On The Cloning,Expression And Bioactivity Of Human Prothymosin α

Goldstein firstly isolated substance from calf thymus, named thymosin which can take on immunity regulation activity in 1966. After 30 years'clinic study, thymosin was applied to clinic as immunity regulator. Several descriptions of its effects arise daily pointing to an essential role in treating or alleviating original immunity lacking, virus or bacteria infection, oneself-immunity disease and cancer. At present, clinical thymosin is thymosin-five extracted from calf thymus, which includes more than 40 seeds, and its relative molecule mass is 1000-15000Da. Prothymosinα(ProTα) belongs to theα-Thymosin family which comprises different polypeptides widely distributed within animal tissues. As immunity modifier, ProTαcould affect several immunodeficiency illness, virus infection and tumor. Besides as nucleus protein, ProTαparticipate in cell multiplication and it could apply to knubbly forecasting, diagnosis and treatment, which is studied by scientists mainly.According to nucleotide acid sequence of reported prothymosinα, prothymosinαwas amplified by RT-PCR with a pair of primers (p1/p2) from the adult human peripheral blood , cloned into pMD18-T Vector, and translated into E. coil DH5α, and sequenced . Then filtered masculine recombined plasmid was identified. The gene fragment cut by EcoRI and BamHI was inserted into pGEX 6p-1 cut by the same enzyme, then was transformed into E.coil BL21. The result indicates that the homology is 99.70% between cloned ProTαgene and referenced amino acid ,and molecular weight of the expressed fusion production ,which is dissolved ,is 37KDa by SDS-PAGE electrophoresis after it was induced by IPTG at 37℃for 5 hours. Also the fusion protein was expressed in the form of soluble. The fusion protein was isolated and purified through GST Agarose Fast Flow. The highly purified protein was obtained, as immune antigen. Rabbits were immunized and polycolonal antibody to ProTαwas prepared. The titer of antibody was more than 1:51200 by ELISA detection. Western bolt analysis showed that the antibody can bind with ProTαspecifically. The prepared antibody against ProTαcan be used to detect the expression products of transgenic which expressed ProTαgene. By anti-tumor experiments, the results showed that the fusion protein can inhibit S180 tumor grown in mice, especially prolong mice'life-span at the over 1μg dosage. By antioxidation experiments, the results indicated that the fusion protein can improve thymus index and enhance mice antioxidation ability.In this research, ProTαwas successfully cloned and expressed in E.coli, and obtained polycolonal antibody against ProTα. By experiments, the results showed that the fusion protein can inhibit tumor, resist oxidation and prolong mice life-span compared with control group, and the difference is significantly. We obtained the fusion protein that takes on biological activity, which laid the foundation for study of its function.