Cloning and expression of human erythropoietin in E. coli
- Author:
Dang Thanh Nam
- Publication Type:Journal Article
- Keywords:
Genetics, gene
- MeSH:
Escherichia coli;
Human
- From:Journal of Vietnamese Medicine
2005;315(10):19-24
- CountryViet Nam
- Language:Vietnamese
-
Abstract:
Cloning and determining the nucleotide sequence and expression of human erythropoietin (h-epo) gene in E. coli cells were presented. A gene encoding for mature h-EPO was amplified from human kidney total RNA by RT-PCR using specific primers. The product was cloned into the pCR2.1 TOPO cloning vector and the nucleotide sequence containing 498 bp encoding for mature h-EPO with 166 amino acids was determined by ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosysterms). To express h-EPO in E.coli cells, the h-epo gene was subcloned into the pET21 (+) vector (Novagen) by ligase reaction and was transformed into BL21 (DE3) cells. The cells harboring the recombinant plasmid were grown in liquid LB medium supplementing ampicillin (final concentration of 100g/ml) at 370C. When the OD600 nm of the culture reached 0.5, IPTG (final concentration of 0.4mM) was added. The expression of the h-EPO was analyzed by SDS-PAGE and confirmed by Western Blotting using anti-h-EPO antibody
