Recognized And Regulation Assembled In Vitro Of Human Papillomavirus Capsid Protein By Molybdenum-Containing Polymetallic Oxygen Clusters

Polyoxometalates?POMs?are transition metal-oxygen nanoclusters with a special three-dimensional structure.They are mainly nano-sized metal oxides made from the V A and VI A metals?mainly V,Mo and W,Nb and Ta are rare?under the high oxidation state?usually d₀ or d₁?.Their sizes range from 0.8 nm to 6 nm.POMs have excellent water solubility,oxidative reducibility and thermal stability,which make them have many practical and potential applications in the fields of catalysis,molecular magnetism,life sciences,sustainable energy,electronics,sensors,and radionuclide capture.Molybdenum-containing polyoxometalates are high-valence molybdenum mixed giant clusters with a large number of negative charges on the surface,which are composed of molybdenum atoms in different valence states.The selected research objects are spherical?NH₄?₄₂[Mo₁₃₂O₃₇₂?CHCOO?₃₀]?H₂O?₇₂(abbreviated as[Mo₁₃₂])and the giant disk-shaped Na₁₅[Mo₁₅₄O₄₆₂H₁₄?H₂O?₇₀]·400H₂O(abbreviated as[Mo₁₅₄]),which has the largest number of derivatives in the molybdenum family.[Mo₁₃₂]is a brown Keplerate molybdenum anion with a diameter of 2.9 nm,containing132 Mo atoms,of which 72 are in Mo?VI?and the rest 60 are in Mo?V?.[Mo₁₅₄]is a large anion cluster in a reduced state of its own.The diameter of a single[Mo₁₅₄]is about 3.6 nm.Influenced by Van der Waals gravity and electrostatic repulsion force,[Mo₁₅₄]will form a bubbly hollow ball in aqueous solution?the cluster and the hydrogen bond from water molecules play a further stabilizing role?,so it has a strong self-assembly ability.When[Mo₁₅₄]is put under the irradiation of 808 nm near-infrared laser,the light energy can be converted into heat energy,achieving the photothermal treatment effect.Human papillomavirus?HPV?is a kind of small,non-enveloped DNA virus whose genetic material is made up of circular,double-stranded DNA that causes lesions through infection and replication in the skin and mucous membranes.The proteins that coat the viral genome are called capsid proteins,which are responsible for the safe transport and delivery of the virus's genetic material to the host cell.The surface of HPV is covered by two capsid proteins L1 and L2.L1 is the main capsid protein,with a molecular weight of about 53 kDa.It consists of five monomers to form a pentamer?L1-p?,and then 72 L1-p will provide the complete viral structure when the virus is mature.Even in vitro or without DNA involved,L1 proteins can spontaneously assemble into viroids?VLPs?by 72 pentamers.Because capsid proteins play a key role in viral infection and replication,especially in the later stages of the viral life cycle,they are of great significance in the development of new antiviral agents.There are several types of cancers associated with human papillomavirus?HPV?,including cervical,vulvar,vaginal,penile,anal,rectal and oropharyngeal cancers.Currently,the prophylactic HPV vaccine on the market consists of viroid-like particles called VLPs,which are synthesized by L1,the main capsid protein of HPV.But they are unstable and expensive,and cannot be widely used in the world.This paper mainly studies how L1,the main capsid protein of HPV,identifies and assembles[Mo₁₃₂]and[Mo₁₅₄]respectively from two parts:Firstly,[Mo₁₃₂]produces contrary color response to HPV 16 L1-p and VLPs:Due to its instability,[Mo₁₃₂]will show a weak subtractive effect when exposed to oxygen and natural lights.However,?1?when L1-p is mixed with[Mo₁₃₂],it can accelerate the color reduction effect of[Mo₁₃₂]under the same conditions,which is mainly attributed to the electrostatic interaction between the two to promote the transformation from Mo?V?to Mo?VI?.After the assembly of L1-p into VLPs,the color recovery of[Mo₁₃₂]will be induced,which indicates that there is a reversible weak interaction between them.?2?After mixing the assembled VLPs with[Mo₁₃₂],it can effectively prevent[Mo₁₃₂]from losing color.In this chapter,the thesis proposes a simple and rapid colorimetric method to directly assess the existence state of HPV capsid proteins.This method may also be extended to other Mo-containing polyoxometalates,and is expected to be a quality detection method for future HPV vaccines.In this chapter,CsCl gradient centrifugation,SDS-PAGE,dynamic light scattering?DLS?,transmission electron microscopy?TEM?,UV-vis spectrophotometry and X-ray photoelectron spectroscopy?XPS?and other technologies are used to study the above processes and mechanisms at length.Secondly,the research on the assembly,regulation and performance of[Mo₁₅₄]and HPV 16 L1 proteins:We've successfully constructed two new heterozygous assemblies by using HPV 16 L1 and[Mo₁₅₄].The assembly is purified by CsCl gradient centrifugation and SDS-PAGE is used to determine the presence of L1 protein in the assembly.DLS and TEM are used to determine the assembly size and element fraction?EDX?to determine the assembly composition.All of these results have proved the successful assembly of L1-p with[Mo₁₅₄].Later,DLS is used to detect the thermal,pH and time effects of the assembly.Through cell experiments,it has found that the appropriate concentration of[Mo₁₅₄]can have a killing effect on cancer cells without damaging the normal cells.After the assembly of L1-p with[Mo₁₅₄],the biocompatibility of VLPs helps[Mo₁₅₄]to enter the cells and enhance its lethality to cancer cells.In this process,if combined with infrared laser irradiation to carry out photohyperthermia,[Mo₁₅₄]can greatly enhance the killing effect on cancer cells.Therefore,this part of the study demonstrates that the successful construction of the assembly not only improves the stability of VLPs,but also lays a foundation for improving the performance of the vaccine.Moreover,it also effectively improves the ability of[Mo₁₅₄]to kill cancer cells,which shows a good application prospect for the further development of polyacids as anticancer drugs.