Molecular Cloning, Primary Sequence Characterization and Phylogentic Analysis of Cathepsin B cysteine from Sheep (Ovis aries) Ovarian

Abstract

Background: CathepsinB (CTSB) is a lysosomal cysteine protease which has been implicated in a variety of physiological and pathological processes. However, the sequence and structure characteristics of sheep CTSB gene are still unclear, which severely restricts the further study about genetic variation, molecular evolution.Methods: Therefore the coding sequence (CDS) of sheep CTSB gene from ovary was cloned by reverse transcription PCR (RT-PCR) amplification and regular sequencing technology. The characteristics of nucleotide and amino acid sequence of CTSB gene were analyzed through bioinformatics and the phylogenetic tree was constructed employing the Bayesian inference based on amino acid sequence of different species in this study.Result: A complete coding sequence of 1008 bp of CTSB was obtained from ovary, which encoded 335 amino acids. Functional prediction of CTSB protein suggested that containing five conserved sequence motif sites in which the site of amino acids between 158 and 205 was the most conserved region. Secondary structure analysis showed that á-helices were the major structure at the N-terminus, while random coils were the major structure at the C-terminus. Additionally, the motif II in the random coils, in which two active sites of His278 and Asn298 were located therein, was more conserved. The high similarity of CTSB amino acids alignment between sheep and other species recommended that CTSB was highly conservative in the evolutionary process.