The Original Expression Of The Prokaryotic Metallothionein Of Sinopotamon Henanense

Metallothioneins (MTs) are a unique class of ubiquitious proteins that are characterized by low molecular weight, high cysteine content, complete abssence of aromatic residues, and cooperative metal binding. The functions of MT include protection against heavy metal toxicity, oxidant demage, radiation and association with cell proliferation, apoptosis, tumorigenesis and chemotherapy resistance. To further study of the bidogical function and develope the product of MT proteins, many scientists extracted them from animals livers. But this methed is of high production cost, time consuming and demanding. So, Sinopotamon henanense were selected as the experimental animals. Using recombinant DNA technology, the protein in vitro prokaryotic expression system was established to explore the expression of different expression vectors Sinopotamon metallothionein. This study has laid foundation for making metallothionein recombinant polyclonal antibody and for studying MT at the protein level of tissue distribution and cellular location.The first, this experiment was based on the cDNA of the metallothione in from Sinopotamon henanense. The cDNA of MT is stablely preserved in the T vector. The experiment used the T vector as carrier to introduce the MT gene into E.coli DH-5a to gain a large number of fragmentsof the T vector. The plasmid pGEM-T-MT from E.coli DH-5a was used as the template. The target fragment of MT gene was amplified using PCR, digested with restriction nucleases, purified by gel electrophoresis, and then bound to the vector pGEM-T for amplification.The amplified fragment of the MT gene was subcloned into the prokaryotic non-fusionexpression vector pQE31 and then transferred to E.coli BL21. It showed that the size of the PCR products met our expectation. After, this experiment was based on the cDNA of the metallothione from the Sinopotamon henanense. The cDNA of MT was stablely preserved in the pQE31 vector. The experiment introduced the pQE31-MT vector into E.coli DH-5a to gain a large number of fragmentsof the pQE31-MT. The plasmid pQE31-MT from E.coli DH-5a was used as the template. The target fragment of MT gene was amplified using PCR, digested with restriction nucleases, purified by gel electrophoresis, and then bound to the vector pGEM-MT for amplification. The amplified fragment of the MT gene was subcloned into the prokaryotic expression vector pGEX-6p-1 and then transferred to E.coli BL21. It showed:The size of the PCR products met our expectation, the expression condition was studied using the concentration of IPTG, the induction time of IPTG and metallic ion concentration. A fusion protein with molecular weight about 35 kD was obtained.