| β2 microglobulin(β2M)is the light chain of the major histocompatibility complex(MHC)class I molecule,which participates in immune response and mutual recognition between cells.It is widely present on the surface of nucleated cells.After normal metabolism,it is degraded and cleared by the kidneys.For patients with renal failure who have lost kidney function,β2M cannot be eliminated by normal metabolism,nor can it be effectively eliminated by conventional dialysis.As β2M accumulates in blood vessels,tissues,and joints for a long time,it will further induce dialysis-related amyloidosis(DRA),leading to osteoarthritis and cardiovascular complications,seriously affecting the quality of life and survival of patients with renal failure.At present,there is a consensus in clinical practice that β2M deposits leads to DRA and causes mid-and long-term complications.It is generally believed that while dialysis treatment,strengthening the active clearance of β2M in the blood and tissue fluid of patients with renal failure will help reduce the occurrence of amyloid deposition,which is an effective way to control DRA.However,due to the fast synthesis speed and wide distribution of body fluids of β2 microglobulin,effective blood purification and removal methods have been lacking in clinical practice.In response to urgent clinical needs,this thesis puts forward an idea to solve the problem of developing high-efficiency and specific β2 microglobulin blood purification adsorbents based on anti-β2 microglobulin nanobodies.The thesis carried out systematic and in-depth research from four following aspects:the recombinant preparation of human β2M,the recombinant preparation of nanobody affinity ligand,and the synthesis and performance evaluation of specific blood purification adsorption materials.(1)The expression and refolding of human β2M.In order to screen high-affinity antihuman β2M nanobodies and subsequently evaluate the effect of adsorbents,it is necessary to prepare a sufficient amount of high purity and active β2M.First,a prokaryotic recombinant expression system for human β2M was constructed.Due to the β sheet secondary structure ofβ2M,it is particularly prone to aggregation during intracellular expression,making it mostly in the form of inclusion bodies.Therefore,the inclusion body must be refolded to obtain solubleβ2M monomer with the correct structure and activity.Systematic and in-depth research has been carried out on how to avoid the aggregation during the refolding process and increase the refolding rate.N-propanol was discovered to be a refolding cofactor in the solubilization and refolding of β2M inclusion bodies,which can improve the hydrophobicity of the refolding solution.The results show that the addition of 4 mol/L n-propanol to the 2 mol/L urea solution can significantly increase the solubility of β2M inclusion bodies,and contribute to the correct folding of the secondary structure during the renaturation process of β2M.The refolding rate increased to 82%,which is highest renaturation rate as we konw.Furthermore,the refoldedβ2M has been validated to be immunological active and can be used for alpaca immunization and nanobody screening.(2)Screening and preparation process of specific anti-β2M nanobodies.A high-affinity clones were obtained from the non-immune library,and the recombinant expression and purification method of the anti-β2M nanobody were further studied.The prokaryotic expression system of anti-β2M nanobody was established,and it was found that its intracellular soluble expression was low,and most of it existed in the form of inclusion bodies.Subsequently,the nanobody inclusion body refolding was studied.Through multi-factor response surface analysis,the refolding conditions were optimized,and the refolding rate could reach 74%.Purified by a Ni-NTA column,anti-β2M nanobody with a purity of greater than 95%was obtained.The equilibrium dissociation constant(KD)of the anti-β2M nanobody to the β2M was determined to be 1.2×10-7 mol/L,which can be used as an affinity ligand for the preparation of β2M specific blood purification adsorbent.(3)Synthesis of human β2M specific adsorbent and evaluation of its adsorption performance.The directional immobilization of the nanobody is realized by the site-directed coupling of the sulfhydryl at the C-terminus of the nanobody sequence with the epoxy group on the surface of the cross-linked agarose beads.The results show that under weakly alkaline conditions(pH=9),which are beneficial to the covalent coupling reaction,the immobilization capacity could reache 5 mg nanobody per mL gel.The adsorption kinetics study showed that the adsorbent can achieve saturated adsorption of β2M within 30 minutes.Isothermal adsorption thermodynamics studies showed that the dissociation constant KD of the adsorbent for human β2M is 8.6×10-6 mol/L,the maximum adsorption capacity is 1.04 mg/mL,and the adsorbent could achieve selectivity of more than 90%.In a system in which the volume ratio of adsorbent to serum was 1:40,for patient serum samples containing 32.07 mg/L β2M,the removal rate was more than 92%under static adsorption.(4)Evaluation of the biological safety of the adsorbent.According to the national standards for hemoperfusion medical devices,the biological safety of adsorbent materials has been systematically evaluated through hemolysis experiments,toxicity experiments and animal experiments..The hemolysis rate of the prepared adsorbent is only 0.8%,which is far less than the 5%stipulated by the standard.The results of partial prothrombin activation experiments,thrombosis experiments,platelet adhesion experiments,and complement activation experiments were not significantly different from those of the commercially available reference products,and all met the national standards,indicating that the adsorbent has good blood compatibility.The results of the acute systemic toxicity test showed that the components released by the adsorbent during use have a potential systemic toxicity risk in a safe range.The results of animal experiments on simulated adsorption also prove that the adsorbent has good biological safety,which lays a good foundation for the further development of the adsorbent as a medical device product.In summary,this study showed that the use of β2M specific nanobodies as affinity ligands to prepare immunoadsorbents for β2M blood purification treatment is a promising way.The prepared adsorbent has the outstanding advantages of high β2M removal ability,high biological safety,low cost,etc.,and has a good application prospect in the blood purification treatment of ESRD. |
PDF Full Text Request