Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation of low-immunogenicity recombinant hirudin mutant

A recombinant hirudin, immunogenic technology, applied in the preparation method of peptides, from leech inhibitors, protease inhibitors, etc., can solve the problems of affecting clinical drug effect, inactivation of hirudin, loss of pharmacological activity, etc.

Inactive Publication Date: 2012-07-18
CHINA PHARM UNIV
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, after hirudin was launched on the market, many foreign clinical users found that because hirudin is a heterologous protein with strong immunogenicity, it is easy to produce neutralizing antibodies after injection, which seriously affects other medicines. Clinical drug effect [Fischer, K.G.et al. (2003) Thromb Haemost 89 (6), 973-982; Huhle, G. et al. (2001) Thromb Haemost 85 (5), 936-938; Greinacher, A.et al. (2003)]
Among them, Fischer, K.G. etc. reported that up to 74% of patients could detect anti-hirudin antibodies in their blood after five days of using hirudin, and Huhle, G. etc. reported that the hirudin antibodies produced by them could be directly neutralized. Inactivate hirudin and lose its pharmacological activity

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation of low-immunogenicity recombinant hirudin mutant
  • Preparation of low-immunogenicity recombinant hirudin mutant
  • Preparation of low-immunogenicity recombinant hirudin mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] The identification of embodiment 1 recombinant hirudin III epitope

[0011] Using Ph.D. from New England Biolabs TM -7 Phage Display Peptide Library Kit for epitope panning. First, the plate was embedded with an appropriate dilution of guinea pig anti-rHV3 antibody and incubated overnight. Each plate was filled with blocking solution, reacted at 4°C for at least 1 hr, and the plate was quickly washed 6 times with TBST (TBS+0.1% [v / v] Tween-20) buffer. Dilute 2 x 10 with 1 ml of TBST buffer 11 The phage (that is, 10 μl of the original library) was then added to the coated plate and shaken gently at room temperature for 10-60 min. Pour to remove unbound phage, wash the plate 10 times with TBST buffer. Competitive elution was performed with 1 ml of 0.1-1 mM rHV3 in TBS solution. The eluate was spliced ​​into an overnight culture of E. coli ER2738. Incubate vigorously at 37°C for 4.5 hr, centrifuge, and take the supernatant. Add 1 / 6 volume of PEG / NaCl solution, preci...

Embodiment 2

[0016] Example 2 Alanine (Ala) scanning technology determines the key binding residues of dominant antigenic epitopes

[0017] 1. According to the principle of overlap extension PCR site-directed mutagenesis ( Figure 6 ), designed a pair of common outer primers and 6 pairs of mutation primers (Table 1) with the plasmid pTASH (Tan, S., et al. (2002). Protein Expression and Purification, 25, 430-436.) as a template, the first Amino acid residues 53, 54, 55, 56, 57, and 58 were point-mutated into Ala respectively. The specific operation is as follows: use the outer primer f and each mutation primer r to amplify fragment A by PCR, then use each mutation primer f and outer primer r to amplify fragment B, then mix fragments A and B, and use this mixture As a template, the outer primer f and the outer primer r are used to carry out PCR, thereby obtaining gene C fragments in which the 53rd, 54th, 55th, 56th, 57th, and 58th amino acid residues are mutated.

[0018] Table 1. Primer s...

Embodiment 3

[0028] Embodiment 3 animal experiments identify the immunogenicity of Q53A hirudin mutants

[0029]Take 20 guinea pigs, half male and half male, and randomly divide them into 2 groups, ie wild-type experimental group and mutant experimental group. Wild-type hirudin and Q53A hirudin mutant were administered subcutaneously on the back, administered twice a day, dose 1.6mg / kg / day, blood was taken after 10 days of administration, and the antibody titers of the two groups of animal serum were detected by ELISA ( Figure 5 ). The results showed that the immunogenicity of the Q53A hirudin mutant was significantly reduced.

[0030] The results of the present invention show that the Q53A hirudin mutant basically maintains the original anticoagulant specific activity, but its immunogenicity is significantly reduced, overcomes the immunogenicity problem of existing hirudin drugs, improves the clinical drug effect of hirudin drugs, and fully It is of great significance to exert its clin...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the field of biotechnology and particularly relates to a low-immunogenicity recombinant hirudin mutant and a preparation method thereof. The method comprises the following steps of: identifying hirudin dominant antigen epitopes by a phage peptide library and a Pepscan (SPOTS) technology; scanning critical combining residues of dominant antigen epitopes by alanine (ALA) canning technology and then positioning accurately; and removing immunogenicity by using a site-specific mutagenesis technology so as to obtain hirudin mutant, wherein according to obtained hirudin mutant of which the anticoagulant rate activity is not changed basically but the immunogenicity and the immunogenicity are obviously reduced. The low-immunogenicity recombinant hirudin mutant and a preparation method thereof have important signification for overcoming the immunogenicity problem of the conventional hirudin medicaments, improving the clinical treatment effect of hirudin medicaments, and fully exerting clinical using values of the hirudin medicaments.

Description

technical field [0001] The invention belongs to the field of bioengineering pharmacy or the field of protein polypeptide medicine, and relates to a preparation method and an application thereof for developing novel low-immunogenicity recombinant hirudin mutants by using technology in the field of protein engineering. Background technique [0002] Hirudin is a kind of polypeptide substance composed of 65-66 amino acids, which was originally isolated from the salivary gland of medical leeches. There are mainly three isomers with high homology (HV1, HV2, and HV3). Hirudin can effectively inhibit the formation of arterial and venous thrombosis and disseminated intravascular coagulation (DIC) by directly inhibiting the activity of thrombin. Therefore, hirudin is effective in preventing and treating deep venous thrombosis, coronary artery thrombosis, and angina pectoris. It has high application value. [0003] At present, there are two recombinant hirudin products approved for ma...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K14/815C07K1/107
Inventor 谭树华张学瑞赵芳塞拉郭享邑
Owner CHINA PHARM UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com