The Interaction Between Survivin Protein And Antibody,Cerium (?) Was Studied By Atomic Force Microscopy

Objective1.The interaction between the key protein survivin and the antibody was studied by the atomic force microscopy?AFM?molecular level imaging technique,and the three-dimensional structure and scope of the antibody were revealed under the natural condition of the protein,so that we can more intuitively understand the molecular mechanism of survivin in the occurrence of glioma and the recovery of the deity injury.2.The tumor cell apoptosis suppressor protein survivin was selected to act in the near physiological environment with the rare earth ion cerium(Ce³⁺),and then the fine structure of the target was reconstructed by atomic force microscopy?AFM?,and the scope of the small rare earth molecules and target objects was studied at the single molecular level.To reveal the intrinsic relationship between the way of rare-earth inhibition and the apoptosis of tumor cells.Methods1.The survivin protein was selected and the high purity water was diluted to 0.2 ng/mL,and the survivin rabbit polyclonal antibody was diluted to 400 ng/mL,and the survivin protein of 5?L 0.04 ng/mL was dropped to the newly stripped mica tablet,and the sample was observed with AFM,and the survivin rabbit polyclonal antibody of 5?L 40 ng/mL was dropped to the mica.The survivin protein of 20?L 0.2 ng/mL was taken by the micropipette,and the survivin antibody of 10?L 400 ng/mL,and diluted with 70?L high pure water,after shaking 2 min,at 25?,37?,45?,inoculated at the corresponding time,the mixture of 5?L survivin protein and survivin antibody was dropped to the mica to be observed with AFM.The sample test uses the knocking mode of AFM.2.The survivin protein was selected and the high pure water was diluted to 0.2 ng/mL,and the standard solution of cerium was diluted to100?g/mL and 10?g/mL respectively.The survivin protein of 5?L 0.02ng/mL was dropped to the newly stripped mica tablet by the micropipette.The Ce³⁺aqueous solution of 5?L 10?g/mL was dropped to the newly stripped mica tablets.After setting the sample with AFM,the survivin protein of 10?L and 0.2 ng/mL is taken with a micropipette,and the corresponding volume of the corresponding volume of the Ce³⁺aqueous solution of 100?g/mL or 10?g/mL is obtained with a proper amount of high pure water,and then mixed into the required concentration of the solution,and after shaking 2 min at 25?,37?and 45?,the corresponding time will be used.The mixture of 5?L survivin protein and Ce³⁺was used to make AFM observation on the newly stripped mica tablets,and the samples were detected by the knocking mode of AFM.Results1.Atomic force microscopy?AFM?observation of survivin protein homologous two polymer showed that in aqueous solution,survivin protein mainly existed with 7-8 nm size dispersions,and high resolution AFM diagram could clearly see the domain of survivin protein and two alpha helix.2.The high resolution AFM images.antibody three-dimensional structure showed that the IgG of the Y shaped structure was distributed in the bulbous bovine serum albumin,and the two in the AFM diagram could be easily distinguished by the structural difference.The high-resolution AFM diagram reveals three relatively independent and flexible Y link Y composite structures of IgG,which can get the more detailed domain of IgG.3.The effect of temperature on the interaction between survivin protein and survivin protein antibody is different.At 25?,the three region of the survivin-antibody binding unit is composed of Y shaped structure and spherical or heart shaped particles;at 37?,there is a single spherical BSA except for the Y survivin-antibody binding unit.At 45?,a lot of different lengths are different.A large number of chain structures appeared,and the structure of single BSA protein also became loose.The high resolution AFM image shows that at 45?,the survivin-antibody binding unit has only part of the two grade structure,and the whole has become a chain,and the chain structure of various length and length is distributed around it.4.The effect of temperature on the structure of survivin protein is different,the aggregation of protein at 25?is more serious,the size of 6-8nm at 37?is obviously increased,the number of small particles and large particles decreases obviously,and the total number of 5-12 nm particles in the solution at 45?is reduced obviously,and the large particles with more than 15 nm in the solution are not seen,and the boundary ratio between the particles is not visible.It is obvious that the protein is damaged and has chain structure,and the survivin protein is more in the form of 6-8 nm double particle binding at 37?,and at 45?,the crystal structure of 10 nm around the mica sheet is uniform,and the edge is smooth.5.The influence of different Ce³⁺concentration on the structure of survivin protein is different.At 37?,the survivin protein is distributed evenly in the water solution.The edge of the particle can be observed to the extended side chain structure.After the Ce³⁺action of 1?g/mL,the particles are aggregated into larger and more uniform particles,and the edges of the particles are similar to the villi.In the side chain structure,the total number of particles after 5?g/mL Ce³⁺increases,some larger particles begin to appear,the edges of the particles begin to smooth and the side chain structure of the villi is no longer clear.The survivin protein particles after the action of 10?g/mL continue to become larger,the edges of the particles are more smooth and compact,and the small particles become loose.A small amount of line peptide appeared.After 50?g/mL Ce³⁺,there was a larger particle assemblage and a regular accumulation of cerium nitrate crystals on the substrate.6.The effects of different time and temperature on the structure.Ce³⁺and survivin protein were different.After the action of 10 min at 25?,survivin protein and Ce³⁺formed different size particles in the aqueous solution.After the action of 20 min,the size of 8-10 nm particles in the aqueous solution increased obviously,and the particles exceeding 5-9 nm occupied the main position,The reunion of 20-30 nm was also increased.After 30 min,more different combinations were formed,some more than60 nm,and similar changes after 10,20 and 30 min at 37?.At 45?,the polymerization increased with the increase of action time,with the change of the structure and the appearance of the broken peptide.Conclusions1.The suitable temperature is important for the retention of protein three structure.The results show that the combination of survivin protein and antibody is more suitable at 37?,which can prevent the reunion at low temperature and avoid the destruction of the tertiary structure at high temperature.2.Low temperature is unfavorable to the dispersion of survivin protein aggregation,and low temperature helps to maintain the presence of survivin protein in the form of monomers,which is not conducive to the rapid formation of the two polymer.Higher temperature can rapidly disperse the original aggregate,but its side effect is to a certain degree of destruction of the structure of survivin protein three,and the three stage structure develops into peptide chain.Under the condition of 37?near physiological condition,these two effects can get better balance.3.Low concentration of Ce³⁺has two effects on the function of survivin protein:one is to reduce the density of survivin protein in the solution through the formation of four polymer.Two is to reduce the effective density of survivin protein in the solution by depolymerization the survivin two polymer in the solution,and the influence of the high concentration of Ce³⁺on the function of survivin protein is main.If inhibition or damage occurs,in addition to reducing the effective density of survivin protein in solution by polymerization,there is also a change and destruction of its functional domain.4.,The effect of time is as follows:as time goes on,Ce³⁺connects survivin protein two mer to become larger structure and increase the number of polymers.The temperature effect is shown in two aspects:on the one hand,the temperature can accelerate the polymerization process of Ce³⁺to survivin protein,on the other hand,the temperature can increase the change and destruction of Ce³⁺to the three grade structure of the protein.At low temperature,the conversion of survivin protein two polymer to survivin protein polymer is the main process.Under the near physiological temperature,there is a one way polymerization process above,and this process can make the transition of survivin protein two polymer to polymer in a short time.There are also the changes in the structure of the survivin protein and the depolymerization of the survivin protein polymer.At a higher temperature,Ce³⁺can not only change the three structure of the survivin protein,but also damage the peptide chain,making it part of the peptide segment.