Design,Preparation And Property Analysis Of Elastin-Like Polypeptides

Elastin-like polypeptides(ELPs)are constructed by genetic engineering techniques.The peptide contains repeated Val-Pro-Gly-Xaa-Gly(VPGXG)n pentapeptide sequence.Xaa is called guest residue,which can be any amino acid other than Pro.When Xaa and the number of repetitions "n" are different,the ELPs are different.Similar to natural elastin,ELPs have good cytocompatibility and low immureactivity.Futher,ELPs can be degraded into natural amino acids in vivo.When the outside temperature is lower than the phase transition temperature(Tt),the ELPs are in a dissolved state.Whereas when the outside temperature is higher than Tt,the ELPs automatically aggregate and precipitate from the solution to form a precipitation.This property is called phase transition property and has been widely used in tissue engineering materials,targeted drug delivery and protein purification studies.In the preliminary work,our research group has constructed a variety of sequence-constituting ELPs,but their Tt is relatively high,which impairs their potential as a drug delivery carrier or tissue engineering material.Meanwhile,it is difficult to achieve inexpensive inverse transition cycling(ITC)purification.Therefore,the purpose of this project is to design,construct and screen ELPs with low Tt and good biocompatibility on the basis of previous studies,which may lay a foundation for the development of drug-targeted delivery carriers and tissue engineering material.Objective:To screen out ELPs with phase transition temperature close to body temperature and good biological activity.Methods:(1)Design ELPs with different amino acid sequences and peptide chain length according to literature reports;(2)Construct and screen out the prokaryotic expression vectors of ELPs(50-fold and 90-fold)using recursive directional ligation(RDL)technology;(3)Express different ELPs by E.coli expression system;(4)Analyze the in vitro activity of different ELPs by proliferation and migration experiment of mouse fibroblast cell line L929;(5)Analyze the in vivo injury repair activity of different ELPs by a trauma animal model.Results:(1)The results of double enzyme digestion showed that different coding sequences of ELP50s and ELP90s were successfully inserted into the mutated cloning vector pUC18 and subcloned into the expression vector pET28a(+)between Nco I and Xho I sites respectively;(2)SDS-PAGE analysis showed that 18 different ELPs could be soluble expressed and accumulated in E.coli BL21(DE3)cells under the induction of isopropyl p-D-thiogalactoside(IPTG);(3)The purity of recombinant ELPs is about 90%after inverse transition cycling(ITC)purification;(4)Proliferation and migration experiments of L929 cells showed that ELP?-50 and ELP?-50 can significantly stimulate the proliferation of L929 cells.EL?-50,ELP?-90,ELP4?50,ELP?-90,ELP?-50,ELP?-90,ELP?-50 and HLC can significantly promote the migration of L929 cells;(5)Mouse skin wound repair experiment showed that ELP?-50 and ELP?-90 accelerated the repair of mouse skin wounds,with a phase transition temperature of 40? and 35? at 1%concentration,respectively.Conclusion:Two kinds of ELPs(ELP?-50 and ELP?-90)with low phase transition temperature and good biological activity were obtained,which laid a foundation for the development of targeted drug delivery vehicles and tissue engineering material.