The Secreted Expression And Purification Of Small Molecular Functional Metallothionein Of Fresh Water Crab (Sinopotamon Henanense) And Identification Of Its Function

Metallothioneins(MTs) are a group of ubiquitous low molecular mass,cysteine-rich intracellular metal-binding proteins. MTs are involved in trace metal homeostasis and metabolism,the detoxification of toxic metals,scavenging of free radicals. Recently,the relationship between MT and tumors has become a focus of research. MTs are known to play putative roles in cell proliferation,apoptosis,differentiation,drug resistance and prognosis.In the National Natural Science Foundation (No.30640051),our group not only studied the relationship of major organizations of Sinopotamon henanense, condition of MT expression and grand total of heavy metals but also studied the cloning of full-length sequence MT cDNA of metallothionein induced by cadmium in Sinopotamon henanense.On this basis, We are aiming to obtain soluble metallothionein (MT) of Sinopotamon henanense. Therefore, a recombinant phoA plasmid containing MT open reading frame fused with a 6his tag was constructed. The plasmid possesses a promoter and signal sequence of alkaline phosphatase gene of E.coli.The recombinant phoA-MT vector was transformed into E.coli BL21 (DE3) strain for expression subsequently. The fusion protein was induced at low concentration of phosphate and purified by Ni2+ affinity chromatography and analyzed by SDS-PAGE and western blot. Then we identificated its function by UV absorbance spectrometry, determination of hydroxyl radical scavenging. The results showed that the fusion protein was expressed with high purity in monomer and dimmer, with the molecular weight about 7.5 KD and 15 KD respectively.After the completion of this thesis,we achieved the expression of recombinant Sinopotamon henanense MT protein, laid the foundation for the preparation and characterization of monoclonal antibodies against metallothionein of freshwater crab (Sinopotamon henanense),and the further study of the biological function of MT and its large-scale applications in environmental monitoring and medicine, health products and cosmetics area.