Construction Of The Chicken Thymosin B₄ Concatemer Fusional Vector And Prokaryotic Expression

Thymosin beta 4(Tβ4)was originally isolated from the bovine thymus gland, it consists of 43amino acid residues and is found in most other tissues of the body. Tβ4 is the major actin regulating molecule in most mammalian cells, it is neither a growth factor nor a cytokine,but, Tβ4 plays an important role in the regeneration, remodeling, and healing of injured or damaged tissues. In addition, the study also confirmed that Tβ4 involved in regulating epithelial formation, angiogenesis, antiinflammatory and other processes, and can promote healing, used to treat infections caused by endotoxin shock and multiple organ failure associated with, is important in medicine. Although Tβ4 is likely to be used widely, it cannot be expressed in E.coli directly because of its low molecular weight. Furthermore, Tβ4 used in the laboratory investigation and veterinary clinical trials now is mainly produced synthetically with a high cost, meanwhile, isolating Tβ4 from the mammalian tissue is a complex technics and with low yield, so the application of Tβ4 is restricted. Extracted thymosin peptide is a multimolecular mixtures, there is poor treatment, side effects and many other serious problems, so the application of Tβ4 is restricted.In the study, we synthesize Tβ4 gene using E.coli preferred codon and connect with PMD-18-T vector to construct PMD-18-Tβ4. Transform Tβ4 gene restriction sites by PCR, and connect PMD-18-Tβ4, construct chicken Tβ4 tandem gene cloned plasmid PMD-18-2Tβ4. After restriction enzyme digestioning, the recombinant plasmid was transformed into the prokaryotic expression vector PET-32a(+) to construct fusion protein expression vector PET-32a(+)-2Tβ4. After restriction enzyme digestioning, transform recombinantplasmid to E.coli BL21(DE3)and induced with IPTG.The expressed protein 2Tβ4 can be purified by nickel chromatography, and identified by HPLC and Western blot, and measured its activity by splenic lymphocyte proliferation. The aim is to use a more economical way of obtaining such peptides. Conclusion:1 Synthesize Tβ4 gene using E.coli preferred codon, combination of PCR technology, construct chicken Tβ4 tandem gene cloned plasmid PMD-18-2Tβ4, then, cloned it into fusion expression vector PET-32a(+).The result of identification by electrophoresis and gene sequencing shows that fusion protein expression vector PET-32a(+)-2Tβ4 was successfully constructed.2 Expression conditions of the PET-32a(+)-2Tβ4 was explorated by groping with IPTG. The optimum conditions for the expression is 37℃, 72 h, IPTG concentration is 0.5mmol/L.3 The results of SDS-PAGE shows that Tβ4 expressed product was soluble, almost ever per gram bacteria will receive 11.3mg protein.The purity of protein that was tested by HPLC is 95.56%. 4 Determined by MTT expressed Tβ4 effect on lymphocyte proliferation, the results show that compared the adding 8μM Tβ4 group with the control group not added Tβ4, lymphocytes were significantly increased yield, p <0.01. Show that the expression of Tβ4 can stimulate lymphocyte proliferation and differentiation.In conclusion, this study expressed chicken Tβ4 in vitro by genetic engineering, explore a low cost, efficient expression and simple purification method of the Tβ4 expression. The result of activity detection shows that Tβ4 tandem as similar biological activity as Natural Tβ4. But other biological activity would have further studies.