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

authors:

avatar Reza Nasr , avatar Mohammad Reza Akbari Eidgahi ORCID , * , avatar Azar sadat RezaPorJavaShak , avatar Ahmad Reza Bandegi , avatar Saeid ValiZadeh

Koomesh: Vol.14, issue 4; 382-388
published online: October 25, 2013
article type: Research Article
received: February 02, 2012
accepted: September 04, 2013

how to cite: Nasr R, Akbari Eidgahi M R, RezaPorJavaShak A S, Bandegi A R, ValiZadeh S. Construction of pcDNA3.1-FSHβ expression vector in order to transfer FSHβ gene into a mammalian cellline. koomesh. 2013;14(4):e152591. 

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.