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Amphioxus galactose lectin AmphiGAL13-gene and its use

A technology of gene and gene coding, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2009-09-02
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Lectin microparticle drug delivery system still has many problems to be solved before it can become a mature drug delivery system

Method used

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  • Amphioxus galactose lectin AmphiGAL13-gene and its use
  • Amphioxus galactose lectin AmphiGAL13-gene and its use
  • Amphioxus galactose lectin AmphiGAL13-gene and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Extraction of total RNA from the digestive tract of amphioxus and synthesis of cDNA

[0029]Extraction of total RNA and synthesis of cDNA; the digestive tract of amphioxus was collected, the total RNA of the digestive tract was extracted by Trizol reagent, and the protein was extracted by phenol / chloroform extraction, and the total RNA of the digestive tract of amphioxus was obtained. 260 / A 280 = 1.828, two clear bands of 18s and 28s can be seen through 1% formaldehyde denaturing gel electrophoresis detection, the ratio> 1 (see figure 2 ), indicating that the total RNA has better integrity and higher purity. Take 1ug of total RNA, with SMART III olignuclotide (5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3') and CDSIII / 3'PCR primer (5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30 N -1 N-3') was reverse-transcribed to synthesize the first strand, and 10 μl of the first-strand cDNA product was obtained.

[0030] The 2ulcDNA one-strand product was taken, and the seco...

Embodiment 2

[0031] Example 2: Determination and Analysis of AmphiGAL13 Gene Sequence in Amphioxus Gastrointestinal Tract

[0032] The second-strand cDNA was connected to the pcDNA3.0 vector (purchased from Invitrogen), transformed into DH5α Escherichia coli, and the recombinant clones were selected and sequenced. Blast homology analysis showed that the EST sequence encoding the full-length AmphiGAL13 gene was obtained. The length of the gene was 465bp, encoding a Galectin protein with 154 amino acids, which was a new Galectin protein.

[0033] Using the ClustalW analysis software to compare the reported human prototype Galectin protein sequences, the results are as shown. From figure 1 It can be seen that the sugar recognition domain of Galectin is relatively conserved among all Galectins.

Embodiment 3

[0034] Embodiment 3: Construction of recombinant PGAL13 expression plasmid

[0035] A pair of primers were synthesized based on the sequences at both ends of the AmphiGAL3 gene, the upstream primers contained BamH I and Eenterkinase cleavage sites, and the downstream primers contained Not I cleavage sites.

[0036] Upstream primer (P1):

[0037] 5′-CG ACGACGACGACAAA ATGG CGT ACC CAG GAT T-3′;

[0038] BamH I Eenterkinase

[0039] Downstream primer (P2):

[0040] 5′-ATA AGA AT TT ATG TGA AGC GGA TCT GCT-3';

[0041] Not I

[0042] Using the pcDNA3.0 plasmid (purchased from Invitrogen) containing the AmphiGAL 13 gene as a template and P1 and P2 as primers for PCR amplification, a specifically amplified single band was obtained with a product size of about 460 bp. The PCR amplified product was cloned into the prokaryotic fusion expression vector pGEX-4T (purchased from Amersham Bioscience Company) to obtain the recombinant expression vector pGEX-AmphiGAL13 (its constru...

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Abstract

The invention relates to amphioxus AmphiGAL13 gene and its coded protein, and the application of the protein in preparing a new bioadhesive agent. In the present invention, the amphioxus AmphiGAL13 gene is isolated from the digestive tract of amphioxus by means of cDNA library construction and large-scale sequencing, and its DNA sequence is shown in the <400>1 sequence in the sequence table. The amino acid sequence of the protein (PGAL13) encoded by the gene is shown in the <400>2 sequence in the sequence listing. The invention expresses the recombinant amphioxus GST-PGAL13 protein through GST fusion, and the protein obtained after being digested by EK enzyme has the general function of lectin, and can be used as a natural small molecule bioadhesive particle preparation for development and application.

Description

technical field [0001] The invention relates to amphioxus galectin (AmphiGAL13) gene and protein P encoded by it GAL13 , and the protein as a potential bioadhesive microparticle formulation. Background technique [0002] Amphioxus (Branchiostoma belcheri tsingtauense), belonging to Chordata and cephalochordate, is the closest living invertebrate to vertebrates, and is also the model of vertebrate ancestors. It is the most primitive species in the ocean. of animals. [0003] Lectins are proteins or glycoproteins that bind specific carbohydrates. The phenomenon of lectin-mediated bioadhesion is produced by "lectin-glycosyl" interaction. This interaction can be divided into two categories: (1) Exogenous lectins combine with endogenous glycosyl groups. Epithelial cell A cell with polysaccharide-protein complexes on its surface, so many sugar residues can be exposed on its surface. Lectin is a protein or non-immunogenic glycoprotein that can specifically recognize sugar mole...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/46C07K1/14A61K38/17
Inventor 徐安龙禹艳红余英才元少春董美玲
Owner SUN YAT SEN UNIV
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