The Preparation Of Dual-Display Phage Nanomaterials And Their Applications On The Diagnosis And Therapy Of Candida Albicans

Filamentous bacteriophages are one kind of viruses with long and filament structure,which can produce progenies without lysing the host cells.The filamentous phage contains a single-strand circle DNA molecule that can encode 5 coat proteins which can be displayed on the surface of phage.This integrates the genotype with phenotype and makes it possible to display foreign peptide or protein on the surface of phage,which named “phage display”.Based on this technology,a variety of phage labraries were constructed.Through screening or biopanning,some new peptides binding with different proteins,cells or materials can be selected,which provide new research methods and tools for modern medical science.As a nanoscale biomaterial,the filamentous phage has great prospect in diagnosis and therapy of many diseases.There are many advantages of phages: First,simple preparation,low cost and no virulence are all good characteristics of filamentous phage.Seconed,the filamentous phage is thermally and chemically robust and can assembly inorganic nanoparticles to form new bio-nano-materials.The last but the most important is that the filamentous phage could naturely produce large amounts of progenies through E.coli without intensive fabrication.Candida albicans(C.albicans)is a normal commensal fungi existing in mucosal surface of oral cavity,respiratory tract,intestinal and vagina,and produce no damage to the host under normal conditions.However,once the human body balance is destroyed,particularly in the patients of acquired immune deficiency syndrome(AIDs)and organ transplant,C.albicans will invade the blood,diffuse into the tissues/organs and cause systemic infection which leading to death.C.albicans infection has been the fourth cause of blood infection in USA.Meanwhile,according to previous studies,C.albicans infection is also an important reason of death in cancer patients.So in order to improve patient survival rates,early starge diagnosis and early treatment would be an efficient way.At present,as a gold standard in the diagnosis of C.albicans infection,blood culture needs long culture time and is hard to achieving early diagnosis.So an advanced detection system using for early diagnosis is necessary for monitoring therapeutic response and improving the efficacy of the treatment.The f88-4 phage vector using for p? display and fUSE55 phage vector using for p? display were integrated into a new dual-display phage vector f388-2,which can display foreign peptides on both p? and p? simultaneously.Based on this new dual-display vector,a kind of new magnetic nano fiber was prepared and applied for the detection of C.albicans infection.On the one hand,VKYTS peptide,one epitope of Sap2 antigen,was displayed on the surface of p? and used to bind with anti-Sap2-IgG.On the other hand,magnetic nanoparticles(MNPs)-binding peptide,PK peptide,was selected by biopanning and displayed on p?.So the MNPs can assembly along filamentous phage and form nano fiber which endowing the phage particles with magnetic characteristic.This magnetic nano fiber could not only bind with anti-Sap2-IgG in serum but also enrich the bound anti-Sap2-IgG by external magnetic field.The concentrated anti-Sap2-IgG was detected using new prepared dual-display magnetic nano fiber and Sap2 antigen,respectively.The results showed that the detection limit of nano fiber-based method is approximately 1.1 pg/ml,about 2 orders of magnitude lower than the traditional antigen-based method.The nano fiber-based detection system could detect anti-Sap2-IgG at low concentration,improve the detection sensitivity and achieve the early stage diagnosis of C.albicans infection.Then,the anti-Sap2-IgG in cancer patients was also detected using nano fiber-based detection system and indicated that the detection rate was increased 10.81% and its specificity reached to 97.9%,compared to the traditional detection method using Sap2 antigen.At the same time,phage can also be applied to the treatment of C.albicans.With the enhancement of antibiotic resistance,the exploitation of new antimicrobial drug is attracting more and more attention and favor.Antimicrobial peptides(AMPs),as a new nature antimicrobial drug,can interact with membrane or penetrate into the cell to cause cell death by changing membrane peneabilization or interfering cell life activities.However,due to expensive price and no specificity,AMPs are not suitable for extensive use.So a new dual-display phage which can not only specifically bind with C.albicans spores but also interact with C.albicans was prepared.Firstly,C.albicans-binding peptide(LL)was selected through biopanning and displayed on p?,making the phage can specific recognize and bing with C.albicans.Seconedly,the antimicrobial peptide,Tat and Bac peptide,was displayed on p?,together with LL peptide,to construct a dual-display phage,which using for the therapy of C.albicans.The results showed that the dual-display phage can improve the antimicrobial efficiency of AMPs.What's more,simple preparation,low cost,and high specificity make it possible to be widely used,which provide a new avenue to the exploitation of new antimicrobial drug.