CN-122011213-A - Fusion polypeptide of horseshoe crab extract antibacterial peptide and carrier protein and preparation method thereof

CN122011213ACN 122011213 ACN122011213 ACN 122011213ACN-122011213-A

Abstract

The invention belongs to the technical field of biological medicines, and discloses a fusion polypeptide of a limulus antibiotic peptide and a carrier protein and a preparation method thereof. The fusion polypeptide is formed by sequentially connecting an N-terminal horseshoe crab antibiotic peptide, a middle microenvironment response type connecting module and a C-terminal carrier protein, wherein the connecting module is GFLGVR sequences or chemical equivalent structures thereof with acid sensitivity and MMP-9 cleavable property. The invention enables the fusion polypeptide to be highly stable under physiological conditions through the double-response connection module of bionic design, efficiently releases active ingredients in an infection microenvironment, remarkably improves therapeutic indexes, prolongs the in vivo half-life of the limulus antimicrobial peptide from minute level to hour level by utilizing the natural long circulation characteristic of carrier protein, overcomes the defect of quick clearance of the limulus antimicrobial peptide, realizes focus targeted activation depending on infection microenvironment specific signals, reduces whole body exposure and potential toxicity, has mature and controllable preparation process, is suitable for large-scale production and lays a foundation for clinical transformation.

Inventors

HONG JUN
FU PENGFEI
WANG XUE
HU JIANYE
WANG YONGQIANG
ZHU TAO
WANG YUANYUAN
LI HONGCHAO
GUO JIANLAI
YE YANXIN
LU MIN

Assignees

河南城建学院
河南省华维素饲料有限公司

Dates

Publication Date: 20260512
Application Date: 20260302

Claims (10)

1. A fusion polypeptide of a limulus antimicrobial peptide and a carrier protein, which is characterized in that the fusion polypeptide is formed by sequentially and covalently connecting the following components: An N-terminal horseshoe crab element antibacterial peptide; a central microenvironment responsive connection module; The carrier protein at the C end is formed by covalent connection in sequence.
2. The fusion polypeptide according to claim 1, wherein the horseshoe crab antibacterial peptide is horseshoe crab antibacterial peptide I derived from Tachypleus orientalis or a functionally equivalent variant thereof, and the amino acid sequence thereof is KWCFRVCYRGICYRRCR.
3. The fusion polypeptide of claim 1, wherein the carrier protein is recombinant human serum albumin having an amino acid sequence identical to that of native human serum albumin and comprising 585 amino acid residues.
4. The fusion polypeptide of claim 1, wherein the microenvironment-responsive linking moiety is a polypeptide sequence of 6-12 amino acids in length, has dual response characteristics of acid sensitivity and MMP-9 cleavage ability, and has cleavage efficiency of 80% or more under the synergistic condition of pH6.0-6.5 and MMP-9 concentration of 50ng/mL or more, and cleavage efficiency of 20% or less under the condition of independent pH6.0 or MMP-9 concentration of 50ng/mL or more. .
5. The fusion polypeptide of claim 1, wherein the microenvironment-responsive linking module remains structurally intact at physiological pH7.4 and normal MMP-9 concentrations, undergoes conformational loosening and is specifically cleaved by MMP-9 under conditions of an infectious microenvironment pH of 6.5 or less and an elevated MMP-9 concentration, thereby releasing the horseshoe crab antimicrobial peptide.
6. The fusion polypeptide of claim 4, wherein the amino acid sequence of the microenvironment-responsive linking module is GFLGVR.
7. The fusion polypeptide of claim 6, wherein the peptide bond between Gly at position 4 and Val at position 5 in the GFLGVR sequence is the specific recognition and cleavage site for MMP-9, and the peptide bond between Phe at position 2 and Leu at position 3 undergoes cis-trans isomerism transformation at a pH less than or equal to 6.5, so that the alpha-helix content of the linking module is reduced by more than 40%, the local hydrogen bond network is broken, and accessibility of the cleavage site for MMP-9 is improved.
8. The fusion polypeptide of claim 1, wherein the N-terminus of the carrier protein is linked to the microenvironment responsive linking module via a flexible linker, the flexible linker being (GGGGS) 3.
9. The fusion polypeptide of claim 1, wherein the N-terminus of the fusion polypeptide further comprises a 6 xhis tag for affinity purification.
10. A method for preparing the fusion polypeptide according to any one of claims 1 to 9, wherein a genetic engineering expression method is adopted, a fusion gene encoding the limulus antimicrobial peptide, a microenvironment responsive connection module and a carrier protein is cloned to a prokaryotic expression vector pET-28a (+) and is transformed to escherichia coli BL21 (DE 3) cells, the fusion polypeptide is induced by IPTG to be expressed for 16 hours at 18 ℃, and then the target fusion polypeptide is obtained by Ni-NTA affinity chromatography purification, 2 glycine residues are introduced between a limulus antimicrobial peptide coding sequence and a connection module coding sequence in the fusion gene as a spacer, the response activity of the connection module is prevented from being influenced by steric hindrance, and the MMP-9 cleavage site exposure rate of the purified fusion polypeptide is more than or equal to 90 percent.

