Construction And Functional Evaluation Of K31-PGLa Hydrogel For Wound Healing Against Drug-resistant Bacteria Infection

Due to exposure to air during the healing process of skin wounds,It often face the risk of bacterial infection.With the gradual emergence of drug resistance in the abuse of antibiotics,the treatment of skin tissue damage caused by drug-resistant bacteria has become a clinical problem.Antibiotics are the main method to treat bacterial infections,but the development of new antibiotics has the characteristics of long cycle and high cost,and once resistant bacteria is applied to the clinic,human still face the problem of drug resistance.Therefore,based on the drug resistance mechanism of bacteria,a method to effectively inhibit drug resistance is proposed to improve the antibacterial effect of traditional antibiotics,and the combination of biological materials to promote wound repair will help the wound repair treatment of drug-resistant bacteria infection.In this thesis,the antimicrobial peptide PGla can inhibit the synthesis of efflux pump by inhibiting the expression of related genes(acr A,acr B,tol C,)that inhibit the synthesis of efflux pump,and improve the sensitivity of bacteria to antibiotics;and keratin promotes cell proliferation and migration To accelerate the function of wound repair,the keratin K31-PGla fusion protein was designed and expressed,and a photopolymerized hydrogel was formed by grafting PEGDA.Laboratory evolution was used to obtain drug-resistant bacteria,and the inhibitory effect of K31-PGla fusion protein on different drug-resistant bacteria was investigated.Then,the in vivo inhibition of K31-PGla hydrogel was evaluated by constructing a mouse model of full-thickness skin defect infection by drug-resistant bacteria.Bacteria and skin repair effect.The main research content and results of this article:(1)The K31-PGLa fusion protein was designed and an expression plasmid was constructed for prokaryotic expression in E.coli.After purification,the fusion protein was subjected to SDS-PAGE,FT-IR,CD,UV-vis and other physical and chemical characterizations.The results of SDS-PAGE showed that the protein band was single,and the molecular weight was consistent with the theoretical value(50.87k Da);the FT-IR spectrum showed that the characteristic absorption peak of K31-PGLa fusion protein was similar to K31,and most of them were peptide bond characteristic peaks;the CD spectrum results proved that K31-The secondary structure of the PGLa fusion protein and K31 is mainlyαhelix,and theαhelix of K31-PGLa is higher than that of K31;and the UV-Vis spectrum shows that the maximum absorption peak of K31-PGLa is around 258nm,where it is human hair keratin.The maximum absorption peak.In summary,the experimental results proved that the K31-PGLa fusion protein was successfully expressed,and its structure and properties were similar to K31.(2)E.coli and S.aur were used as experimental subjects to investigate the antibacterial effect of K31-PGLa fusion protein.The results show that K31-PGLa has a certain antibacterial effect.When its concentration is 256μg/m L,the antibacterial rates against E.coli and S.aur are 38.33%and 36.79%,respectively,but they are weaker than the positive drugs ampicillin and ampicillin.Ciprofloxacin.Furthermore,the laboratory has evolved to obtain drug-resistant bacteria,and experiments have found that the antibacterial effect of the combined use of K31-PGLa and antibiotics is significantly better than that of a single drug or protein;taking AMP-resistant E.coli as an example,the antibacterial rate of 200μg/m L AMP is about When combined with K31-PGLa,the bacteriostatic rate was about 89.53%.In addition,it was found that the expression of genes(acr A,acr B,tol C)related to efflux pump synthesis in drug-resistant bacteria treated with K31-PGLa was down-regulated.It shows that the antibacterial mechanism of K31-PGLa mainly reduces the pumping of drugs by inhibiting the synthesis of efflux pumps.(3)K31-PGLa/PEGD photopolymerized hydrogel was synthesized by the reaction between the sulfhydryl group of K31-PGLa and the carbon-carbon double bond of PEGDA.A mouse full-thickness skin injury model infected by drug-resistant bacteria was established to investigate the repair effect of K31-PGLa hydrogel.The total number of white blood cells and the content of neutrophils in BALB/c mice are about 6～9×10~9/L,which is 4 to 5 times higher than that of normal mice,indicating that the infection model has been established successfully.The wound healing rate of the K31-PGLa hydrogel group reached 79.86%at 14 days,which was significantly higher than that of the blank group(55.98%);and H&E staining of tissue sections showed that the K31-PGLa hydrogel group had a thinner epidermal layer and more Skin appendages.In addition,compared with the blank group,the levels of white blood cells and neutrophils in the K31-PGLa hydrogel group increased significantly on day 3 and decreased significantly after 7days,proving that K31-PGLa can stimulate the immune system in the early stage of treatment Synergistic antibacterial effect.The biocompatibility was investigated by subcutaneous implantation of K31-PGLa in SD rats.Experiments have found that it can be completely degraded 21 days after implantation,and there is no inflammation such as redness and swelling on the skin surface.The ELISA test did not find a significant increase in IL-1β,IL-6,and TNF-α,indicating that K31-PGLa has good biocompatibility.In summary,this project is based on the major need for clinical treatment of skin tissue damage caused by drug-resistant bacteria.The K31-PGLa fusion protein is designed to combine with commonly used antibiotics to achieve a new strategy of antibacterial-repair synergistic treatment.The implementation of this paper can provide a new strategy for the clinical treatment of wounds infected by drug-resistant bacteria.The design and construction of the keratin K31-PGla fusion protein can provide guidance for the design and development of new antibacterial materials for keratin.