Cloning of rat galectin-9 gene and construction of its eukaryotic expression vector*,☆

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Abstract

BACKGROUND:

Galectin-9 is considered to play a key role in cell differentiation, apoptosis, adhesion, cell aggregation and regulation of inflammatory response. However, its mechanism is still not clear.

OBJECTIVE:

To clone rat galectin-9 gene fragment and construct its eukaryotic expression vector pDC316-GFP-Galectin-9, further investigate the function and reaction mechanism of galectin-9.

DESIGN, TIME AND SETTING:

Single sample observation was performed in the Beijing AGTC Gene Technology Company LTD between November 2008 and January 2009.

MATERIALS:

One male Lewis rat of SPF grade.

METHODS:

Rat Galectin-9 gene was amplified by RT-PCR method from rat liver tissue and reconstructed into eukaryotic expression vector pDC316-GFP using gene reconstruction technique to construct pDC316-GFP-Galectin-9 recombinant plasmid. The recombinant DNA was identified by PCR amplification, enzyme digestion and DNA sequencing.

MAIN OUTCOME MEASURES:

Identifications of recombinant plasmid.

RESULTS:

After RT-PCR amplification and agarose gel electrophoresis, an obvious band was observed at 1 kb, which accorded with the predicted galectin-9-cDNA length. The recombinant plasmid pDC316-GFP-Galectin-9 digested with Not I /HindIII or without digestion were presented with two bands (980 bp: Galectin-9-cDNA product, 5.8 kb: pDC316-GFP plasmid) or one band (6.7 kb: recombinant plasmid pDC316-GFP-Galectin-9), respectively. This was a preliminary marker of successful construction. Sequencing results were analysis by pairs using NCBI BLAST, its nucleotide sequence completely matched the galectin-9 gene mRNA coding sequence (NM_012977) in GeneBank, which was then confirmed as rat galectin-9-cDNA.

CONCLUSION:

Galectin-9 gene is successfully cloned and its eukaryotic expression vector pDC316-GFP-Galectin-9 is constructed.

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