| Tβ4(thymosin β4) is an important G-actin sequestening factor in eukaryotic cells, which has multifariously physiological characteristics and cell functions to promote cell migration, angiogenesis, cell survival and regulate cell cytokines, chemotactic factor, specific protease, gene expression and so on.Tβ4 is often used to cure dermatosis, corneal injury, myocardial damage, central nervous system disease in clinic. Currently, the Tβ4 protein was basically obtained from two ways, which are extracted from thymus tissue of calf and synthesized by chemic method. The cost of Tβ4 protein is very high due to limitation of thymus tissue and lower content of Tβ4 protein via extraction approach, and this way also has risk due to zoonosis. Similarly, synthesized Tβ4 protein also has disadvantage in production costs because of complicatedly synthetic process. Therefore, The limitation of the Tβ4 protein application in clinic is due to the reasons above mentioned. Now, with developing of genetic engineering methods and ripeness of technique, it is possible to produce Tβ4 protein via genetic engineering approach, it is becoming a new way.Plants are highly efficient and stable expression systems to express foreign protein. It has several advantages in producing therapeutic proteins, such as high safety and low-cost, easy to carry out large-scale production. In this study, the 4×Tβ4 protein was expressed in the transgenic tobacco, as well as was also expressed in Escherichia coli(the purpose is providing a reference and positive protein for plant expression system). And results obtained were as follows:(1) A 167 bp gene encoding Tβ4 was designed and synthesized according to tobacco codon usage bias, and an 4×Tβ4 concatemer (four copies of a DNA sequence arranged end-to-end in tandem) was created via genetic enaineerina approach. in order to purify target protein, a DNA sequence which is encoding 6×His was introduced at 5 end of the 4×Tβ4, and a fused gene of the 6×his-4×Tβ4 was created.(2) A prokaryotic expression vector plasmid of the pET28a-6×his-4×Tβ4 was constructed, and subsequently transformed into E.coli (strain BL21) via thermal stimulation method. The 4×Tβ4 protein was successfully expressed by IPTG induction in E.coli, and the induction parameters are including temperature, time and IPTG concentration were preliminary optimized, and 4×Tβ4 protein was obtained by Ni-NTA affinity technique.(3) The plant expression plasmid of the 35S::6×His-4×Tβ4(backbone pCAMBIA 2300) was constructed, and then the 35S::6×His-4×Tβ4 was transformed into EHA105 using freeze-thaw method.(4) The explants which are from discs of tobacco leaves(Bairihong) were transformed by EHA105 with 35S::6×His-4×Tβ4, and 127 plants ware obtained through kanamycin(75 mg/L) screen, and 8 plants of which have been found that the 4×Tβ4 was integrated into genome by PCR and Southern blotting.(5) The results which are from Western blotting and ELISA showed that the 4×Tβ4 protein was successfully expressed in leaves of the transgenic tobacco.(6) It was confirmed that the tobacco-derived 4×Tβ4 protein has function to promote cell proliferation in vitro by the MTT assay. The percentage of proliferation reaches 28.59±4.97%, and significantly higher than E.coli-derived 4×Tβ4(18.13±3.65%)。(7) The results of healing wound demonstrated that tobacco-derived 4×Tβ4 protein can promote wound healing and vessel increasing in vivo. The results showed that reepithelialization is increasing with days of the applied Tβ4, and has a trend that reepithelialization rate and the numbers of blood vessels in wound are higher in tobacco-derived 4×Tβ4 than that in both E.coli-deriwed 4×Tβ4 and standard Tβ4. The results suggested that tobacco-derived 4×Tβ4 has a function to promote reepithelialization of wound vessel increasing in vivo, and better than E.coli-derived 4×Tβ4. |
PDF Full Text Request