Construction of Synthetic Intact Human Parathyroid Hormone Gene and Testing the Transformation Efficiency and Expression of it in E. coli strains

  • Adnan Al-Badran University of Basra, College of Science, Department of Biology, Basra, Iraq
  • Rafeef A. Abdul-Jabbar University of Basra, College of Pharmacy, Department of Clinical Laboratory Science, Basra, Iraq
Keywords: synthetic gene, synthetic human parathyroid hormone gene, SOE PCR.

Abstract

 human parathyroid hormone gene (hPTH gene) is responsible for preproparathyroid hormone production (biologically inactive hormone) which further modified to form the biologically active hormone, a synthetic hPTH gene with 289bp which cod for active intact hPTH was constructed by Five long primers by splicing overlap extension PCR (SOE), the gene contain a de generated codon, and have two recognition site of restriction enzymes in both ends of the Gene, and a stop codon. The synthetic hPTH gene was transformed in two E. coli strains DH5α and BL21 (DE3) with transformation efficiency6.6* 106 and 3.4* 106 for both isolatesrespectively. And the transformation of gene was detected by extraction of constructed plasmid and amplification of cloning gene on it, the expression of the cloning gene in the BL21 (DE3) was detected by performing Real Time PCR which gave a Ct value of about (23.46). That makes the synthetic gene suitable for direct expression of human active protein inside the bacterial cells.

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Published
2017-01-01
Section
Articles