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CN-121975778-A - Recombinant A-type botulinum toxin chimeric protein and application thereof

CN121975778ACN 121975778 ACN121975778 ACN 121975778ACN-121975778-A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a recombinant A-type botulinum toxin chimeric protein and application thereof. The invention is based on wild type BoNT/A by substituting only the C-terminal receptor binding domain or the whole receptor binding domain in its receptor domain to construct a recombinant botulinum toxin type A chimeric protein. The recombinant A-type botulinum toxin chimeric protein has substrate cleavage activity equivalent to that of wild-type toxin in vitro, can be effectively ingested in Neuro-2a nerve cells and realize substrate cleavage, shows obviously reduced toxicity level in animal bodies, and has good application prospect. The AAAF chimeric protein has the characteristics of quick response and short acting time, is suitable for the situation requiring short-term intervention on nerve activity, such as short-term muscle tension regulation or local injection treatment, and AAFF chimeric protein has longer neuromuscular blocking duration and higher safety, and is suitable for long-term treatment of chronic nervous system diseases.

Inventors

  • WANG JUFANG
  • PAN SIHAN
  • YE YUANZHI

Assignees

  • 华南理工大学

Dates

Publication Date
20260505
Application Date
20260109

Claims (10)

  1. 1. A recombinant A-type botulinum toxin chimeric protein is characterized in that the recombinant A-type botulinum toxin chimeric protein is mutant AAAF or a mutant AAFF, wherein the amino acid sequence of the mutant AAAF is shown as SEQ ID NO.2, and the amino acid sequence of the mutant AAFF is shown as SEQ ID NO. 3.
  2. 2. A product characterized by comprising the following: I. A nucleic acid molecule encoding the recombinant botulinum toxin type A chimeric protein of claim 1, or, II. A recombinant vector comprising a nucleic acid molecule as described in I; III, a recombinant cell containing the recombinant vector described in II.
  3. 3. The product according to claim 2, characterized in that: the sequence of the coding nucleic acid molecule of the mutant AAAF is shown as SEQ ID NO. 6; the sequence of the coding nucleic acid molecule of the mutant AAFF is shown as SEQ ID NO. 7; the carrier framework of the recombinant carrier is from an escherichia coli expression carrier; the chassis cell of the recombinant cell is an escherichia coli expression cell.
  4. 4. A product according to claim 3, characterized in that: The carrier framework of the recombinant carrier is from pET series carrier; The chassis cell of the recombinant cell is E.coli BL21(DE3)、E.coli MG1655、E.coli DH5α、E.coli Top10、E.coli B0013、E.coli ATCC 8739、E.coli W3110(DE3) or E.coli JM109.
  5. 5. The product according to claim 4, wherein: the carrier framework of the recombinant carrier is pET28a; The recombinant cell is a chassis cell E.coli BL21 (DE 3).
  6. 6. Use of a recombinant botulinum toxin type a chimeric protein of claim 1 for the preparation of a botulinum toxin product.
  7. 7. The use of the recombinant botulinum toxin type A chimeric protein according to claim 6 for the preparation of a botulinum toxin product, wherein the botulinum toxin product is a pharmaceutical and/or a medical preparation.
  8. 8. A botulinum toxin product comprising a recombinant botulinum toxin type A chimeric protein of claim 1.
  9. 9. A botulinum toxin product according to claim 8, characterized in that: When the botulinum toxin product is a product for short-term intervention in neural activity, the recombinant botulinum toxin type A chimeric protein is mutant AAAF; When the botulinum toxin product is a longer duration neuromuscular blockade product, the recombinant botulinum toxin type A chimeric protein is mutant AAFF.
  10. 10. A botulinum toxin product according to claim 9, characterized in that: The short-term intervention neural activity is short-term muscle tension regulation or local intervention; the neuromuscular blockade duration is long as a chronic neurological disease.

