Molecular Cloning and In-Silico Analysis of A WGS derived genomic contig of a putative Angiotensinogen from the Teleost Sebastes Schlegelii

Authors Affiliation(s)

  • 1Department of Zoology, University of Sri Jayawardanepura, Gangodawila, Nugegoda, SRI LANKA
  • 2Department Of Chemistry, University Of Colombo, Kumaratunga Munidasa Mawatha, Colombo 00300, SRI LANKA

Can J Biotech, Volume 1 Special Issue,  Page 16,  DOI: https://doi.org/10.24870/cjb.2017-a4

Presenting author: buddhimaperera93@gmail.com

Abstract

Angiotensinogen (AGT) is the major substrate in the Renin-Angiotensinogen system (RAS), the primary hormonal signaling cascade ascribed primarily towards body fluid and blood pressure regulation, with peripheral albeit salient pro-inflammatory immune roles [1]. A WGS derived genomic DNA contig sequence with a presumed angiotensinogen gene (3802 bp with a 1383 bp, 6-exon coding region) was acquired from Sebastes schlegelii (Rock Fish) and subjected to extensive computer-assisted sequence analysis. The polypeptide derived via sequence based prediction tools defined a length of 460 amino acids, with a molecular mass of 51.3 kDa. Furthermore, RFAgt revealed a signal peptide incorporating approximately 19-residues upstream the putative angiotensinogen I signature motif (20NRVYVHPFYL29), with the peptide cleavage site residing between 19AlaAsp20, indicating its secretory nature. RFAgt also demonstrated a Serpin domain (between residues 9-458) with conserved sequence motif (431LSINRPFFFSV441), implicating a sequence-specific non-inhibitory role [1]. Sequence homology and genetic distance based phylogenetic analysis (augmented by 1000-iteration bootstrap analysis) revealed that RFAgt is evolutionary proximate to the AGT’s of Oplegnathus fasciatus, Larimichthys crocea and Rhabdosargus sarba. Validation of the In-silico predicted ORF conducted via PCR amplification using sequence specific primers (F-5ATG CGG TCG CCT CTT CTA GC-3 and R-5- TTA CAG TGT AGG ATT GAT GAT CTT GCC-3), and subsequent visualization via Gel-electrophoresis revealed a concomitant band at 1383 bp. Consecutively, upon purification, an attempt was made to ligate the product into a pGEM®-T Easy vector (size 3015 bp). The experimental component will further expound on the Tissue-specific expression analysis with anticipated highest expression in the liver and a challenge (injury/infection) based expression study with a potential upregulation of RFAgt expression during physiological stress expected [1].

References

  1. Griendling, K., Murphy, T. and Alexander, R. (1993) Molecular biology of the renin-angiotensin system. Circulation 87: 1816-1828. Crossref