Construction Of 3D Immune Probe And Regulation Of The Activity Of Pyridoxine 5'-phosphate Oxidase

The worldwide prevalence of dangerous pathogens,is a serious threat to people's lives,to bring great losses to the economy and property.Although in recent years investment in disease diagnosis and prevention has been gradually increased,the diagnosis of many diseases is cumbersome and expensive,some diseases still lack of effective method in early diagnosis.In this study,the virus are selected as the research objects,we have constructed local detection method based on nanomaterials for the detection of virus.Sup35 prion protein from Saccharomyces cerevisiae,which can be self-assembled into highly ordered nanowire structure,is considered as a good natural nanomaterials.In addition,the nanowires have excellent characteristics,such as large surface area,good physical/chemical stability,and flexible to surface modification,making them a good candidate to accomplish rapid and highly sensitive detection for efficient and rapid detection.Acetylation,as an important post-translational modification of proteins,has been widely involved in physiological processes.Escherichia coli 5'-pyridoxine phosphate oxidase(PNPOx)could be acetylated,catalyze substrate into pyridoxal phosphate(PLP),PLP as a cofactor of 100 kinds of enzymes take part in many important vital movement and it is very essential to cell.Acetylation and deacetylation can regulate the activity of PNPOx.Firstly,three dimensional(3D)immune probe has been constructed based on protein nanowires to local detect virus.In this study,the composite was constructed by displaying the fusion protein(Sup35 prion fused with antigen protein)on the magnetic beads through,based on the biotin-streptavidin interactions and seed-induced self-assembly processes.The obtained immune complexes,namely 3D immune probe,enriches with a large number of antigens,become a new biosensor to improve the capture abilities toward the target molecules,and therefore significantly enhance the sensitivity of virus detection.Here,Sup35 was fused with HIV-1 p24,the green fluorescent protein(GFP)and BAP to form fusion proteins.The binding force of antigen and antibody displayed on the nanowire was measured by isothermal titration calorimetry(ITC).The growth of protein nanowires in situ on the magnetic beads has been investigated,and regulated the growth length of the final nanowires by controlling the assembly time and protein mass ratio.In order to explore the general adaptability of constructing 3D immune probe method,hepatitis C virus(HCV),Lassa virus(LASV),Zika virus(ZIKV)are selected as candidates.The antigen epitope proteins are HCV core protein,LASV nucleoprotein and ZIKV envelope protein,respectively.As a result,all of the fusion proteins have good solubility,and can assemble into nanowires.Secondly,reversible lysine acetylation regulates the activity of Escherichia coli pyridoxine 5'-phosphate oxidase(PNPOx)..Western bolt was used to test the level of acetylated protein and deacetylated protein,and to determine the catalytic activity of acetylated protein and deacetylated protein respectively.The results indicate that the deacetylation of acetylated protein can restore the catalytic activity of the enzyme,verifying acetylation modification is a reversible post-translational modification.