Construction And Application Of Coiled Coil Protein Based On Genetic Engineering

Hydrogels are physically or chemically cross-linked hydrophilic polymer chains with3D network structure.Self-healing and injectable hydrogels have become one of the hotspots of recent research.Especially,hydrogels based on peptide and protein,due to their excellent biocompatibility,adjustable mechanical properties and flexibility environmental responsiveness,have good prospects in biomedical applications.The main work of this paper is as follows:Approaches of genetic engineering were introduced to fabricate the plasmids and induce the expression of artificial protein A,AC₁₀,AC₁₀A,P,PC₁₀,PC₁₀P and PC₁₀A.The expressed proteins were confirmed by SDS-PAGE and mass spectrometry analysis.The microscopic morphology of the proteins was detected by transmission electron microscopy.The results showed that the proteins we constructed were able to form nanoparticles at low concentrations,but P-containing proteins were more likely to form stable and uniform nanoparticles.A fluorescence resonance energy transfer?FRET?system was designed,which consisted of 520 nm-emitting FITC donor and 580 nm-emitting RBITC acceptor.The two fluorescent dyes were covalently conjugated with proteins,and the products were PC₁₀P-FITC,PC₁₀-RBITC and AC₁₀-RBITC.Capillary electrophoresis was adoptedto analyze the self-assembly of peptides.The fluorescence energy resonance transfer efficiency of PC₁₀P-FITC and PC₁₀-RBITC was 0.44,R₀ was 6.12 nm,and the distance between proteins was 6.36 nm,demonstrating the two ploypeptides could self-assemble together.However,FRET was not detected between PC₁₀P-FITC and AC₁₀-RBITC,indicating PC₁₀P and AC₁₀ could not self-assemble through a coiled-coil structure.The PC₁₀A peptide and CdSe@ZnS quantum dot was used to prepare three hybrid gels with different fluorescence emission wavelengths.Scanning electron microscopy results showed that the hybrid hydrogels could be connected to each other by coiled coil to form a stable three-dimensional network structure;the mechanical properties of the hybrid hydrogels were tested by a rheometer.The storage modulus G'was maintained at 1000 Pa and the loss modulus G''was 120 Pa.Compared with protein hydrogels,the storage modulus G'of hydrogels doped quantum dot increased,and their shear thinning and self-healing properties remained unchanged.Finally,we verified the self-healing properties of the hydrogel.The results showed that when the three-segment monochromatic hydrogel was put close,it could be connected as a whole to form a multicolor hybrid quantum dot hydrogel.