Preparation Of Nano Self-assembly Immunomagnetic Beads And Use For Stem Cells Separation

Surface layer protein (S-layer protein, SLP) is a self-assembling protein, which canspontaneously form an orderly and dense nano protein layer in the solid or other kinds ofsurface. We build an expression vector which contains the S-layer protein fragment andfunctional fragment of ProteinAand Protein G. The fusion proteins are SLP-ZZ andSLP-ZZGG which can be coated to the magnetic beads surface. The functional fragments inthe beads are outwardly distributed to bind the FC fragments of antibody. In this way, theactivity and the utilization of the antibody which is binding to the beads will be improved.The surface of these nano-modified magnetic beads can be used for stem cell separation andstem cell therapy.Research targets: first we express the SLP fusion proteins, and coat the proteins in magneticbeads. Then test the binding capacity with the antibody of the magnetic beads, and use thebeads for stem cell separation. The goal of the project is to produce a kind of highly efficientand easy-to-use nano-immunomagnetic beads products and apply it in stem cell separationprogram.Experiment methods: Express SLP fusion protein by E. coli, and then the target proteins arepurified by gel filtration and detect the molecular weight and purity of SLP molecular. Theability of self-assembling and the cytotoxicity of the SLP are tested to find out whether it canbe used for cell research. The cytotoxicity of beads is also tested by293cells to find outwhich are fit for SLP coating and cell research.SLP-immunomagnetic beads are made of magnetic beads which are coated with SLP fusionprotein. The binding capacity with IgG and other kinds of antibody is also be detected. Weseparate the mouse bone marrow mesenchymal stem cells by this immunomagnetic beads andtest the efficiency of the cell separation.Result: The SLP fusion protein purity can reach85%or more, and the SLP can bespontaneously formed in the plastic surface and the surface of the beads. The IgG bindingquantity is2times more than traditional beads under the same condition. The SLP-ZZ and the SLP-ZZGG beads can combine with a variety of antibodies, but we find out that theSLP-ZZGG beads have higher IgG binding rate and stronger binding capacity. We also provethat the SLP fusion protein is cytotoxin-free and which can be applied in cell biology research.This kind of beads can be used for both positive and negative cell sorting. The experiment hasproved that the positive cell screening can improve the proportion of target cell from44.39%to83.47%. We have also proved that the SLP beads screening process can increase the rate ofCD34positive cell from38.67%to56.61%and decrease the rate of CD34negative cell from38.67%to0.3%.