Cloning, Sequencing and Expression of the Gene encoding Heat Shock Protein 70 (HSP70) from Chickens (Gallus gallus)
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Date
2013
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Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana
Abstract
Heat shock proteins (HSPs) are molecular chaperones associated with the folding of proteins which are expressed at normal body temperature as well as under conditions of stress (e.g. thermal stress as in heat shock) and therefore are critical for both normal cell function and survival under stress. Amongst all the HSPs, the HSP70 is the most inducible protein during stress response and its expression acts as a potential indicator of animal adaptation to harsh environmental stress, which is one of the main concerns in the poultry industry. The present study was conducted to amplify, clone, sequence and characterize the intronless gene encoding heat shock protein 70 (HSP70) from chicken (Gallus gallus) The HSP70 gene contains 1905 bp ORF. PCR product was cloned in pGEM®-T Easy cloning vector and sequenced. Sequence analysis using Mega 4 and DNA STAR softwares showed a high sequence homology of the HSP70 gene across different species indicating that the gene is evolutionarily conserved. The amplicon was further cloned in prokaryotic expression vector pPRO ExHTa and expressed in DH5α strain of E. coli to get recombinant His-tagged protein ~70kDa which was detected by SDS PAGE. The recombinant HSP70 was purified by affinity chromatography using Ni-NTA column. The immuno-reactivity of the recombinant protein was checked by western blotting.