Crassostrea gigas interferoid (CgIFN-like) gene recombinant protein, and preparation and application thereof

A technology of genetic recombination and oyster growth, applied in the field of molecular biology, can solve the problems of less research on interferon systems, and achieve the effect of promoting phagocytosis

Inactive Publication Date: 2014-10-08
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the interferon system has been deeply studied in mammals and fish, but there are few studies on the interferon system in molluscs. use

Method used

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  • Crassostrea gigas interferoid (CgIFN-like) gene recombinant protein, and preparation and application thereof
  • Crassostrea gigas interferoid (CgIFN-like) gene recombinant protein, and preparation and application thereof
  • Crassostrea gigas interferoid (CgIFN-like) gene recombinant protein, and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: In vitro prokaryotic recombinant expression of the long oyster interferon (CgIFN-like) gene coding region, comprising the following steps:

[0024] 1. Construction of recombinant vector

[0025] The recombinant vector used in the present invention is pET-30a(+) prokaryotic expression vector. By PCR, using gene-specific primers:

[0026] P1(5'-CGGGATCCATGGAGAGGAAAAAGGAT-3');

[0027] P2(5'-CCCAAGCTTCTAAGTCATTAATTTTCTTTC-3'),

[0028] The reaction conditions were as follows: 94°C pre-denaturation for 5 minutes, followed by the following cycles: 94°C denaturation for 30 seconds, 52°C annealing for 30 seconds, 72°C extension for 30 seconds, a total of 35 cycles, and finally 72°C extension for 10 minutes. The PCR product was purified and recovered, and ligated with the pET-30a(+) vector after double digestion. Transformation, colony PCR screening and sequencing, extraction of positive clone plasmids, and completion of vector construction.

[0029] 2. Expre...

Embodiment 2

[0048] Example 2: Functional analysis of long oyster interferon-like (CgIFN-like) prokaryotic recombinant protein promoting apoptosis and phagocytosis

[0049] (1) Dilute the above-mentioned recombinant protein with seawater to different concentrations: 10ug / ml, 1ug / ml, 100ng / ml, use seawater as the control group, inject 10ul per oyster

[0050] (2) Take blood cells at 0h, 3h, 6h, and 12h respectively, and after centrifugation, wash and suspend them with L-15 (gibco) medium. In order to maintain the balance of osmotic pressure, add the final concentration of KCl (0.54g / L), NaCl (20.2g / L), CaCl 2 (0.6g / L), MaCl 2 (3.9g / L), MaSO 4 (1g / L), glucose (20.8g / L), according to the Beyontian Annexin V-FITC detection kit, use flow cytometry to detect the immune system-related indicators activated by the recombinant protein.

[0051] (3) The experimental results are as follows figure 1 with figure 2 shown.

[0052] apoptosis by figure 1 Shown:

[0053] After treatment with high ...

Embodiment 3

[0057] Example 3: Analysis of prokaryotic recombinant protein of long oyster interferon (CgIFN-like) on L929 and A549 cell proliferation inhibition function

[0058] (1) Human lung cancer cells A549 were cultured in 1640 medium containing 10% (volume ratio) fetal bovine serum, and mouse fibroblasts (L929) were cultured in DMEM medium containing 10% (volume ratio) fetal bovine serum, The culture conditions were set at 37°C, 5% CO 2 Cultured in an incubator, and the logarithmic phase cells were used for the experiment.

[0059] (2) The above cells were divided into 5×10 3 -1×10 4 Inoculate in a 96-well cell culture plate at a density of 100ul / well in 5% CO 2 Cultivate in the incubator for 12-24 hours, and replace the respective medium after the cells adhere to the wall.

[0060] (3) Add recombinant interferon (rCgIFN-like) recombinant protein to the culture wells of the above cells and incubate the cells. The dosage concentration of interferon (rCgIFN-like) recombinant prote...

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Abstract

The invention belongs to the field of molecular biology research, and relates to a Crassostrea gigas CgIFN-like gene in-vitro recombinant expression technique and function identification thereof. The in-vitro recombinant expression technique is utilized to obtain the Crassostrea gigas interferoid recombinant protein and detects that the Crassostrea gigas CgIFN-like gene in-vitro recombinant protein can activate the immune system of the Crassostrea gigas, cause apoptosis, promote the phagocytic function of the cells for Vibro Splendidus and inhibit the proliferation of the human lung cancer cell A549. The recombinant protein can be used for development of novel immunopotentiators, and development and utilization of marine natural anticancer drugs.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a long oyster interferon-like (CgIFN-like) gene recombinant protein and its preparation and application. Background technique [0002] Interferon (IFN) is a highly biologically active glycoprotein secreted by recipient cells when cells are infected by viruses, or under the action of nucleic acids, bacterial endotoxins, and mitogens. Anti-tumor, immune regulation and other biological functions. The interferon system is the main component of the innate immune system to resist virus infection, including the cell system that secretes and synthesizes interferon and the cell system that accepts the action of interferon, that is, all cells that can activate interferon synthesis after external stimuli such as virus infection And cells that respond to the action of interferon and establish an anti-viral state are an important defense system for the body against viral infection....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/555C12N15/20C12N15/70A61K38/21A61P35/00A61P37/04A23K1/16A23K10/16A23K20/147A23K40/00
CPCA61K38/00C07K14/555C12N15/70
Inventor 宋林生张冉王玲玲张峘刘瑞
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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