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Cloning and co-expression 3 subunits of human gonadotropins ( FSH, LH)

Tavasoli, Afsaneh and Gheibi, Nematollah and Kazemi, Bahram and Piri, Hossein and Bandehpour, Mojgan (2016) Cloning and co-expression 3 subunits of human gonadotropins ( FSH, LH). Masters thesis, Qazvin University of Medical Sciences and Health Service, Qazvin, Iran.

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Abstract

Background: Human gonadotropins including LH, FSH, and HCG those secreted from ‎anterior pituitary which has an important role in the reproductive system, and belong to glycoprotein hormone families with heterodimeric ‎structure. Disruption in the endocrine secretion of these hormones, put an ‎adverse effect on reproductive glands and sometimes lead to infertility‎. On trends related to infertility treatment, such as assistant reproductive technology administration ‎external gonadotropins consider as a basic principle of ‎treatment. Different types of gonadotropins in which derived from urea ‎‎(HMG or HP-HMG) or recombinant forms ‎are commercially available. In most cases many infertility treatment ‎centers administered, HMG or HP-HMG because contains a‏ ‏‎ 1:1 ratio of LH ‎and FSH and synergistic effects of these hormones induce appropriate response. ‎ Material and methods: The aim of this study is cloning and coexpression three subunits of human gonadotropins. In this study genes encoding β subunits of each LH and FSH hormones ‎as well as gene encoding common α subunit between two desire hormones were amplified by ‎PCR reaction and these fragments were joined together by SOEing PCR techniques. SOEing ‎PCR product was primary cloned into cloning pTG19 vector by TA cloning techniques. Afterward this product was subcloned in pEGFP-N1expression shuttle vector.‎ In order to expression, this construct transfected to the CHO cells. Gene expression evaluated in both cellular and molecular levels. Finally, Ni-NTA affinity chromatography was performed for purification of recombinant protein and confirmed by western blotting. Result: The cloning accuracy of three subunits of human gonadotropins in pEGFP-N1 expression shuttle vector confirmed by PCR, enzymatic digestion and sequencing.‎ The successfully simultaneouse expression of three subunits of human gonadotropins in CHO transfected cells was approved by SDS-PAGE and western blotting analysis. Purification of the heterotrimeric protein was done by Ni-NTA affinity chromatography and accuracy of this protein approved by western blotting. Conclusion: Recombinant heterotrymeric protein of simultaneous expression of three subunits of human gonadotropins approved by invitro analysis and this protein produce in lab scale.

Item Type: Thesis (Masters)
Subjects: R Medicine > RP Paramedical > RP105 Biotechnology
Divisions: University Thesis > Faculty of Paramedicals > Emergency
Depositing User: Paramedicals School Students
Date Deposited: 22 Nov 2017 05:21
Last Modified: 22 Nov 2017 05:21
URI: http://eprints.qums.ac.ir/id/eprint/6967

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