Cloning and expression of osteonectin gene from rats

Abstract

Total cellular RNA was extracted from the osteoblast cells of newborn rats' calvarial bones, and the cDNA containing open-reading frame of osteonectin was amplified by reverse transcription polymerase chain reaction (RT-PCR). The obtained product was named On. The On fragment was inserted into pBT-T vector. Then, the On was subcloned, in-frame fused to 3′-end of the GST gene of the prokaryotic expression vector pGEX-KG, and the resulting recombinant plasmid was transformed into E. coli BL21 (DE3) pLysS competent cells. A 60 kD fusion protein was expressed after IPTG induction. The On fragment was sequenced, and the sequencing result shows that it shares 99.8% homology with the sequence published in GenBank. The On SDS-PAGE analysis exhibits and the On was expressed with the GST gene. There is 10% fused protein in the total E. coli proteins, and the fusion protein is a soluble protein. These experimental results imply that On from Wistar rats was cloned successfully and expressed efficiently.

Keywords

wistar rat / osteonectin gene / cloning / expression

References

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