CN114672476A

CN114672476A - Dominant conformation epitope of human PCSK9 protein and application thereof

Info

Publication number: CN114672476A
Application number: CN202210040413.7A
Authority: CN
Inventors: 刘超; 李雪宁; 徐红蓉; 苑菲
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2022-01-14
Filing date: 2022-01-14
Publication date: 2022-06-28

Abstract

The invention discloses a dominant conformation epitope of human PCSK9 protein and application thereof. The invention uses gene recombination technology to construct a nucleic acid fragment library covered with a mature human PCSK9 gene reading frame based on an amino acid sequence and a protein structure of a mature human PCSK9 protein, expresses and displays each nucleic acid fragment at the N end of a P III protein on the surface of an M13 bacteriophage, uses the phage display technology to carry out affinity screening of epitopes on the PCSK9 library through a specific antibody so as to screen out a dominant sequence of the human PCSK9 protein combined with the antibody, recombines the dominant sequence and constructs the dominant conformation epitope of the PCSK9 protein. The epitope can be used for detecting serum monoclonal antibody medicines in the clinical medication process, interfering peptides, monoclonal antibodies and vaccine medicines in the research and development of new medicines, and the like.

Description

Dominant conformation epitope of human PCSK9 protein and application thereof

Technical Field

The invention relates to a dominant conformation epitope of human PCSK9 protein and application thereof, belonging to the technical field of biological medicine.

Background

Cardiovascular disease is one of the most lethal, low-density lipoprotein cholesterol (LDL-C) has been identified as one of the independently effective risk factors for cardiovascular disease development, and lowering LDL-C levels can be effective in reducing the incidence of cardiovascular adverse events. At present, cholesterol-lowering drugs used clinically are mainly statins, but many uncertain factors are generated on clinical medication selection and patient tolerance due to continuous rising of adverse events such as transaminase rise, myalgia, rhabdomyolysis and the like generated after patients take the drugs.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a member of the subtilisin family of serine proteases, has become an important new target for the prevention and treatment of hypercholesterolemia and cardiovascular diseases in recent years. The enzyme is mainly expressed in the liver, and after secretion, the enzyme is directly combined with a low-density lipoprotein receptor (LDL-R) to cause the post-translational down-regulation of the LDL-R on the surface of liver cells, so that the circulating LDL-C level is increased. Thus blocking or inhibiting the expression or function of PCSK9 is effective in reducing circulating LDL-C levels. At present, research directions aiming at PCSK9 protein targets mainly comprise monoclonal antibodies, small molecule inhibitors, interference peptides, small molecule interference RNAs, antisense oligonucleotides, gene editing, antibody protein simulating medicines, vaccines and the like, wherein the monoclonal antibody medicines are the fastest advancing ones, monoclonal antibody medicines evolocumab (developed by ANINGK) and alirocumab (developed together by Seinuffy and the Probiotics) are sold on the market in Europe, America and China in sequence, and global clinical tests show that the two monoclonal antibody medicines can reduce the LDL-C level of various patient groups by 40-60% and show very similar and strong LDL-C reduction effects. However, the clinical wide popularization and application of the monoclonal antibody are restricted due to the defects of high price, limited drug effect time, high immunogenicity (generation of anti-drug antibodies) and the like of the monoclonal antibody. Compared with monoclonal antibodies, the vaccine has the advantages of small administration dosage, low preparation cost, low economic burden of medicines, long action duration and the like, so that the development of PSCK9 vaccine research is a very potential choice and can be used for long-term treatment and prevention of cardiovascular diseases such as hyperlipidemia, atherosclerosis and the like. And the searching of effective epitopes of the protein is a first consideration of vaccine development.

The human Pcsk9 gene is located on chromosome 1p32.3, contains 12 exons and encodes a glycoprotein of 692 amino acid residues. The PCSK9 protein initially forms a 75kDa zymogen during endoplasmic reticulum synthesis, comprising an N-terminal signal peptide, a prodomain (residues 31-152), a catalytic domain (residues 153-452) and a cysteine-rich C-terminal domain (residues 453-692). Followed by intramolecular autocatalytic cleavage at residues 152 and 153 after removal of the signal peptide sequence from PCSK9 zymogen in the endoplasmic reticulum, to form a 14kDa prodomain fragment and a 63kDa mature fragment, the prodomain remaining tightly linked to the catalytic domain in a non-covalent bond and forming a complex, blocking the substrate binding site of the enzyme, so PCSK9 is protease inactive and the prodomain serves as both a folding partner and a catalytic activity inhibitor. Finally, the protein is transported to Golgi apparatus from endoplasmic reticulum, and finally secreted into blood to play a role after a series of modifications such as acetylation and the like.

Disclosure of Invention

The purpose of the invention is: the dominant peptide sequence capable of being combined with the marketed monoclonal drug is obtained by screening through a phage display technology, and the dominant peptide sequence is recombined through a gene recombination technology to obtain the dominant conformation epitope of the human PCSK9 protein, wherein the dominant epitope can be used for detection of serum monoclonal antibody drugs, screening of monoclonal antibody drugs, research and development of PCSK9 interference peptide new drugs and vaccines and the like.

