Preparation And Studying The Activity Of The Elastin-like-basic Fibroblast Growth Factor Fusion Protein

Basic fibroblast growth factor (bFGF) plays a key role in the process of wound healing and tissue repair. Recombinant bFGF has been used in the clinical treatment of various trauma including bone injury, cartilage damage or skin vulnus. However, bFGF need a long-time to interact with its receptor to exert its function, during which bFGF is prone to be degraded by proteases and the time of drug action is reduced. For the clinical application of bFGF, the pre-ssing problem is to develop the stability of bFGF to prolong the action time.Elastin-like peptide (ELPs) is genetically derived from the sequence bio-inspired by which found in natural elastin. The most striking feature of ELPs is that of a thermally responsive reversible phase transition which has been studied extensively in the field of drug delivery with a promising expectation. The fusion protein of ELPs and biological factors keeps the self-assembly and the phase transition characteristics of ELPs as well as the bioactivity of the biological factors. Therefore, it is assumed that the fusion protein of ELPs and bFGF may exhibit the properties of bFGF and ELPs.Objective:The aim of this study was to construct the expression vector pET28a-ELP50 and pET28a-ELP50-bFGF in which the ELP50-coding sequence was composed of the repeat unit eNcoding (VPGVG)10S. These plasmids were transformed into E.coli BL21 (DE3) competent cells and induced to overexpress the recombinant protein ELP50 and fusion protein ELP50-bFGF. The bioactivity of the purified recombinant proteins were analysed through the proliferation and migration experiments of mouse fibroblast cells and the skin defect repair of KunMing mice models, by which it was assessed whether it was practicable to fuse bFGF to the C-terminus of ELP50 to maintain or improve the stability of bFGF.Methods: ? Recursive connection technology (RDL) was used to extend the gene eNcoding (VPGVG)10S to obtain the gene coding for [(VPGVG)10S]5 cloned invector pMD19-T-Simple. At the same time, the coding sequence of bFGF was synthesized and cloned into the vector pMD19-T-Simple to construct pMD19-T-Simple-bFGF. After the cloning vector pMD19-T-Simple-bFGF was digested by BamHI and XhoI and 1% agarose gel electrophoresis, the coding sequence of bFGF gene was recovered and ligated with the linear pMD19-T-Simple-ELP50 treated by BamHI and XhoI. The recombinant vector pMD19-T-Simple-ELP50-bFGF was detected by Ncol and XhoI treatment. The coding gene of ELP50 and ELP50-bFGF were cleaved out and recovered from pMD19-T-Simple-ELP50 and pMD19-T-Simple-ELP50-bFGF after digestion with Ncol and XhoI, which then were recombined with linear pET28a (+) to construct the expression vector pET28a-ELP50-bFGF and pET28a-ELP50; ?The titer of antiserum was prepared from the rabbits immunized with purified ELP50 and detected by ELISA; ? Preparation of recombinant ELP50 and ELP50-bFGF was conducted in fermentation scale and the target proteins were purified by cation exchange chromatography, anion exchange chromatography, reversed phase chromatography and gel chromatography. The purity of ELP50 was more than 95% and that of ELP50-bFGF was approximate 90%; ? The silk fibroin membrane was prepared by soaking in the recombinant ELP50 and ELP50-bFGF solution respectively, and then several characteristics was analysed using fourier infrared spectrum and universal material tensile machine; ? The proliferation and migration experiments of mouse fibroblast cell line L929 and the skin defect repair of KunMing mice models were employed to evaluate the biological activity of the purified ELP50 and ELP50-bFGF.Results: ? The coding gene of ELP50 and the ELP50-bFGF were properly inserted into the site between the NcoI and XhoI recognition sequence of the prokaryotic expression vector pET28a (+) respectively; ? The results of SDS-PAGE showed that the recombinant ELP50 and ELP50-bFGF were overexpressed in E.coli BL21 (DE3), which was confirmed by western blotting using ELP50 antiserum; ? After cation exchange chromatography, anion exchange chromatography, reversed phase chromatography and gel chromatography, the ELP50 (purity> 95%) and ELP50-bFGF (purity> 90%) were obtained respectively; ?The proliferation of mouse fibroblast cell line L929 showed that the group coated with ELP50 could promote L929 cell attachment, spreading and migration, and that coated with ELP50-bFGF could promote cell proliferation and migration. There was no significant difference bewteen ELP50 and ELP50-bFGF; ? From 4 d to 20 d, the two groups of the silk fibroin membrane coated with ELP-50 and ELP50-bFGF remarkably enhance the skin repair to silk fibroin group and control.Conclusion: ? The prokaryotic expression vector pET28a-ELP50 and pET28a-ELP50-bFGF were successfully constructed; ? The purification methods of the recombinant ELP50 and ELP50-bFGF were established; ? The recombinant ELP50 showed positive effects on the cell proliferation and migration mouse fibroblast cell line L929, as well as the skin defect repair assay.