Amphioxus galactose lectin AmphiGAL13-gene and its use

A technology of galectin and amphioxus, applied in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2007-06-20
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 prob

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

[0029] Example 1: Extraction of total RNA from the digestive tract of amphioxus and synthesis of cDNA

[0030] 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 to obtain the total RNA of the digestive tract of amphioxus, and its A 260 / A 280=1.828, two clear bands of 18s and 28s can be seen by 1% formaldehyde denaturing gel electrophoresis, 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.

[0031] The 2ulcDNA one-strand product was taken, and the second-strand cDNA was...

Embodiment 2

[0032] Example 2: Determination and Analysis of AmphiGAL13 Gene Sequence in Amphioxus Gastrointestinal Tract

[0033] 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 for sequencing. 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.

[0034] Using the ClustalW analysis software to compare the reported human prototype Galectin protein sequences, the results are as shown. It can be seen from Figure 1 that the sugar recognition domain of Galectin is relatively conserved among all Galectins.

Embodiment 3

[0035] Embodiment 3: Construction of recombinant PGAL13 expression plasmid

[0036] 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.

[0037] Upstream primer (P1):

[0038] 5′-CG ACG ACG ACG ACA AA A TGG CGT ACC CAG GAT T-3';

[0039] BamH I Eenterkinase

[0040] Downstream primer (P2):

[0041] 5′-ATA AGA AT TT ATG TGA AGC GGA TCT GCT-3';

[0042] Not I

[0043] Using the pcDNA3.0 plasmid containing the AmphiGAL13 gene (purchased from Invitrogen) 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) to obtain the recombinant expression vector pGEX-Am...

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Abstract

Chinese amphioxus galactose agglutinin AmphiGAL13 gene and its production are disclosed. The process is carried out by constructing cDNA library, large-scaled sequencing, and separating out AmphiGAL13 gene from digestive tube of Chinese amphioxus. It expresses recombinant amphioxus GST-PGAL13 protein by GST fusion and obtains agglutinin by EK enzyme digestion. It can be used to develop and research for natural small molecule biological granular preparation.

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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IPC IPC(8): C12N15/12C07K14/46C12N1/21C07K1/14A61K38/17
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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