Logo-ipp
Submitted: 25 Mar 2024
Accepted: 02 Jul 2024
ePublished: 31 Aug 2024
EndNote EndNote

(Enw Format - Win & Mac)

BibTeX BibTeX

(Bib Format - Win & Mac)

Bookends Bookends

(Ris Format - Mac only)

EasyBib EasyBib

(Ris Format - Win & Mac)

Medlars Medlars

(Txt Format - Win & Mac)

Mendeley Web Mendeley Web
Mendeley Mendeley

(Ris Format - Win & Mac)

Papers Papers

(Ris Format - Win & Mac)

ProCite ProCite

(Ris Format - Win & Mac)

Reference Manager Reference Manager

(Ris Format - Win only)

Refworks Refworks

(Refworks Format - Win & Mac)

Zotero Zotero

(Ris Format - Firefox Plugin)

Immunopathol Persa. 2025;11(1): e40643.
doi: 10.34172/ipp.2025.40643
  Abstract View: 132
  PDF Download: 82

Original

Cloning and expression of UbiA human gene using innovative methodologies for recombinant protein production in PUAST vector

Maytham Abdulkahdim Dragh 1* ORCID logo, Zainab Sabeeh Al-Allak 2 ORCID logo, Zainab Zamil Gataa Allami 1 ORCID logo

1 Department of Biology, College of Science, University of Misan, Amarah, Maysan, Iraq
2 College of Dentistry, University of Misan, Amarah, Maysan, Iraq
*Corresponding Author: Maytham Abdulkahdim Dragh, Email: maithamdragh@uomisan.edu.iq

Abstract

Introduction: A systematic library of Gal4/UAS-regulated transgenes has proven to be a powerful genetic system for identifying genes and defining pathways in development. This system offers valuable insights that highlight the evolutionary conservation between animals and humans.

Objectives: The objective of this study was to clone, express, and characterize the UbiA gene. The research presents a highly effective method for cloning genes, using the UbiA-pcDNA3 gene as a model for mammalian cloning. These genes were then integrated into the PUAST vector of Drosophila, an expression vector and eukaryotic cell system commonly used for producing recombinant proteins.

Materials and Methods: UbiA was isolated from human cells, and complementary DNA was synthesized. An oligonucleotide primer pair was designed based on the UbiA gene sequence, incorporating XhoI and Xbal restriction sites at the 5´ end of the forward and reverse primers, respectively. The UbiA gene was then amplified by PCR, cloned into the pcDNA3 plasmid, and the resulting recombinant plasmid was sequenced. Subsequently, the gene was sub cloned into the PUAST vector and expressed in S2 cells as a eukaryotic cell system. Protein determination and verification were conducted through western blotting techniques.

Results: Confirmation of UbiA gene cloning into the PUAST vector was achieved through colony-PCR and digestion by enzymes. Cloning and sub cloning techniques validated by enzymatic digestion, along with gene sequencing. The identity between cloned UbiA gene and the identical gene exhibited 99%. We revealed a singular band purified protein through western blotting with 60 kDa size.

Conclusion: More protein synthesis of the UbiA gene can be achieved by using the eukaryotic expression system provided by the PUAST vector. This technique has been proven to be a suitable platform and can be instrumental in various applications such as therapeutics, pharmacology, and vaccine development.

Keywords: UbiA, cloning, PUAST, pcDNA3, GAL4/UAS

Citation: Abdulkahdim Dragh M, Sabeeh Al-Allak Z, Zamil Gataa Allami Z. Cloning and expression of UbiA human gene using innovative methodologies for recombinant protein production in PUAST vector. Immunopathol Persa. 2025;11(1):e40643. DOI:10.34172/ipp.2025.40643.
First Name
 
Last Name
 
Email Address
 
Comments
 
Security code


Abstract View: 133

Your browser does not support the canvas element.


PDF Download: 82

Your browser does not support the canvas element.