Detection Of Early Protein E6 And Controlled Assembly Of Late Protein L1 Of Human Papillomavirus

Human papillomavirus(HPV)is a small,double-stranded DNA virus that infects human skin and mucosal basal layer squamous epithelium.HPV can cause a variety of cancers in women(cervix,vagina,vulva,anus and oropharynx)and men(oropharynx,anus and penis),and can induce many benign diseases such as genital warts and flat warts.Worldwide,there were about 500,000 cases of cervical cancer occur each year,which leads to more than 250,000 deaths.Cervical cancer is the second largest killer in women’s health.Therefore,through the identification to high-risk HPV,we can use immunization or other targeted therapy ways to prevent the occurrence of cancer.HPV oncoprotein E6 is essential for initiating HPV-associated malignancies and can be expressed consistently and retain transcriptional activity in transformed cells of cancer and precancerous lesions caused by HPV.The E6 protein assures replication of viral DNA by targeting many cell pathways,which is a major oncogenic protein in HPV-associated tumors.E6 and E7 are the only expression of HPV protein in cervical cancer cells and are often seen as signs of cervical cancer.The HPV L1 recombinant expression vector is expressed in vaccinia,baculovirus or yeast systems capable of forming virus-like particles(VLPs).VLPs are shells similar to native viruses formed by assembly of approximately 72 L1 pentamers.At present,VLPs are mainly used for vaccine research,but in gene therapy and drug delivery and other biomedical aspects,VLPs also showed great potential.The polymetallic oxygen cluster is a class of pre-transition element-oxygen anion clusters with a clear size and configurable charge,and also has the ability to produce organic-inorganic hybridization.These advantages,making the multi-metal clusters in the anti-cancer,anti-virus,anti-tumor and anti-bacterial,and so has great potential.In addition,some polymetallic oxygen clusters have special optical properties,so they can be used as a new type of probe for biomarkers and recognition.In this paper,we study the Eu-like polyoxometalates as a molecular probe to identify HPV early carcinogen E6 and virion-like double-stranded DNAs based on intermolecular interactions.We hope that this study can provide a new idea for early identification and treatment of HPV.In the first part of this study,we selected a polyoxometaline EuW10 containing nine negatively charged nuclei as a fluorescent probe to detect HPV early oncogene E6,using the sensitive fluorescence properties of Eu-containing polymetallic oxygen clusters.First,based on the previous work of this group,we selected a small peptide of amino acid-rich amino acid on HPV E6 protein and constructed a platform for the interaction of small peptide and EuW10,thus realizing the detection of E6 protein by EuW10.In the study of the interaction of E6 small peptides with EuW10,we found that E6 small peptides were self-assembled with EuW10 as a regular sphere,and E6 small peptides could enhance the fluorescence of EuW10 significantly.Then we use the time-resolved fluorescence,isothermal titration calorimetry to reveal the mechanism of the two,and found that the main driving force of the interaction between the two is electrostatic interaction.After confirming that there was a strong interaction between E6 small peptides and EuW10,we further detected E6 protein using EuW10.It was found that E6 protein could also be self-assembled with EuW10 as a sphere,and the fluorescence of EuW10 could be enhanced obviously.This shows that we use EuW10 to detect E6 protein method is feasible.Through this part of the study,we have a practical support for the application of polymetallic oxygen clusters as biological probes,which is of great significance for HPV-induced early detection of cervical cancer.In the second part of this study,we used HPV16 L1 pentamer to encapsulate double-stranded DNA into VLPs by in vitro self-assembly.By introducing a fluorescent small molecule probe with AIE effect-DSAI labeled target dsDNA,To achieve the target DNA fluorescence monitoring and characterization.In addition,we also characterize the particle size and composition of the assembled virions with a series of biological and chemical means,thus demonstrating that DSAI-dsDNA can indeed be encapsulated into assembled VLPs.The results of this study will be of vital importance for the further development of gene delivery studies with VLPs as carriers.In particular,the fluorescent small molecule probes we introduced have the ability to monitor target dsDNA in real time.The monitoring method is convenient,rapid and low cost,which is convenient for the further study of gene transfer with VLPs as carrier.