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CN-121991881-A - Affinity filler and preparation method and application thereof

CN121991881ACN 121991881 ACN121991881 ACN 121991881ACN-121991881-A

Abstract

The invention discloses an affinity filler and a preparation method and application thereof. The affinity filler of the invention can separate EVs from biological samples such as cell culture fluid, and the separated EVs can be used as a drug delivery carrier for treatment and diagnosis. Compared with the standard ultracentrifugation separation method, the method for separating the affinity filler has the advantages of high purity, high recovery rate, short time, low cost, simple and convenient operation, capability of being amplified to large-scale GMP production and the like.

Inventors

  • LI KECHUN

Assignees

  • 李克春

Dates

Publication Date
20260508
Application Date
20241104

Claims (10)

  1. 1. The affinity filler is characterized by comprising a matrix, a spacer arm and an affinity ligand, wherein one end of the spacer arm is connected with the matrix, the other end of the spacer arm is connected with the affinity ligand, the affinity ligand comprises a polypeptide or a protein, the amino acid sequence of the polypeptide has at least 80% -100% sequence identity with SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and the amino acid sequence of the protein has at least 80% -100% sequence identity with SEQ ID NO.7 and SEQ ID NO. 8.
  2. 2. The affinity filler of claim 1, wherein the spacer arm comprises a polymer or a combination of polymers.
  3. 3. Affinity filler according to claim 2, characterized in that the polymer comprises (PEG) a, (GGGGS) b or (GGGS) c, wherein the number of aggregation a = 2-40, the number of aggregation b = 1-15, and the number of aggregation c = 1-20.
  4. 4. An affinity filler according to claim 1, wherein the matrix comprises a high molecular polymer or a magnetic material, preferably the matrix is a microsphere or a column, preferably the spacer arm is modified with one or more of a free thiol group, a free amino group, an epoxy group, an N-hydroxysuccinimide group, a hydrogen bromide group, biotin or streptavidin.
  5. 5. The method for preparing an affinity filler according to any one of claims 1 to 4, comprising the steps of: 1) Preparing a polypeptide or protein containing a spacer; 2) The polypeptide or protein containing a spacer is linked to the matrix.
  6. 6. The method according to claim 5, wherein the polypeptide containing a spacer in step 1) is prepared by Fmoc solid phase synthesis.
  7. 7. The method for producing an affinity filler according to claim 5, wherein the spacer-containing protein of step 1) is obtained by constructing and expressing a target gene sequence in a prokaryotic expression system.
  8. 8. The method of claim 5, wherein the attachment means in step 2) comprises coupling a group on the affinity ligand to a reactive group on the substrate.
  9. 9. Use of an affinity filler according to any one of claims 1-4 for isolating extracellular vesicles in a biological sample.
  10. 10. The use according to claim 9, wherein the biological sample comprises a cell culture fluid, blood, urine or other body fluids.

Description

Affinity filler and preparation method and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to an affinity filler, a preparation method and application thereof, and particularly relates to an affinity filler for separating extracellular vesicles. Background Extracellular vesicles (extracellular vesicles, EVs) are self-nonrepeatable extracellular vesicles (Welsh JA,Goberdhan DCI,O'Driscoll L,etal.Minimal information for studies of extracellular vesicles(MISEV2023):From basic to advanced approaches.J Extracell Vesicles.2024Feb;13(2):e12404.). that contain lipid bilayers released by cells, and almost all cell types release extracellular vesicles. EVs can be divided into exosomes, microbubbles and apoptotic bodies, according to the biogenesis pathway and size of the vesicles. The exosomes are released after the fusion of the multi-vesicular bodies and cell membranes, the size of the exosomes is 30-200nm, the exosomes belong to small extracellular vesicles (small extracellular vesicles, sEVs), the micro-vesicles are formed by directly sprouting cell membranes, the size of the micro-vesicles is 100-1000nm, the apoptotic bodies are vesicles formed by splitting cell membranes when the cells undergo apoptosis, and the EVs generated by the donor cells with the size of 500-2000nm(Pegtel DM,Gould SJ.Exosomes.Annu Rev Biochem.2019Jun 20;88:487-514.Wu Q,Fu S,Xiao H,Du J,Cheng F,Wan S,Zhu H,Li D,Peng F,Ding X,Wang L.Advances in Extracellular Vesicle Nanotechnology for Precision Theranostics.Adv Sci(Weinh).2023Jan;10(3):e2204814.). can transfer substances containing biological information, such as encapsulated proteins, lipids, nucleic acids, metabolites and the like, to the receptor cells, so that the transfer of the information among the cells is realized. EVs have a bilayer lipid structure similar to that of producer cells, mainly containing phosphatidylcholine, cholesterol and sphingomyelin (Hallal S,Grau GE,Buckland ME,Alexander KL.Understanding the extracellular vesicle surface for clinical molecular biology.J Extracell Vesicles.2022 Oct;11(10):e12260.Skotland T,Sagini K,Sandvig K,Llorente A.An emerging focus on lipids in extracellular vesicles.Adv Drug Deliv Rev.2020;159:308-321.); The surface has lipid raft structure (Skryabin GO,Komelkov AV,Savelyeva EE,Tchevkina EM.Lipid Rafts in Exosome Biogenesis.Biochemistry(Mosc).2020Feb;85(2):177-191.);sEVs with high membrane curvature (Nepal B,Leveritt J 3rd,Lazaridis T.Membrane Curvature Sensing by Amphipathic Helices:Insights from Implicit Membrane Modeling.Biophys J.2018 May 8;114(9):2128-2141.); due to its small size, and the EVs surface is also coated with protein crown (Buzas EI.Opportunities and challenges in studying the extracellular vesicle corona.Nat Cell Biol.2022 Sep;24(9):1322-1325.)。 Based on the characteristics of EVs, the method has very wide application prospect in the medical field, the method can be mainly used for three major directions of disease diagnosis, disease treatment and drug delivery. Especially small extracellular vesicles, which, due to their small nanoscale nature, allow them to pass through the tiny interstices of the physiological barrier. For example, the blood brain barrier consists of tightly linked endothelial cells sEVs can cross the barrier (Alvarez-Erviti L,Seow Y,Yin H,Betts C,Lakhal S,Wood MJ.Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes.Nat Biotechnol.2011Apr;29(4):341-5.). through the intercellular gap or through intracellular transport mechanisms while the membrane structure of the bilayer lipid protects the membrane molecules from degradation, with good stability and biocompatibility, making sEVs a very promising drug delivery vehicle. However, lack of an effective means for isolating and purifying EVs severely hampers its clinical transformation. Currently, the most common method for isolating EVs is ultracentrifugation (Ultracentrifugation, UC), which can obtain medium purity EVs by differential ultracentrifugation of samples, but is time consuming, small in sample throughput, low in yield, and unsuitable for mass production. Other methods such as Size Exclusion Chromatography (SEC) based on particle size, immunoaffinity based on surface tag molecules and precipitation based on solubility have also been commercially available, but these methods have their advantages and disadvantages as shown in table 1. TABLE 1 separation method commonly used for EVs, and advantages and disadvantages Therefore, there is a need for a new separation method that can separate EVs rapidly, efficiently, at low cost, and can be scaled up to large-scale GMP production to produce EVs that meet the number and quality of clinical needs. Disclosure of Invention The invention aims to solve the technical problem of providing an affinity filler with high separation performance. The invention also solves the technical problem of providing a preparation m