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CN-121974998-A - TNFRSF13C mutant, humanized fusion protein and application thereof

CN121974998ACN 121974998 ACN121974998 ACN 121974998ACN-121974998-A

Abstract

The invention belongs to the technical field of biopharmaceutical and protein engineering, and in particular relates to a TNFRSF13C mutant, a humanized fusion protein thereof and application thereof. The invention provides a TNFRSF13C mutant and a TNFRSF13C mutant-Fc fusion protein, the amino acid sequences of which are respectively shown as SEQ ID NO.3 and SEQ ID NO. 10. The mutant and the fusion protein have higher binding affinity with BAFF, have stable structure and simple preparation, can specifically bind with BAFF and participate in regulating and controlling BAFF-mediated immune signals, have important application value in regulating B cell functions and maintaining immune homeostasis, and provide novel molecules and technical supports for relevant research and development of immune regulation.

Inventors

  • ZHANG SHUANGQUAN
  • ZHANG JIAXIN
  • YU PING

Assignees

  • 江苏双全生物医药科技有限公司

Dates

Publication Date
20260505
Application Date
20260408

Claims (10)

  1. 1. A TNFRSF13C mutant is characterized in that the amino acid sequence is shown as SEQ ID NO. 3.
  2. 2. A TNFRSF13C mutant-Fc fusion protein is characterized in that the amino acid sequence is shown in SEQ ID NO. 10.
  3. 3. A nucleic acid molecule encoding the TNFRSF13C mutant of claim 1 or the TNFRSF13C mutant-Fc fusion protein of claim 2.
  4. 4. A recombinant expression vector comprising the nucleic acid molecule of claim 3.
  5. 5. A host cell comprising the nucleic acid molecule of claim 3 or the recombinant expression vector of claim 4.
  6. 6. The method for preparing a mutant of TNFRSF13C according to claim 1, wherein the nucleic acid encoding the mutant is introduced into a host cell to be expressed, and purified.
  7. 7. The method for preparing a TNFRSF13C mutant-Fc fusion protein according to claim 2, wherein the nucleic acid encoding the fusion protein is introduced into a host cell for expression, and purified.
  8. 8. The use of TNFRSF13C mutant according to claim 1 in the preparation of a medicament for modulating BAFF-mediated immune signaling.
  9. 9. The use of TNFRSF13C mutant-Fc fusion protein according to claim 2 in the preparation of a medicament for modulating BAFF-mediated immune signaling.
  10. 10. Use of the TNFRSF13C mutant according to claim 1 or the TNFRSF13C mutant-Fc fusion protein according to claim 2 in the preparation of a medicament for modulating B cell function and immune homeostasis.

Description

TNFRSF13C mutant, humanized fusion protein and application thereof Technical Field The invention belongs to the technical field of biological pharmacy, and in particular relates to a TNFRSF13C mutant, a humanized fusion protein and application thereof Background B cell activating factor BAFF (B-CELL ACTIVATING factor, also called BLyS) is an important member of tumor necrosis factor superfamily, and is mainly involved in regulating proliferation, differentiation, survival and maturation process of B lymphocyte, and has key effect for maintaining humoral immune homeostasis of organism. TNFRSF13C (BAFF-R) is a specific receptor of BAFF, is mainly expressed on the surface of B lymphocytes, and can initiate downstream signaling through specific binding with BAFF to participate in the regulation of immune cell functions and the maintenance of immune balance. The extracellular domain of TNFRSF13C is a core region that realizes specific binding to BAFF, and its amino acid sequence directly determines intermolecular binding capacity and signal initiation efficiency. Under physiological conditions, the binding strength of TNFRSF13C and BAFF directly influences the regulatory efficiency of B cell immune signals. The TNFRSF13C-Fc fusion protein disclosed in the prior art is mostly directly fused by adopting an extracellular domain sequence of wild TNFRSF13C, the binding domain is not subjected to targeted optimization, so that the binding affinity of the fusion protein and the ligand BAFF is low, the excessive ligand BAFF is difficult to neutralize efficiently, partial researchers try to screen high-affinity TNFRSF13C mutants through random mutation, but the screening efficiency is low, the randomness of mutation sites is strong, the structure of the TNFRSF13C protein is easy to be unstable, the preparation process of the subsequent fusion protein is complex, the purification difficulty is high, the production cost is high, the practicability is poor, and the large-scale production cannot be realized. In addition, although the binding affinity of a few optimized TNFRSF13C fusion proteins can be improved to a certain extent, the problems of excessive mutation sites, low homology with wild TNFRSF13C sequences, unknown biological safety and the like exist, and the application prospect is limited. Based on the defects of the prior art, the development of the TNFRSF13C mutant-Fc fusion protein with high binding affinity, stable structure, simple preparation and strong practicability becomes a technical problem to be solved urgently by the current technicians in the field, and is the core research and development of the invention. Disclosure of Invention The invention aims to: Aiming at the defect of insufficient binding affinity of the TNFRSF13C fusion protein in the prior art, the invention designs site-directed mutagenesis through ProteinMPNN artificial intelligent model, provides a TNFRSF13C mutant-Fc fully humanized fusion protein with high affinity activity, and simultaneously provides related biological materials, a preparation method and application thereof, solves the defect of the prior art, and meets the clinical treatment requirement in the future. Technical proposal A TNFRSF13C mutant is characterized in that the amino acid sequence is shown as SEQ ID NO. 3. A TNFRSF13C mutant-Fc fusion protein is characterized in that the amino acid sequence is shown in SEQ ID NO. 10. A nucleic acid molecule encoding said TNFRSF13C mutant or said TNFRSF13C mutant-Fc fusion protein. Recombinant expression vectors comprising said nucleic acid molecules. A host cell comprising said nucleic acid molecule or said recombinant expression vector. The preparation method of the TNFRSF13C mutant is characterized in that nucleic acid for encoding the mutant is introduced into a host cell for expression, and the TNFRSF13C mutant is obtained through purification. The preparation method of the TNFRSF13C mutant-Fc fusion protein is characterized in that nucleic acid encoding the fusion protein is introduced into a host cell for expression, and the TNFRSF13C mutant-Fc fusion protein is obtained through purification. The TNFRSF13C mutant is applied to the preparation of medicines for regulating and controlling BAFF-mediated immune signals. The application of the TNFRSF13C mutant-Fc fusion protein in preparing medicines for regulating and controlling BAFF-mediated immune signals. The TNFRSF13C mutant or the TNFRSF13C mutant-Fc fusion protein is applied to the preparation of medicines for regulating B cell functions and immune homeostasis. The invention has the following research ideas: 1. structural sources of TNFRSF13C mutants 1. The origin of the defined mutants was based on the amino acid sequence at positions 2-71 of the wild-type TNFRSF13C protein 2. Mutation characteristics that only 1-3 conservative amino acid mutations exist, no amino acid is deleted or added, and the homology with the amino acid sequence of the 2-71 positions of the wil