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Recombinant carp Nrf2 (NF-E2-related factor 2) gene, protein, preparation and detection methods and application of recombinant carp Nrf2 gene

A detection method, carp technology, applied in the field of recombinant carp Nrf2 gene, protein and its preparation and detection, can solve the problem that there is no full-length nucleotide sequence of carp cDNA, no research report on Nrf2, and no research on signal regulation molecules Report and other issues, to achieve the effect of improving the antioxidant capacity of cells, preventing oxidative damage of cells, and improving the survival rate of cell culture

Inactive Publication Date: 2013-05-01
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whether Nrf2 is a signaling regulator of Cu / ZnSOD gene transcription in aquatic animal intestinal cells has not been reported, and it is necessary to carry out research
[0003] So far, studies on the regulatory role of Nrf2 in the synthesis of antioxidant proteins in mammalian cells have been carried out in human, rat, mouse and other mammalian cells, and many achievements have been made, but there is no relevant research on lower vertebrate fish. There is no report on the study of Nrf2 in cells, and there is no report on the full-length cDNA nucleotide sequence and corresponding amino acid sequence of carp Nrf2 gene

Method used

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  • Recombinant carp Nrf2 (NF-E2-related factor 2) gene, protein, preparation and detection methods and application of recombinant carp Nrf2 gene
  • Recombinant carp Nrf2 (NF-E2-related factor 2) gene, protein, preparation and detection methods and application of recombinant carp Nrf2 gene
  • Recombinant carp Nrf2 (NF-E2-related factor 2) gene, protein, preparation and detection methods and application of recombinant carp Nrf2 gene

Examples

Experimental program
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Effect test

Embodiment 1

[0038] The recombinant carp NRF2 gene in this embodiment is the gene sequence shown in SEQ ID No: 1 in the sequence listing. As a person skilled in the art, this sequence can also be the gene sequence shown in SEQ ID NO: 1 through one or several base substitutions, deletions or additions and expressed with the gene sequence shown in SEQ ID No: 1 A gene sequence with the same amino acid sequence. If A is replaced by G at position 610, the amino acid sequence encoded by the sequence is the same.

[0039] The recombinant carp NRF2 amino acid sequence of the present embodiment is the amino acid sequence shown in SEQ ID NO: 2; As a person skilled in the art, this sequence can also be the amino acid sequence shown in SEQ ID NO: 2 through one or several A protein derived from the sequence of SEQ ID NO: 2 having the same activity as the amino acid sequence of SEQ ID NO: 2 by substitution, deletion or addition of amino acid residues. If A is replaced by G at position 123, the biologi...

Embodiment 2

[0041] In order to obtain the recombinant carp NRF2 gene sequence described in Example 1, the method of the present embodiment can be used, specifically as follows:

[0042] Firstly, carp (Common carp, Cyprinus carpio ) of various tissue samples. Immediately after the carp was slaughtered on site, tissue pieces such as kidney, intestine, muscle, spleen and hepatopancreas were taken, put into cryovials and immediately stored in liquid nitrogen, and brought back to the laboratory and stored in a -80°C refrigerator for later use. Then use TaKaRa’s total RNA extraction kit (TaKaRa RNAiso Reagent) and follow the operating procedures in the instruction manual to extract total RNA from carp intestinal tract, muscle, kidney, hepatopancreas, and spleen. Finally, according to the mammalian NRF2 gene reported in the literature, According to the expression in various tissues and the extraction results of total RNA, the total RNA of intestinal tissue was selected as the template for reve...

Embodiment 3

[0062] This example is the application of the recombinant carp NRF2 protein described in Example 1 and Example 2 in determining the relative expression of signal molecule Nrf2 mRNA in different intestinal segments of carp.

[0063] In this example, a pair of Nrf2-specific quantitative amplification primers was designed based on the recombinant carp Nrf2 gene sequence cloned in the present invention to detect the expression of Nrf2 mRNA in different carp intestinal segments. The relative expression of Nrf2 mRNA in each intestinal segment was represented by the relative ratio of the fluorescence value of Nrf2 and the housekeeping gene β-actin in the same system×100.

[0064] Test results such as Figure 4 As shown, the results showed that there were significant differences in the relative expression of Nrf2 mRNA in different intestinal segments of carp. Among them, the relative expression of Nrf2 mRNA in the first and third intestinal segments was significantly higher than that...

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Abstract

The invention discloses a recombinant carp Nrf2 (NF-E2-related factor 2) gene, a recombinant carp Nrf2 protein, preparation and detection methods and application of the recombinant carp Nrf2 gene. The recombinant carp Nrf2 gene has a sequence shown by SEQIDNO:1, and the recombinant carp Nrf2 protein has a sequence shown by the SEQIDNO:2. By the nucleotide sequence of the carp Nrf2 gene complementary deoxyribonucleic acid (cDNA) disclosed by the invention, the tissue specific expression of the Nrf2 gene can be further detected through a reverse transcription-polymerase chain reaction (RT-PCR) or hybridization method, and the full-length cDNA of the carp Nrf2 gene can be further cloned. The complete Nrf2 protein may be expressed through recombining the cDNA of the recombinant carp Nrf2 gene onto an expression vector, so that the Nrf2 protein can be used for producing antibodies, and the Nrf2 gene expression states of cells, tissues, early embryos and individuals of carps under different states are detected.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant carp Nrf2 gene, protein, preparation and detection method and application thereof. Background technique [0002] Nuclear factor-related factor 2 (NF-E2-related factor 2, Nrf2) is a key signaling molecule that regulates the expression of intestinal mucosal glutathione-S-transferase (glutathione S-transferase, GST) gene. The Antioxidant Response Element (ARE) is the 5′-TGACnnnGC-3′ sequence of the promoter region of the GST gene. Studies have shown that the signaling molecule Nrf2 binds to the ARE sequence of the GST gene and activates GST transcription. There are ARE sequences in the upstream region of Cu / ZnSOD gene promoter in both mouse and human. However, there are great differences in the full sequence characteristics of Cu / ZnSOD genes among different species. The human Cu / ZnSOD genome sequence is 8419bp (with 5 exons and 4 introns), but the full seque...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10C07K14/435C12Q1/68
Inventor 周小秋姜维丹冯琳姜俊刘扬李树红
Owner SICHUAN AGRI UNIV
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