Clone And Expression Of Thymosinb4 Gene And Biological Activity Of Expressive Product

Thymosinβ4 (Tβ4) is a small multifunctional peptide with a molecular weight of 4982 Da and an isoelectric point of 5.1, and exists in many tissues of lots of animals. It is comprised of 43 amino acid residues abundant in glutamic acid and lysine. The NH2 terminus of thymosinβ4 was found to be acetylated. The cDNA sequence and amino acid sequence have been demonstrated. The amino acid sequence has twoα- located on from the 4th to the 16th and from the 30th to the 40th amino residues. The structure is related to actin binding. Tβ4 with a variety of biological activities is present in a number of mammals such as humans, sheep, pigs, mice and other vertebrates such as birds, amphibians tissues. Tβ4 is an important mediator of biology reaction and can adjust and increase human immunity. It also promotes lymphocytic mature, endothelial cell migration and adhesion, angiogenesis,nerve regenesis, tumor metastasis, cardiac repair,accelerates wound healing and reduces inflammation.The purpose of this study is to express, isolate and purify Tβ4 expressed by genetic engineering and detects the biological activity of Tβ4 in order to gain a overexpressed, simple method of producing Tβ4.1. The sense and anti-sense chain of the Tβ4 coding sequence is dissected into 5 equal fragments and the 10 fragments are synthesized. The entire Tβ4 DNA is amplified applied the 10 fragments by two-step method successfully. Secondly, we design a double of specific primers of pTXB1 with a multi-cloning restriction sites - Nde I and Xho I according to Tβ4 gene sequence. The target fragment is connected to the pMD18-T vector and the connection is transform into the E.coli DH5αcompetent cell. The recombinant plasmid pMD18-T-Tβ4 is constructed successfully through positive clone screening, digestion identification, sequencing, gene comparison.2. The pMD18-T-Tβ4 and prokaryotic expression vector pTXB1 are digestion with Nde I and Xho I restriction enzyme. The pMD18-T-Tβ4 vector was cleaved into two fragments. The smaller fragment encoding human TB4 was recovered using a gel extraction kit and the pTXB1 vector was also digested and the larger fragment was recovered. Then the ligation product is transformed into E.coli DH5αcompetent cell. The expression vector, pTXB1–Tβ4, was constructed through a ligation reaction between the two fragments using the DNA ligation kit Ver2.1 after the positive clone screening, digestion identification, gene sequencing, and sequence analysis. The E. coli strain BL21 codon plus competent cell was transformed with pTXB1–TB4.The successfully transformed E. coli was induced by adding isopropylthio–b-D-galactoside (IPTG) to the transformed E. coli. After evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).3. Subjected to sonication and purified with Chitin-affinity column, the degree of expression was evaluated by tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Tricine-SDS–PAGE). The study demonstrated that the Tβ4 was expressed highly in E.coli BL21 codon plus and gained more pure Tβ4 with expression. After desalted using HiTrap Desalting column, dried by vacuum centrifuge enrichment, weighed and dissolved into PBS, Protein concentration was determined by using the BCA protein assay kit according to the manufacturer's instructions.The biological activity of recombinant and synthetic human Tβ4 was evaluated by its promotion of T lymphocyte proliferation and differentiation through MTT method and the most befitting concentration is 1.6ug/mL. The mice full-thickness cutaneous wound healing assay showed that recombinant Tβ4 exhibited the ability to promote wound healing.In the study, we gained the overexpressed E. coli strain BL21- pTXB1- Tβ4 and high pure Tβ4. In conclusion, our expression system is found to be a good approach for production of a biologically active and difficultly expressed peptide such as Tβ4 and laid the foundation for further study of other functions and the mechanism.