Description

Fusion polypeptide of horseshoe crab extract antibacterial peptide and carrier protein and preparation method thereof Technical Field The invention belongs to the technical field of biological medicines, and relates to a fusion polypeptide of a limulus antibiotic peptide and a carrier protein and a preparation method thereof. Background The antibacterial peptide is taken as a natural or synthetic small molecular polypeptide with broad-spectrum antibacterial activity, has unique advantages in the clinical difficult problems of coping with multi-drug resistant bacterial infection and the like, and the limulus element antibacterial peptide has the advantages of stable structure, unique sterilization mechanism and difficulty in inducing bacterial drug resistance and is paid attention to. The unmodified free antibacterial peptide is extremely easy to be degraded by protease in blood plasma after systemic administration, and simultaneously is easy to be rapidly cleared by reticuloendothelial system due to the fact that the molecular weight is small, the charge characteristic is obvious, and the unmodified free antibacterial peptide is easy to be combined with blood plasma protein in a nonspecific mode, so that the half-life in vivo is extremely short, and the bioavailability is low. And the molecules lack active recognition capability on infection focus, are difficult to effectively enrich in inflammation or infection sites, and severely restrict the therapeutic index. In the prior art, a strategy of fusion expression or chemical coupling of antibacterial peptide and carrier protein (such as albumin, transferrin or immunoglobulin fragment) is generally adopted, so that the circulation time is prolonged and the stability is improved. Such fusion proteins improve the pharmacokinetic behavior of antimicrobial peptides to some extent by increasing the molecular size, shielding protease cleavage sites and exploiting the natural long circulation properties of the carrier proteins. Some studies have further attempted to introduce targeting ligands (e.g., mannose, folic acid, etc.) into fusion structures in an attempt to enhance their affinity for specific cells or tissues. Although the carrier protein can effectively prolong the blood plasma residence time of the fusion polypeptide, the stable covalent connection mode of the carrier protein often leads to the fact that the antibacterial peptide cannot be timely and fully dissociated and has sterilization effect after reaching an infection position. Although the infection microenvironment has the characteristics of low pH and high concentration matrix metalloproteinase or specific proteinase secreted by bacteria, the conventional fusion protein lacks the sensing and response capability to such biochemical signals, so that the antibacterial active ingredients are locked on a carrier, and the space-time controllable intelligent release cannot be realized, thereby causing the delay and even failure of the drug effect. Disclosure of Invention To achieve the above object, the present invention provides a fusion polypeptide of a limulus antibiotic peptide and a carrier protein and a method for preparing the same. The fusion polypeptide leads the fusion structure to keep high stability in the systemic circulation by introducing a bionic microenvironment response type connecting module between the horseshoe crab antibiotic peptide and the carrier protein, and specifically triggers the release of the antibiotic peptide in the infected focus microenvironment, thereby solving the fundamental conflict between the long-acting circulation and the focus high-efficiency release in the prior art. The fusion polypeptide is formed by sequentially and covalently connecting three parts, namely an N-terminal horseshoe crab element antibacterial peptide, a middle microenvironment response type connecting module and a C-terminal carrier protein. The horseshoe crab antimicrobial peptide is a natural horseshoe crab antimicrobial peptide I sequence derived from horseshoe crab or a functionally equivalent variant thereof, the amino acid sequence is KWCFRVCYRGICYRRCR, the total amino acid residues are 17, the carrier protein is recombinant human serum albumin obtained through human serum albumin genetic engineering, the amino acid sequence is consistent with that of the natural human serum albumin, the carrier protein comprises 585 amino acid residues, the carrier protein has the characteristics of long circulation half-life and low immunogenicity, and the microenvironment response type connecting module is a polypeptide sequence with the length of 6-12 amino acids, and the sequence has the dual response characteristics of acid sensitivity and protease cleavable. Specifically, the amino acid sequence of the microenvironment responsive connecting module is GFLGVR, wherein the peptide bond between Gly at the 4 th position and Val at the 5 th position is the specific recognition and cleavage site of MM