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Advanced conformational epitope of human PCSK9 protein and application of dominant conformational epitope

A technology of advantageous conformation and protein, applied in anti-enzyme immunoglobulin, introduction of foreign genetic material and peptides using vectors, etc., can solve problems such as restrictions on wide popularization and application, high immunogenicity, and limited efficacy time.

Pending Publication Date: 2022-06-28
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high price of monoclonal antibody, limited drug effect time and high immunogenicity (production of anti-drug antibodies) and other defects, it restricts its wide clinical application.

Method used

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  • Advanced conformational epitope of human PCSK9 protein and application of dominant conformational epitope
  • Advanced conformational epitope of human PCSK9 protein and application of dominant conformational epitope
  • Advanced conformational epitope of human PCSK9 protein and application of dominant conformational epitope

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Construction of PCSK9 polypeptide fragment phage display library

[0061] 1. Experimental method

[0062] (1) Preparation of PCSK9 reading frame sequence

[0063] The human PCSK9 gene information was obtained from the NCBI website, and the open reading frame sequence was read from the mature mRNA sequence (NM_174936.4), and the 498th base C was designed to be mutated to T to eliminate the KpnI restriction site in the sequence , which is convenient for the selection of enzyme cleavage sites during subsequent cloning, but does not change the post-translational amino acid sequence. After the sequence is designed, the whole gene is synthesized and sequenced to confirm the correctness of the sequence.

[0064] (2) Primer design

[0065] Combined with the open reading frame sequence of the whole PCSK9 gene and the division of each domain and the differences of each functional region, the signal peptide sequence and stop codon were removed, and the remaining gene ...

Embodiment 2

[0082] Example 2 Affinity Screening of PCSK9 Polypeptide Fragment Phage Display Library

[0083] In this example, two kinds of phage display library affinity screening designs are mainly used, one is phage overload design, the purpose is to quickly find the optimal binding sequence among 21 kinds of PCSK9 short peptides, and the other is monoclonal antibody overload design, the purpose is to The 21 PCSK9 short peptides were given sufficient opportunities to combine to investigate the competitive relationship between different peptide fragments, and to analyze and obtain the optimal conformational epitope of PCSK9.

[0084] 1. Experimental method

[0085] (1) Affinity screening of phage display library designed by phage overloading

[0086] In order to dilute the molecules of the two monoclonal antibodies evolocumab and alirocumab to 10 μg / mL with pH9.6 carbonate buffer, add 100 μL to each well to coat on the enzyme labeling strip, block overnight with protein-free blocking so...

Embodiment 3

[0102] Example 3 shows the preparation and identification of recombinant phages with four PCSK9 conformational epitopes

[0103] This example is based on the analysis of the results of affinity screening in Example 2, combined with the spatial structure of the PCSK9 protein, and the use of gene recombination technology to recombine the base fragments of the dominant sequence obtained in the first aspect above, and a flexible link is added between the dominant fragments The base sequence of the peptide (Gly4Ser)3 can better display the dominant conformational epitope of PCSK9.

[0104] 1. Experimental method

[0105] (1) Primer design

[0106] The overlapPCR method was used to recombine the dominant base sequence to obtain four recombinant sequences of SNRP1, SNRP2, LNRP1 and LNRP2. The primer sequences are shown in Table 6:

[0107] Table 6 Primer Sequence

[0108]

[0109] The single underline in the primer sequence in Table 6 is the restriction enzyme recognition site Sf...

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Abstract

The invention discloses a dominant conformation epitope of a human PCSK9 protein and application of the dominant conformation epitope. A nucleic acid fragment library covered with a mature human PCSK9 gene reading frame is constructed on the basis of an amino acid sequence and a protein structure of a mature human PCSK9 protein by applying a gene recombination technology, and each nucleic acid fragment is expressed and displayed at an N end of a PIII protein on the surface of an M13 bacteriophage; the epitope affinity screening is carried out on the PCSK9 library through a specific antibody by applying a phage display technology so as to screen out a dominant sequence of the human PCSK9 protein combined with the antibody, and the dominant sequence is recombined to construct the dominant conformation epitope of the PCSK9 protein. The epitope can be used for detection of serum monoclonal antibody drugs in the clinical medication process, research and development of interference peptides, monoclonal antibodies and vaccine drugs in research and development of new drugs and the like.

Description

technical field [0001] The invention relates to a dominant conformational epitope of human PCSK9 protein and its application, and belongs to the technical field of biomedicine. Background technique [0002] Cardiovascular disease is one of the diseases with the highest fatality rate. Low-density lipoprotein cholesterol (LDL-C) has been confirmed as one of the independent and effective risk factors for the development of cardiovascular disease. Reducing the level of LDL-C can effectively reduce cardiovascular adverse events. Incidence of events. At present, the clinically used cholesterol-lowering drugs are mainly statins. However, due to the continuous rise of adverse events such as elevated transaminases, myalgia and rhabdomyolysis, the choice of clinical drugs and the tolerance of patients have produced many problems. Uncertainties. [0003] Proprotein convertase subtilisin / kexin type 9 (PCSK9), a member of the subtilisin family of serine proteases, has become an importa...

Claims

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Application Information

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IPC IPC(8): C12N9/56C12N15/10C12N15/70C12N7/01C07K16/40
CPCC12N9/54C12Y304/21061C12N15/1037C12N15/70C12N7/00C07K16/40C07K2319/00
Inventor 刘超李雪宁徐红蓉苑菲
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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