Expression And Purification Of Crassostrea Hongkongensis Recombinant HSP70 Protein, And Preparation Of Polyclonal Antibody Against The Protein As Well As Its Application

Heat shock protein 70s (HSP70s) play an important role in folding, assembling, transfering proteins and degradation, regulation polypeptides of living things, and contribute to the protection of cellular proteins by functioning as molecular chaperones. Moreover, they take part in immune activity against pathogens, sensitively response to various pollutants and so on. Therefore, HSP70s are key molecules of cells. The oyster, Crassostrea hongkongensis, is one of the most important economic mollusc and alongshore inhabits in South China Sea. It has become an important reared edible species, and a monitoring animal for early warning of marine environment pollution in shore. Because the oyster HSP70 is involved in controlling diseases of the oyster and monitoring polluted status of marine environment in which the oyster inhabited, it is necessary to get the HSP70 protein to produce a kind of monoclonal antibody against the oyster HSP70 protein for further research of the HSP70 function.The recombinant expression plasmid pQE30/HSP70 was constructed by means of linking the oyster HSP70 cDNA with prokaryotic expression vector pQE30. It was identified by endonuclease digestion and DNA sequencing of the recombinant plasmid. Then, the recombinant plasmid pQE30/HSP70 was transformed into E. coli M15 and induced to express the fusion protein with 0.2 mmol/L IPTG. The fusion protein was identified by SDS-PAGE and Western blot. We gained the recombinant HSP70 protein by means of.affinity chromatography and gel-filtration chromatography. The purity of the recombinant HSP70 protein is over 80%. We acquired anti-HSP70 polyclonal antibody by means of immuning New Zealand white rabbit. The result of the Enzyme-linked Immunosorbent Assay (ELISA) showed the highest titer of anti-serum was 1:128 OOO.Using this polyclonal antibody detected muscles,mantles,digestive glands and muscles of C. hongkongensis injected with live V. alginolyticus. The resules showed the polyclonal antibody not only has reaction with HSP70, but also has obvious cross reaction with HSC70. It can detect two kinds of HSPs proteins in C. hongkongensis.