Molecular and Immunological Characterization of Heat Shock Protein 70 (HSP70) Gene from Buffalo

Heat shock protein 70 (HSP70) is a predominant member of the HSP family of proteins, which play a variety of functions in the cells and are responsible for cytoprotection under stress conditions. The present study was characterized by HSP70 (orf) in buffalo (Buablus bubalis). Genomic DNA was isolated from lymphocytes and that was used for PCR amplification of HSP70 gene. Polymerase chain reaction product (1,926 bp) was cloned in pGEM-T easy vector and sequenced. Sequence analysis revealed 1,926 bp long open reading frame of HSP70 gene encoding 641 amino acids in buffalo. The amino acid sequence showed 98 % identity with Bos taurus, Bos indicus, Yak, Capra hircus and 90–95 % identity with Camelus dromedaries, Felis catus, Canis familiaris, Sus scrofa, and Homo sapiens. The expression of this gene was in prokaryotic expression vector pPRoExHTa producing a recombinant protein of ~70 kDa, which is detected by SDS-PAGE. The tagged protein purified by Ni-NTA affinity chromatography under denaturing conditions was confirmed by western blotting using Ni-NTA HRP conjugate and 4 chloro-1-naphthol as substrate. Recombinant HSP70 was injected in mice and antiserum contained polyclonal antibody, which was detected by western blot. The recombinant HSP70 protein obtained may be used for the development of an assay for detection of thermal stress.