Cloning,expression Of Tandem Repeat Thymosin α₁ Gene And The Purification Of The Peptide And Its Biological Function Research

Thymosin, which was firstly purified from noncellular extraction solution of bovine thymus by Goldstein in 1965, has been researched for more than 150 years. Thymosin alphal (Ta1), originally isolated from thymosin fraction 5(TF5), is a heat stable, highly acidic polypeptide composed of 28 amino acid residues. It's molecular weight is 3.1 O8Ku. Secreted by the epithetical cells of animal thymus, Ta1 is widely distributed. It is an immunoregulatory peptide with multiple biological activities such as modulate cell-mediated immunity, anti-aging and anti- virus activity and so on. Up to the present, Ta1 has been used in treatment of diseases such as primary and secondary immunodeficiency, aplastic anemia, acute and chronic viral hepatitis etc. What's more, there's no allergy and side effect. However, the molecular weight of Ta1 is too small to be obtained by using gene engineering method directly. Nowadays, the clinically used Ta1 is chemical synthesized or extracted from thymus. As a result, the application is severely restricted because of the expensive price or complex component. In order to prepare Ta1 in large scale and research the potential biological activity of the tandem repeat Tal, we synthesized the gene fragment of Ta1 according to the preferential codons of Escherichia co/i and obtained the tandem repeat gene of Ta1-Ta1 by amplifying the annealing product of synthesized fragment with PCR. Then Ta1 was cloned between EcoRI and PstI sites of the cloning vector-pGEM-3zf(-) and sequenced. The correct tandem repeat gene was then cloned into the fusion protein expression vector lhioHisA and the recombinant plasmid plhioHis- Ta1 was transformed into TOP 10 strain. The fusion protein Thioredoxin-Ta1? with a molecular weight of about 3 lkD, was expressed when induced at 370C by IPTG for about 4 hours. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the fusion protein band constituted more than 30% of the total bacterial protein and was soluble. The fusion protein can be isolated and highly purified by heat treatment at 80~C and anion exchange chromatography. After CNBr cleavage of the fusion protein, Ta1?was obtained by ion exchange chromatography. It has been proved that the tandem repeat a1 has similar biological activity to that of the artificial synthesized Ta1. It could increase the proliferative response of the mitogen ConA-stimulated spleen lymphocytes and upregulate the JL-2R expression level on I cell surface. b order to make full use of Ta1, it is essential to improve the investigation to industrial production scale by drawing assistance from high cell density fermentation. Pilot test of tube culture and flask shaking culture were done to get optimized culture conditions. Finally, we reached the goal of high cell density fermentation and high level expression of recombinant human Ta1 (E. co/i Top 10/pThioHisA-Ta1. The research was no doubt the useful bases for further study and large scale production of Ta1.