Construction of pcDNA3.1-FSHβ expression vector in order to transfer FSHβ gene into a mammalian cellline

Abstract

Introduction: Follicle stimulating hormone (FSH) is one of the glycoprotein hormones of pituitary gland that consists of two subunits, alpha and beta. Beta subunit has 111 amino acids and a signal peptide, which is consisted of 18 amino acids and it is responsible for biological activity of the hormone. The aim of this study was to construct a pcDNA3.1FSHβ expression vector in order to transfer FSHβ gene into a mammalian cell line.Materials and Methods: To sub-cloning of beta chain from T.vector, a pair of primer was designed that they had a site for EcoRI and HindIII in addition of the start and stop site of the beta chain gene. Amplified beta chain was cloned in pcDNA3.1 at the site of the mentioned enzymes anvd the recombinant plasmid was transformed into E.coli cell. The resulting colonies were checked by PCR re-amplification. The plasmid was purified from some positive colonies. The accuracy of purified plasmids were confirmed by both enzyme digestion and sequencing.Results: Enzyme analysis and sequencing demonstrated that pcDNA3.1-F390β had both correct construction and gene sequence which had an exact correlation with reported FSHβ gene in GenBank.Conclusion: This recombinant plasmid structurally is correct and is proper for transmitting into mammalian cell culture.

Keywords

• Follicle stimulating hormone
• β-subunit
• Cloning
• Mammalian cell line