in the art; not departing from spirit and scope of the invention; various changes and modification can be done; therefore, what protection scope of the present invention should define with claims is as the criterion.

Claims (8)

1. a deer oxidization-reduction enzyme 4 gene PRDX4, it is characterized in that, nucleotide sequence is as shown in SEQIDNO.1.

2. an albumen for genes encoding described in claim 1, is characterized in that, aminoacid sequence is as shown in SEQIDNO.2.

3. a cloning process for gene described in claim 1, is characterized in that, step is as follows:

1) take the bone knob periosteum Sensitizing area cell of spotted deer and cultivate, obtaining deer horn handle periosteum Sensitizing area cell;

2) extraction step 1) in obtain deer horn handle periosteum Sensitizing area cell in mRNA, according to gained mRNA synthesize cDNA;

3) pcr amplification primer is designed;

4) with step 2) cDNA of gained is template, utilizes step 3) primer that designs carries out pcr amplification, utilize electroresis appraisal and reclaim target amplification product;

5) to step 4) the target amplification product of gained carries out analysis of biological information qualification.

4. method described in claim 3, is characterized in that, step 1) described cultivation, culture condition is: utilize containing 10% foetal calf serum, the DMEM substratum of 1% mycillin, at 37 DEG C, 5.00%CO ₂condition under cultivate 3 days.

5. method described in claim 3, is characterized in that, step 3) described amplimer, its nucleotide sequence is as shown in SEQIDNO.3-SEQIDNO.4.

6. method described in claim 3, is characterized in that, step 4) described pcr amplification, amplification condition is: 95 DEG C of denaturation 5min, 94 DEG C of 30s, 67.3 DEG C of 30s, 72 DEG C of 150s, 30 circulations, 72 DEG C of 1min after loop ends.

7. method described in claim 3, it is characterized in that, step 5) described Analysis and Identification utilizes nucleic acid electrophoresis to judge to detect in swimming lane whether contain the band that size is 800-900bp, again by being analyzed with known species sequence, determine fragment for the purpose of object fragment whether.

8. method described in claim 3, is characterized in that, concrete steps are as follows:

1) take the bone knob periosteum cell of spotted deer, and utilize containing 10% foetal calf serum, the DMEM substratum of 1% mycillin, at 37 DEG C, 5.00%CO ₂condition under cultivate 3 days, obtain deer horn handle periosteum Sensitizing area cell;

2) extraction step 1) in obtain deer horn handle periosteum Sensitizing area cell in mRNA, according to gained mRNA synthesize cDNA;

3) pcr amplification step 2 is designed) primer of described cDNA, obtain the primer as SEQIDNO.3-SEQIDNO.4;

4) with step 2) cDNA of gained is template, utilizes step 3) primer that obtains carries out pcr amplification, amplification condition is: 95 DEG C of denaturation 5min, 94 DEG C of 30s, 67.3 DEG C of 30s, 72 DEG C of 150s, 30 circulations, 72 DEG C of 1min after loop ends, obtain amplified production;

5) nucleic acid electrophoresis determining step 4 is utilized) by contrast marker, the DNA band whether containing a 800-900bp in gained amplified production, then by being analyzed with known species sequence, determines fragment for the purpose of object fragment is whether.