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Iranian journal of biotechnology2015; 13(2); 10-17; doi: 10.15171/ijb.1004

Cloning and Expression of Iranian Turkmen-thoroughbred Horse Follicle Stimulating Hormone in Pichia pastoris.

Abstract: Follicle stimulating hormone (FSH) plays an essential role in reproductive physiology and follicular development. Objective: A new variant of the equine () gene was cloned, sequenced, and expressed in ) GS115 yeast expression system. Methods: The full-length cDNAs of the and chains were amplified by reverse transcription polymerase chain reaction (RT-PCR) using the total RNA isolated from an Iranian Turkmen-thoroughbred horse's anterior pituitary gland. The amplified chains were cloned into the pPIC9 vector and transferred into The secretion of recombined eFSH using expression system was confirmed by Western blotting and immunoprecipitation (IP) methods. Results: The DNA sequence of the chain accession number JX861871, predicted two putative differential nucleotide arrays, both of which are located in the 3'UTR. Western blotting showed a molecular mass of 13 and 18 kDa for and eFSHβ subunits, respectively. The expression of desired protein was confirmed by protein G immunoprecipitation kit. Conclusions: eFSH successfully expressed in . These findings lay a foundation to improve ovulation and embryo recovery rates as well as the efficiency of total embryo-transfer process in mares.
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Publication Date: 2015-06-01 PubMed ID: 28959285PubMed Central: PMC5435000DOI: 10.15171/ijb.1004Google Scholar: Lookup
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Summary

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The research article discusses a study in which a new variant of the equine follicle stimulating hormone (eFSH) was cloned, sequenced, and expressed using the Pichia pastoris GS115 yeast expression system. This work could potentially improve ovulation and embryo recovery rates in mares.

Objectives and Methods

  • The main objective of this research was to clone and sequence a new variant of the equine follicle stimulating hormone (eFSH) gene using the deoxyribonucleic acid (DNA) extracted from an Iranian Turkmen-thoroughbred horse’s anterior pituitary gland.
  • The researchers used reverse transcription polymerase chain reaction (RT-PCR), a common method in molecular biology, to amplify the full-length cDNAs of the FSH and LH chains.
  • These amplified chains were then cloned into a pPIC9 vector and subsequently transferred into Pichia pastoris, a species of yeast often used in genetic research because of its ability to grow on simple culture conditions.

Results

  • The DNA sequence of the FSH chain showed two potential differential nucleotide arrays, both of which were discovered in the 3’UTR, a region of messenger RNA (mRNA) that follows the coding region and can potentially influence gene expression and protein function.
  • A technique known as Western blotting revealed the molecular masses of the FSHα and FSHβ subunits, found to be 13 and 18 kilodaltons respectively, confirming the successful expression of these genes in Pichia pastoris.
  • The production of the eFSH protein was also confirmed using an immunoprecipitation (IP) method, specifically a protein G IP kit, which is typically used for the detection and analysis of protein complexes.

Conclusions

  • The eFSH was successfully expressed in Pichia pastoris, marking a potential advancement for equine reproductive research. This could provide a basis for the improvement of ovulation and embryo recovery rates in mares, as well as the overall efficiency of the embryo-transfer process.

Cite This Article

APA
Elyasi Gorji Z, Amiri-Yekta A, Gourabi H, Hassani S, Fatemi N, Zerehdaran S, Vakhshiteh F, Sanati MH. (2015). Cloning and Expression of Iranian Turkmen-thoroughbred Horse Follicle Stimulating Hormone in Pichia pastoris. Iran J Biotechnol, 13(2), 10-17. https://doi.org/10.15171/ijb.1004

Publication

Iranian journal of biotechnology
ISSN: 1728-3043
NlmUniqueID: 101276796
Country: Iran
Language: English
Volume: 13
Issue: 2
Pages: 10-17

Researcher Affiliations

Elyasi Gorji, Zahra
  • Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
  • Department of Animal Breeding, Genetics and Physiology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.
Amiri-Yekta, Amir
  • Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Gourabi, Hamid
  • Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Hassani, Saeed
  • Department of Animal Breeding, Genetics and Physiology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.
Fatemi, Nayeralsadat
  • Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Zerehdaran, Saeed
  • Department of Animal Breeding, Genetics and Physiology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.
Vakhshiteh, Faezeh
  • Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
Sanati, Mohammad Hossein
  • Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
  • Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.

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