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Epinephelus coioides interferon IFNgamma2 and preparation method and application thereof

A technology of slanted grouper and interferon, applied in the field of interferon

Active Publication Date: 2013-07-24
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a new fish interferon IFNγ2 gene and its encoded protein according to the lack of interferon protein IFNγ defect in the existing fish immune control methods

Method used

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  • Epinephelus coioides interferon IFNgamma2 and preparation method and application thereof
  • Epinephelus coioides interferon IFNgamma2 and preparation method and application thereof
  • Epinephelus coioides interferon IFNgamma2 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Cloning of the grouper interferon IFNγ2

[0040] 1. Extraction of total RNA from head and kidney of grouper

[0041] Take healthy whole grouper fish, anesthetize them in ice bath for about 2 minutes, kill the fish and take samples, separate the head and kidney tissue, and extract the head and kidney total RNA of grouper with Trizol reagent, and its OD260 / 280 = 1.91.

[0042] 2. Synthesis of cDNA First Strand

[0043] Take 1 μg of the total RNA sample of grouper head kidney and perform DNase treatment to remove the contamination of genomic DNA, mix it with RNA Oligo dT (sequence shown in SEQ ID NO:3), perform reverse transcription, and place the obtained product in- Store at 20°C for later use.

[0044] 3. Cloning of the complete cDNA sequence of the IFNγ2 gene of the grouper

[0045] According to the information in the grouper genome database, design specific primers at both ends of the spliced ​​sequence of the IFNγ open reading frame, the upstream prime...

Embodiment 2

[0046] Example 2. Expression of the recombinant protein of interferon IFNγ2 from the grouper

[0047] 1. Construction of recombinant expression plasmids

[0048] Using the pTZ57R / T plasmid containing the IFNγ2 coding gene as a template, design a pair of upstream primers SEQ ID NO:6 containing EcoRI restriction sites and downstream primers SEQ ID NO:7 containing XhoI restriction sites, and amplify by PCR A single band with a product size of about 550 bp was obtained, and the electrophoresis results were as follows figure 2 As shown, wherein, M is a DNA Marker, and 1 is an IFNγ2 nucleotide target fragment with a restriction site. The mature peptide coding sequence of grouper interferon IFNγ2 was cloned into the prokaryotic expression vector pET-22b to construct the recombinant expression vector pET22b-IFNγ2, (the construction process is as follows image 3 shown). The exogenous gene sequence in the expression vector was identified by sequencing.

[0049] 2. Prokaryotic expr...

Embodiment 3

[0054] Example 3. Activity analysis of the influence of the recombinant protein of the grouper interferon IFNγ2 obtained in Example 2 on the expression of immune-related genes

[0055] Sampling of rapidly frozen and anesthetized experimental fish, cut head and kidney tissue, soaked in appropriate amount of RPMI1640 medium, and placed on ice; put the head and kidney tissue in an ultra-clean workbench and shredded slightly, after removing the connective tissue, Grind with the frosted surface of the baked frosted glass slide until it is fine; pass the ground cells with the medium through the Cell strainer (BD Falcon, 70 μm, Nylon) and transfer to a 50ml centrifuge tube: Centrifuge, discard the supernatant Add 2 ml of RPMI 1640 medium to resuspend the cells, wash, centrifuge, and resuspend the cells with 2 ml of complete medium (RPMI 1640 containing 2 mM L-glutamine, 10% FBS and 1% penicillin / streptomycin) to adjust the number of cells to 1 x 10 7 / mL, add 2 mL of the above cell...

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Abstract

The invention relates to an epinephelus coioides interferon IFNgamma2 gene and a protein encoded by the sameas well as application of the protein in preparing a novel immunopotentiator. According to the invention, an open reading frame of the IFNgamma2 gene is cloned from epinephelus coioides head-kidney total RNA by specific primers SEQ ID NO:4 and SEQ ID NO:5 by means of a method of a primer amplification gene segment and RACE overall length amplification. The nucleotide sequence is shown as SEQ ID NO:2. The amino acid sequence of encoded protein is shown as SEQ ID NO:1. An initial test shows that the epinephelus coioides interferon IFNgamma2 protein can induce relevant immunogene expression and can be developed to natural fish immunopotentiator or immunologic adjuvant.

Description

technical field [0001] The present invention relates to the field of interferon, and more specifically relates to grouper interferon IFNγ2 and its preparation method and application. Background technique [0002] Interferon (IFN) is a group of active proteins (mainly glycoproteins) with multiple functions, and is a cytokine produced by monocytes and lymphocytes after cells and the body are infected by viruses or other inducers. According to the difference in homology and receptor specificity, three types of interferon have been found so far: type I, type II and type III. Among them, type II IFN (IFNγ), also known as immune interferon, is a major macrophage stimulator and a key signaling molecule that regulates immune responses. It can activate effector cells and increase the activity of natural killer cells (NK) and macrophages. Promote the conversion of immunoglobulins, and have the functions of anti-tumor, anti-virus, regulating and enhancing the immune function of the bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/23C12N15/70C12N15/10C07K14/57C07K1/22A61K39/39A23K1/16
Inventor 卢丹琪孙艳易诗白彭弯张勇林浩然
Owner SUN YAT SEN UNIV
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