Amphioxus endonuclear protein, and expression gene and application thereof

A technology for expressing genes and amphioxus, applied in the field of genetic engineering, can solve problems that hinder the rapid development of seawater aquaculture

Inactive Publication Date: 2013-05-22
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, diseases have seriously hindered the rapid

Method used

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  • Amphioxus endonuclear protein, and expression gene and application thereof
  • Amphioxus endonuclear protein, and expression gene and application thereof
  • Amphioxus endonuclear protein, and expression gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cloning of the gene encoding AmphiAkirin in the nucleus protein of Amphioxus qingdaoensis:

[0034] 1.1 Extraction of total RNA from Amphioxus spp. and synthesis of first strand cDNA (refer to the relevant kit instructions of Takara and TIANGEN for the extraction and synthesis steps).

[0035] 1) Cut 100 mg of fresh tissue (Adult Amphioxus) into a pre-1.5mlep tube.

[0036] 2) Add 1ml Trizol (purchased from Invitrogen) reagent, use a grinding pestle to quickly grind the tissue until there is no large tissue mass, and let it stand at room temperature for 5 minutes to fully lyse the tissue. At this time, the sample can be stored at -80°C for a long time.

[0037] 3) Add 0.2ml of chloroform (chloroform) to every 1ml of Trizol reagent, cover tightly, shake vigorously for 15s, and let stand at room temperature for 15 minutes.

[0038] 4) Centrifuge at 1000g for 15min at 4℃. At this time, the sample is divided into three layers: the red organic phase in the lower layer, the middle la...

Embodiment 2

[0058] Gene sequencing

[0059] 2.1. DNA fragment and cloning vector connection

[0060] The recovered DNA fragments were ligated to the pEASY-T3 vector of Transgene.

[0061] The ligation reaction system is as follows: ligate carrier enzyme mixture 1μl, foreign DNA fragment 4μl, flick the centrifuge tube with fingers, mix the sample, centrifuge for 2 seconds, collect the sample at the bottom of the tube, and ligate at 37°C for 5 minutes.

[0062] 2.2. Prepare E. coli DH5α competence according to the method for preparing E. coli competence in "Molecular Cloning Experiment Guide".

[0063] 2.3. According to the heat shock transformation method in the "Molecular Cloning Experiment Guide", the ligated recombinant vector pEASY-T3 was transferred to E. coli DH5α competent.

[0064] 2.4. The transformed Escherichia coli DH5α was spread on MacConkey agar medium containing 100μg / L ampicillin, incubated overnight at 37°C, white transformants were selected as templates, T7 and SP6 were used as...

Embodiment 3

[0069] Detect the distribution of AmphiAkirin in various organizations

[0070] 1) Cut the adult wenchang fish into tissue segments no more than 1cm in length.

[0071] 2) Immediately put into 4% paraformaldehyde solution (newly prepared in PBS), and fix at room temperature for 3-4 hours or 4°C overnight.

[0072] 3) Rinse with PBS, dehydrate with gradient alcohol, transparent xylene and embed the slices in wax.

[0073] 4) Cut into 10μm thin slices and adhere to the glass slide coated with adhesive, dry the slices overnight in a 40℃ thermostat, dewax with fresh xylene for 2×10min, rehydrate with 100%, 95%, gradient alcohol, 70%, 50%, 30% all levels of alcohol 1×5min, ddH 2 O rinse for 2×5min to block endogenous biotin.

[0074] 5) Incubate the tissue sections in PBS (pH 7.4) for 2×5 min.

[0075] 6) Incubate in PBS containing 0.3% Triton X-100 for 15 minutes; wash with PBS for 2×5 minutes.

[0076] 7) Incubate in 5-20μg / ml TE solution of RNase-free protein kinase K at 37°C for 15-30min ...

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Abstract

The invention relates to an amphioxus endonuclear protein, and an expression gene and application thereof. The nucleotide sequence of the expression gene of the amphioxus endonuclear protein AmphiAkirin is disclosed as SEQ ID NO.1; and the amino acid sequence of the amphioxus endonuclear AmphiAkirin is disclosed as SEQ ID NO.2. The expression gene provided by the invention is beneficial to theoretically enhancing the recognition on immune system composition and immune response mechanism, and lays the foundation for producing and developing new marine culture anti-parasite vaccines on the gene level.

Description

Technical field [0001] The present invention relates to a nuclear protein of Amphioxus and its expression genes and applications, in particular to a marine model organism Amphioxus nuclear protein Akirin and its expression genes and applications, belonging to the technical field of genetic engineering. Background technique [0002] NF-κB is an important transcription factor, which plays an important role in immune regulation, inflammatory response, stress response and apoptosis, so it has always been a research hotspot (Brian et al., 2009). As a transcription factor, how NF-κB specifically recognizes the target genes required for binding in the huge kB binding site is still a hot and difficult point in the research of NF-κB signaling pathway. After entering the nucleus, NF-κB requires multiple co-activators including Akirin, Nurr1, SIRT6 and other co-activators to help NF-κB co-regulate the expression of target genes (Wang et al., 2010). Therefore, understanding the regulatory m...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N5/10C07K14/435A61K48/00A61P33/00
Inventor 冯力骏闫杰荆韧威李开霖刘凯兴张妍董璇王建峰张长青孔雨刘欢欢
Owner SHANDONG UNIV
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