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Cervus nippon PRDX4 gene, cloning method thereof and encoding protein

A cloning method and gene coding technology, which can be applied in the field of genetic engineering and can solve problems such as difficulty in the sequence of sika deer PRDX4 cDNA

Inactive Publication Date: 2015-11-11
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The significant expression of sika deer PRDX4 protein in the cells of the periosteum sensitized area of ​​sika deer indicates that it may play an important role in the rapid growth and reproduction, differentiation and migration of velvet antler, and the maintenance of tissue cell stability. The cloning and functional studies of sika deer have not been reported, which brings great difficulties to the sequence of sika deer PRDX4 cDNA

Method used

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  • Cervus nippon PRDX4 gene, cloning method thereof and encoding protein
  • Cervus nippon PRDX4 gene, cloning method thereof and encoding protein
  • Cervus nippon PRDX4 gene, cloning method thereof and encoding protein

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Experimental program
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Effect test

Embodiment 1

[0029] The extraction (SunH etc., 2012) of embodiment 1 staghorn periosteal cells and the extraction of mRNA

[0030] After the periosteum of the sika antler peduncle obtained from the experimental deer farm of the Chinese Institute of Special Products of Agricultural Sciences was retrieved, according to SunH et al. (SunH, YangF, ChuW, ZhaoH, McMahonC, LiC. : e47367) method to process the obtained periosteum, and obtain the periosteum sensitized zone cells of the sika deer.

[0031] Place the cells in the sensitized area of ​​the antler horn periosteum on the 75cm of the DMEM medium containing 20ml of 10% fetal bovine serum and 1% penicillin and streptomycin 2 Culture flask, 37°C, 5.00% CO 2 Cultivate for 2 days under the same conditions; discard the old culture medium, replace with 20ml of the same new culture medium as above and continue to culture for 1 day under the same conditions to obtain cells in the sensitized area of ​​the periosteum of Stalk antler.

[0032] The c...

Embodiment 2

[0033] Example 2 Primer Design

[0034] Primers were designed according to the bovine PRDX4 mRNA sequence, and 11 sets of primers were designed, as shown in Table 1. Amplification was performed using these 11 sets of primers.

[0035] Table 111 sets of primer sequences

[0036]

[0037]

[0038] The primers from the first group to the ninth group were all primers designed by Primer software, and the target product was not obtained by using the nine sets of primers for amplification. The tenth set of primers is two sequences cut directly from the two ends of the bovine PRDX4 mRNA sequence as primers, and no target product is obtained when using this primer set to amplify. The tenth set of primers is to select two sequences of upstream and downstream primers near the complementary sequence of the deer PRDX4 mRNA start codon and stop codon, and after comprehensive evaluation of the CG content, annealing temperature, dimer and complementarity of the primers, and then A sm...

Embodiment 3

[0040] Embodiment 3 bioinformatics analysis

[0041]The sequencing results showed that the mRNA sequence of deer PRDX4 was cloned for the first time in this experiment. Using the cDNA of the cells in the periosteum sensitized region of Antlers antlers as a template, and using the sequences shown in SEQIDNO.3-SEQIDNO.4 as primers, PCR amplification was performed, and the amplification results were detected by nucleic acid electrophoresis. The results are as follows: figure 1 shown. from figure 1 It can be seen that there is a bright single band in the nucleic acid electrophoresis lane with a size of about 800-900bp, the size of the PRDX4 fragment amplified by PCR is basically the same as that of other species, and the actual length of the cloned mRNA sequence obtained is 819bp. Through bioinformatics analysis, it was found that some physical and chemical properties and biological processes of deer PRDX4 protein were basically similar to those of cattle, humans and other speci...

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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

technical field [0001] The invention relates to a deer PRDX4 gene, its cloning method and encoded protein, and belongs to the technical field of genetic engineering. Background technique [0002] PRDX4 is an important member of the PRDXs family, localized in the endoplasmic reticulum. There are two main mechanisms of action of PRDX4: one is to participate in the redox reaction of cells to prevent cell oxidative damage; the other is to act as a kind of TRX-dependent peroxidase that activates NF-κB and ICAM-1 amino-terminal kinases. At present, the PRDX4 mRNA sequences of various animals have been successfully cloned, and the PRDX4 sequences of five species including human, Dictyostelium discoideum, mouse, rat and bovine have been identified and reviewed on UNIPROT. The significant expression of the sika deer PRDX4 protein in the cells of the periosteum sensitized region of the sika deer indicates that it may play an important role in the rapid growth and reproduction, differ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/08C12N15/10
Inventor 王桂武杨福合王权威鲍加荣刘琳玲贾博寅刘宗岳
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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