Sub-cloning of alpha subunit of anterior pituitary glycoprotein hormone into a mammalian expression vector

authors:

avatar Mohammad Reza AkbariDidgahi , avatar reza nasr , * , avatar AliAkbar Shabani , avatar Mojtaba GhadamYari

Koomesh: Vol.13, issue 2; 143-150
published online: May 25, 2012
article type: Research Article
received: January 17, 2011
accepted: September 04, 2011

how to cite: AkbariDidgahi M R, nasr R, Shabani A, GhadamYari M. Sub-cloning of alpha subunit of anterior pituitary glycoprotein hormone into a mammalian expression vector. koomesh. 2012;13(2):e152501. 

Abstract

  Introduction: Anterior pituitary glycoprotein hormones include thyroid stimulating hormone, lutinizing hormone, follicule stimulating hormone, and gonadotropine hormone. Each of them contains alpha and beta subunits. The alpha subunit gene is the same in all of these hormones and contains 4 exones and 3 intrones. The beta subunit is responsible for specific function of each hormone. The aim of this study was to clone alpha chain cDNA of glycoprotein hormones in a proper vector for eukaryotic system.   Material and Methods : To clone cDNA, alpha subunit of glycoprotein hormones was amplified by using one pair primers and T.vector as template and cloned in Not I and Bam HI sites of pcDNA3.1 plasmid. The recombinant plasmid transformed to E.coli Top10F΄ cell and colonies that contain plasmid were selected by Colony PCR. The accuracy of extracted plasmid of these clones was approved by enzyme digestion and sequencing.   Results : Enzyme analysis showed that pcDNA3.1-F351 a had correct structure and sequencing confirmed by 100% homology of the gene with reported alpha gene in Gene Bank.   Conclusion : Because of its proper structure, this plasmid is able to transform to Eukaryotic system and translation.