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Galectin-3 binding protein, preparation and application thereof

A protein and bacterial technology, applied in the field of molecular biology, can solve problems such as unknown functions

Inactive Publication Date: 2014-04-09
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far G3BP has only been found in two fish species (zebrafish and Atlantic salmon), but its function is completely unknown

Method used

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  • Galectin-3 binding protein, preparation and application thereof
  • Galectin-3 binding protein, preparation and application thereof
  • Galectin-3 binding protein, preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The galectin-3 binding protein of the present invention is the amino acid sequence in SEQ ID No. 1 in the sequence table.

[0026] The sequence listing SEQ ID No.1 is:

[0027] MQTHQNFCRIWVLLLLLHVADGAFTFDLFENIAAPKEGDVRLAGSRSSSEGRVEVYHDGRWGTVCDDGWDIAEAQVVCRQLRFSGAKGVQVGQPYGEATGPIWMDDMICQGTEKHLLNCAFKSWGVTDCTHKEDVGIICENNDNNSKNAIYSLDHSFSLSDELGQLFDSEVGCDFLIILRSPTGHRFEYETEEMICAHKMILLLVPRFNASQGVSNITVDISQSCKPYFPTFLRYIYTRQIDVDLSSIHCLHWMASMFEVKHLMEGTARLFSEVLPEDTLFHTQVSIYNYAVETEDLVLQENCLQYLSWNFQNLTNSPAWPDISVKLLRAILVRSDLVVPNEYFVLQSVENWITDKDEAISLEDQALILNCLRFPMIPVEKLYVLETNSSLYNNHSKLYSEKILKAFQFNVLLFSDLLTNPKFDREERDYHPRLYVDYPWSVTISPSYSSRSLSTPSHNSLIFRSKTINWEANIYRSHYDCSNRRLQCKSLPMARLASHSHYHGNNIVYQNQLLLSCQGKYICHIQDFKSDLAYVTKNATHGLTYPCPDSQYTYQFVVRPVYVRV

[0028] (a) Sequential features:

[0029] ●Length: 565

[0030] ●Type: amino acid sequence

[0031] ●Chain type: single chain

[0032] ●Topological structure: linear

[0033] (b) Molecule Type: Protein

[0034] (c) Assumption: No

[0...

Embodiment 2

[0039] Preparation method of galectin-3 binding protein G3BP:

[0040] 1) Construction of plasmid pETG 3BP:

[0041] The cDNA of tongue sole was used as a template and PCR amplification was carried out with primers F1 and R1. PCR conditions are: 94°C 60s pre-denatured template DNA, then 94°C 40s, 60°C 60s, 72°C 60s, after 5 cycles, change to 94°C 40s, 65°C 60s, 72°C 60s, After 30 cycles, extend the reaction at 72°C for 7-10min. The PCR product was purified with Tiangen’s PCR product purification kit and ligated with the plasmid T-Simple (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) at room temperature for 2-8 hours. After the ligation mixture was transformed into E. Penicillin (100ug / ml), Xgal (40ug / ml), and isopropyl-β-D-thiogalactoside (24ug / ml) were cultured on LB medium for 18-24 hours, and the transformants were screened to extract plasmids to obtain Recombinant plasmid pTSG3BP.

[0042]pTSG3BP was digested with Sma I, and a 1.6 kb fragment was recovered...

Embodiment 3

[0048] Bacterial binding of the galectin-3 binding protein G3BP

[0049] 1) Bacterial suspension preparation. The Gram-negative bacteria Pseudomonas fluorescens (preserved at CGMCC, date of deposit January 9, 2008, numbered CGMCC 2329), Edwardsiella tarda ( Preserved in CGMCC, date of preservation is January 9, 2008, numbered CGMCC 2330), Escherichia coli (DH5α, purchased from "Tiangen Biochemical Technology (Beijing) Co., Ltd."), Vibrio anguillarum (Preserved in CGMCC, date of preservation is June 21, 2012, numbered CGMCC 6250, depository unit: General Microbiology Center of China Committee for the Collection of Microbial Cultures; depository unit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) And the gram-positive bacteria Streptococcus iniae (Streptococcus iniae) (preserved in CGMCC, numbered as CGMCC1984, date of deposit on March 22, 2007) and Bacillus subtilis (purchased from CGMCC, numbered as CGMCC 1.460) to OD 600 0.8, centrifuge (5000g, 4...

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Abstract

The invention relates to the field of molecular biology, and in particular to a galectin-3 binding protein, and preparation and application thereof. The galectin-3 binding protein is shown as an amino acid sequence SEQ ID No.1 in the sequence table. The preparation method is as below: using Cynoglossus semilaevis cDNA as a template; conducting PCR amplification by primers F1 and R1; connecting PCR products to an expression vector to obtain a plasmid pETG3BP; conversing BL21DE3 to obtain a transformant BL21 / pETG3BP; inducting by isopropyl-beta-D-thiogalactoside; and purifying the recombinant protein with affinity chromatography column to obtain the galectin-3 binding protein. The galectin-3 binding protein has significant binding ability to various bacteria.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a galectin-3 binding protein and its preparation and application. Background technique [0002] Galectin-3b inding protein (G3BP) is a glycoprotein secreted extracellularly, which can bind galectin-3 (galectin-3) and other extracellular proteins, such as collagen protein (collagen), fibronectin (fibronectin), β1 integrin (β1 integrins), etc. Human G3BP is the most researched at present, and it is found that it contains a scavenger receptor (Scavenger receptor) functional domain in its molecular structure, which is related to the binding of target molecules. Human G3BP is expressed in immune tissues and cells, and its expression is up-regulated in the serum of virus infection and some cancer patients, so it may participate in the body's immune resistance, inflammatory response, tumorigenesis and other processes. So far G3BP has only been found in two fish species (zebrafish and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46C12N15/70A61K38/17A61P31/04
Inventor 孙黎陈骋
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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