Description

Recombinant A-type botulinum toxin chimeric protein and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a recombinant A-type botulinum toxin chimeric protein and application thereof. Background Botulinum toxins (Botulinum neurotoxins, boNTs) are a class of bacterial protein neurotoxins produced by Clostridium (Clostridium) and can be classified into types a-G7, the most common of which is type a (BoNT/a), depending on the antigen to be immunized. BoNTs consists of two chains and three functional domains, a light chain (LC, 50 kDa) and a heavy chain (HC, 100 kDa), which are linked by a pair of interchain disulfide bonds. LC is a catalytic domain with zinc-dependent metallopeptidase activity, which is the toxic moiety of toxins, HC can be further divided into two domains, the receptor binding domain (H C) and translocation domain (H N）,HC comprises two subdomains, N-terminal receptor binding domain (H CN), C-terminal receptor binding domain (H CC) BoNTs was initially synthesized as a precursor single chain protein of relative molecular mass 150 kDa, the linkage between LC and HC being cleaved by bacterial or host proteases to convert the inactive protoxin into a double chain active form. Botulinum toxin products have been used in a wide variety of therapeutic areas, such as the treatment of isolated cervical dystonia, relief of spastic cerebral palsy in children's lower limb spasticity, temporary improvement of benign rongeur hypertrophy, and botulinum toxin for injection in the treatment of primary axillary hyperhidrosis and the cosmetic industry. In particular, in recent years, the number of injections of chinese botulinum toxin has shown a significant trend to increase. From 170 tens of thousands in 2017 to 450 tens of thousands in 2021, the growth rate of the composite year is as high as 27.4%. This growth trend is expected to continue, reaching 1270 tens of thousands by 2025, and is expected to climb further to 4030 tens of thousands by 2030. With the continued increase in revenue that can be regulated by both Chinese individuals, the expectations of human consumption of botulinum toxins will continue to rise. In addition, because the average effectiveness of botulinum toxin products currently on the market typically lasts for about four months, consumers often need multiple uses, which further drives the growing demand of the market. With the continuous and deep scientific research, the application prospect of the botulinum toxin product is wider, the application range of the botulinum toxin product is expected to be further expanded, and the botulinum toxin product can positively promote the whole scale growth of the botulinum toxin market. The preparation of BoNT/A by extraction from a culture broth of botulinum is a common method of preparation of BoNT/A products currently on the market. The complete preparation process is completed in 16 steps including culturing botulinum, filtering culture solution, ultrafiltering, gel chromatography and other steps to obtain BoNT/A stock solution. The preparation method has the advantages of complicated extraction steps, long process, 15-20 days for the whole process, and low efficiency. The BoNT/A stock solution HAs low purity, contains components such as HA and NTNH besides BoNT/A, HAs the risk of adverse reactions such as coagulation and allergy, and is not suitable for long-term use. To improve the quality of botulinum toxin products, new production technologies, such as recombinant protein technologies, are being actively explored in the industry. With the development of molecular biology techniques and rapid DNA synthesis, a variety of recombinant BoNT derivatives with alternative functions have been evaluated as potential novel therapeutic agents. Recombinant BoNT derivatives that retain structural features important for the development of therapeutic candidates or useful biological agents have been successfully produced in e.coli, bacillus, yeast and insect cell expression systems. Although natural botulinum toxin has extremely high neurotoxicity and curative effect, the natural botulinum toxin still has the problems of poor targeting, difficult balance between curative effect and toxicity and the like. The design of mutant toxins is an important means of functional modification and mechanism research of botulinum toxins, and through site-directed mutagenesis, inactivation modification or enhancement modification of key structural domains or functional residues, not only can toxicity and functions of toxins be precisely regulated, but also new biological characteristics can be endowed to the toxins, such as changing target cell types, prolonging or shortening action time, reducing immunogenicity, or realizing specific activation. In addition, modular recombinant construction of chimeric proteins of different serotypes of botulinum toxin domains has become an important strategy for th