Cloning and expression of recombinant hirudin in CHO cell line

Subject Areas : Journal of Comparative Pathobiology

, A. Sharifzadeh ¹ , , M. Heidari ² , , F. Aali ³

1 - .Department of Microbiology, Faculty of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
2 - Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
3 - Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

Received: 2020-11-07 Accepted : 2020-11-07 Published : 2020-05-21

Keywords: Hirudn, Cloning, gene expression,

Abstract :

Hirudin is a 65-66 amino acids polypeptide which is secreted as an anticoagulant compound from salivary glands of medical leech. This drug is a very potent inhibitor of thrombin and is so effective for arterial and venous thrombosis prevention. The aim of the present research was to clone and express of hirudin gene in CHO cell line as a eukaryotic host cell. In this experimental study, a 221 bp fragment of hirudin gene was cloned into pcDNA3.1(+) vector. The pcDNA3.1(+)-hirudin recombinant vector was transfected into CHO cells by lipofectamine 2000 reagent. The eukaryotic expression of hirudin was evaluated by RT-PCR method. The pcDNA3.1(+)-hirudin recombinant vector was confirmed by PCR and enzymatic double digestion. Our findings showed that the mammalian expression of hirudin was successful. As, a 221 bp fragment corresponded to hirudin mRNA was observed on agarose gel after RT-PCR. The pcDNA3.1(+)-hirudin recombinant vector was constructed in this research successfully. This new recombinant plasmid can express the hirudin mRNA in CHO cells. Therefore, the recombinant protein that produced from this research can be a recombinant vaccine that could be applied for future research. Additionally, this construct has the potential to be use as DNA vaccine in next experiments.

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