Construction And Expression In Escherichia Coli Of HEGF With High Permeability And Subcutaneous Targeted Release

Epidermal growth factor(EGF)is a single-chain small polypeptide that has 53 amino acids and efficiently promote cell proliferation such as epidermis cell,fibroblast,keratinocyte and many other cells.As a new cosmetic agent and drug,it can increase the amount of new skin cells and endogenous growth factors to repair and regulate skin.However,transdermal efficiency of EGF is extremely low,which limit the practical application.Cell-penetrating peptides have tremendous potential on the intracellular delivery of a variety of exogenous substance.In order to improve penetration through the cell membrane and release under the skin efficiently,N terminal of EGF is introduced by pep-1 using genetic engineering technology and design a matrix metalloproteinase 2(MMP-2)recognition sequence inserted between EGF and pep-1 so that remove pep-1 by MMP-2 of high-efficiency expression in fibroblast.The specific research content as follows:Firstly,the gene of EGF and pep-1 was amplified from the plasmid of pGEX-6p-3-GP-EGF by PCR.Secondly,pep-1,coding DNA sequence of MMP-2 recognition site and hEGF was spliced together by overlapping PCR technology,then construct five fusion expression plasmids using different methods.Finally,the fusion protein was expressed in Escherichia coli.Under the comprehensive consideration,the most of suitable expression system for the protein high-expression was E.coli BL21-TrixB(DE3).The result of optimize conditions showed that the recombinant strain could yield the highest expression quantity when the culture was propagated(37℃,200 r/min)in medium(M9 medium containing 2%glycerin)to an optical density(OD600)of 1.0,then induced by 0.1 mM IPTG at 37℃ for 10 h.The degenerative protein was purified by Ni affinity chromatography and gain bioactivity by urea gradient dialysis after induction and ultrasonic decomposition.Recombinant proteins PME was successfully verified by western blot anlysis.It was examined using MTT assay,immunity fluorescence assay that recombinant protein has transmembrane ability to keratinocyte Hacat and promote the proliferation to fibroblast Balb/c 3T3.For further study,The Construction of 3D Skin Model using Transwell could absolutely simulate physiological barrier of realistic skin after verification result of rhEGF.So,the recombinant proteins PME does pass through the cell membrane of keratinocyte Hacat and effect on fibroblast Balb/c 3T3.In vitro,it turned out that PME was digested with matrix metalloproteinase 2 and the digested efficiency could increase as time goes on.Besides,the best protease digestion system was identified:17.1 μg PME was digested with 40 ng matrix metalloproteinase 2 at 37℃ for 1 h.In conclusion,hEGF with high permeability and subcutaneous targeted release has been expressed and optimized in Escherichia coli,which not only contribute to improve bioavailability of EGF applying to cosmetic and pharmaceutical,but also laid a certain foundation for enhancing the quality of related products.