Study On The Interaction Between DNA-Protected Silver Nanoclusters And Human Papillomavirus-Associated Proteins

DNA-protected silver nanoclusters(AgNCs-dsDNA)usually consist of less than 20 silver atoms,and have the advantages of ultra-small size,low toxicity,and good biocompatibility.Therefore,AgNCs-dsDNA has received extensive attention in the fields of photochemistry,biological imaging,biological detection,and biosensing.Human papillomavirus(HPV)is a small,non-enveloped,encapsidated doublestranded DNA tumor virus that forms squamous epithelial papilloma;in papilloma,the virus can be replicated.More than 200 HPV genotypes have been sequenced and classified into alpha,beta,and gamma genus.The HPV type of the genus ? is mainly infected with mucosal or cutaneous epithelial cells,while the HPV type of other genus mainly infects skin cells.HPV types that infect mucosa can be classified into high-risk type(HR)and low-risk type(LR)according to their carcinogenic ability.HPV is the leading human oncogenic virus and is closely related to the incidence of approximately 4.5% of cancers worldwide.HPV-related carcinogenic problems are still quite severe in the coming decades,and humans urgently need some new treatments and precise detection methods to compete with them.Therefore,HPV related testing is crucial.The HPV L1 protein is the major capsid protein of HPV,and human papillomavirus particles contain two virally encoded proteins,L1 and L2,which are synthesized at a later stage of the viral infection cycle.The LI protein accounts for 90-95% of the virion protein content,which can cause specific neutralizing antibody responses,thereby protecting the vaccinated animals from subsequent infections,so the protein can be used as a primary vaccine target for vaccination.Currently,prophylactic vaccines on the market are based on VLPs formed by the major capsid proteins of HPV L1.Although they are free of DNA,they are similar in immunology and morphology to native virions.In high-risk HPV,E6 is a major class of oncogenic proteins.The E6 protein is about 100 to 150 amino acids in length and is a key factor in maintaining the malignant phenotype of HPV-positive cancer cells.Moreover,E6 and E7 are two oncogenic proteins encoded by HPV,which are directly involved in the process of inducing carcinogenesis.Therefore,the detection of HPV E6 protein is of great significance for the prevention and treatment of cancer.In the first part of this paper,we constructed two assembly systems using AgNCsdsDNA and HPV major capsid protein L1,namely AgNCs@VLPs and VLPs@AgNCs.We used CsCl gradient centrifugation,sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),dynamic light scattering(DLS)and transmission electron microscopy(TEM)to determine the morphology and particle size of the assembly.Among them,the co-assembly system of AgNCs@VLPs can induce the fluorescence enhancement of AgNCs-dsDNA,so it provides a new fluorescence method for monitoring the assembly and disassembly process of VLP in situ;in addition,the process can be distinguished pre-formation and post-maturing of VLP capsids during assembly.Therefore,this part of the study not only proposes an AILE phenomenon of silver nanoclusters,but also provides a novel fluorescent probe for monitoring the assembly/deassembly process of VLPs,which is of great significance for the improvement of HPV-related vaccines based on VLPs.In the second part of this paper,we used AgNCs-dsDNA to perform in vitro detection of HPV E6 protein.And by testing the detection method,we calculated that the detection limit of the method is 0.886 nM.Compared with other methods for detecting E6 protein in vitro,the method has high sensitivity(LOD = 0.886 nM)and simple operation.In addition,the detection method has better anti-interference and selectivity,and can detect E6 in serum samples.Therefore,through this part of the research we not only further broaden the biological application of AgNCs-dsDNA;and because of its high detection sensitivity,it will be of great significance for the early diagnosis of HPVrelated cancer.