In order to achieve the above purpose, the present invention provides a natural dominant conformation epitope of human PCSK9 protein, which is SEQ ID NO: 1-4, wherein the polypeptide sequence can specifically bind to anti-PCSK 9 monoclonal antibody drugs.

Preferably, the native dominant conformational epitope is SEQ ID NO: 1 or SEQ ID NO: 2.

Preferably, the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 3 has an overlapping amino acid sequence: SIPWNLERITPPRYRADEYQPPDGGSLVEV, respectively; the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 4 has an overlapping amino acid sequence: AGIAAMMLSAEPELTLAELRQRLIHFSAKD are provided.

Preferably, the anti-PCSK 9 monoclonal antibody drugs comprise evolocumab and alirocumab monoclonal antibodies.

The invention also provides a recombinant dominant conformation epitope of the human PCSK9 protein, which is SEQ ID NO: 5-8, wherein the recombination sequence is represented by SEQ ID NO: 1-4 and a flexible polypeptide linker, or a polypeptide sequence represented by SEQ ID NO: 1-4 and a flexible polypeptide linker.

Preferably, said recombinant dominant conformational epitope consists of SEQ ID NO: 1. SEQ ID NO: 2 and a flexible polypeptide linker.

Preferably, the recombinant dominant conformational epitope further comprises the amino acid sequence V423-H449 of the PCSK9 protein: VINEAWFPEDQRVLTPNLVAALPPSTH are provided.

Preferably, the flexible polypeptide linker is (Gly)₄Ser)₃A flexible linker peptide having the amino acid sequence: GGGGSGGGGSGGGGS.

The invention also provides an M13 phage display vector pCANTAB5E-Pro which comprises a multiple cloning site and (Gly)₄Ser)₃The pCANTAB5E carrier of deoxynucleotide sequence corresponding to joint, and the multiple cloning site is GGCCCAGCCGGCC and GGTACC.

The invention also provides an M13 recombinant phage comprising the amino acid sequence shown as SEQ ID NO: 9-12, wherein the nucleotide sequence shown in SEQ ID NO: 9-12 are used for coding the recombinant dominant conformation epitope of the human PCSK9 protein.

The invention also provides the use of recombinant dominant conformational epitopes of human PCSK9 protein, excluding use as diagnostic and therapeutic methods.

Preferably, the application comprises the application in the preparation of anti-PCSK 9 monoclonal antibody medicines or vaccines, PCSK9 interference peptide new medicines or vaccines and reagents or kits for detecting anti-PCSK 9 monoclonal antibody medicines.

The technical principle of the invention is as follows:

The invention utilizes a 2018 Nobel chemical prize winning technology phage display technology to insert different peptide segment genes covered with a mature human PCSK9 reading frame into a phage coat protein PIII gene, and an exogenous polypeptide is displayed on the surface of phage particles. Therefore, the genotype and the phenotype of the exogenous polypeptide can be skillfully combined, the exogenous protein displayed on the surface of the phage is directly associated with the coding gene of the exogenous protein, the activity identification and the sequence analysis of the exogenous protein can be directly carried out through phage reproduction and amplification, a target molecule specifically bound can be obtained through screening of a specific antibody, and the phage containing the target protein is enriched by ten thousand times to hundred million times through the repeated process of ' adsorption ', elution ' and ' amplification ', so that a sequence dominantly bound in the PCSK9 sequence is obtained, and the PCSK9 dominant conformational epitope is finally obtained through sequence analysis.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention utilizes the affinity screening technology of phage display, can combine the genotype and phenotype of exogenous polypeptide ingeniously, the exogenous protein displayed on the surface of phage is directly associated with its coding gene, the activity identification and sequence analysis of exogenous protein can be directly carried out through phage reproduction and amplification, the most direct evidence is provided for the dominant target, the defect that the superiority of the target can not be directly proved by adopting computer simulation and artificial speculation for the target screening of the drug acting on PCSK9 protein at present is overcome;

2. The invention takes the monoclonal antibody drug which is clinically proved to have superior curative effect and is on the market as the bait, and the optimal selection of a plurality of epitopes of the PCSK9 protein is selected to obtain the dominant conformation epitope of the PCSK9, thereby providing direct evidence for the selection of subsequent new drug research targets.

Drawings

FIGS. 1A-D show four rounds of affinity screening results for phage display libraries designed for overloading of phage;

FIGS. 2A-D show four rounds of affinity screening results for monoclonal antibody-overloaded phage display libraries;

fig. 3A, 3C, 3E, 3G show the positions of the four recombinant dominant conformational epitopes in the spatial structure of PCSK9, and fig. 3B, 3D, 3F, 3H show the positions of the four recombinant dominant conformational epitopes as viewed from the surface of the PCSK9 protein.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

The first aspect of the invention is to obtain two polypeptides and two polypeptides suboptimal combined, which can be optimally combined with anti-PCSK 9 monoclonal antibody drugs, namely, evolocumab and alirocumab, which are clinically used at present, from a full-length amino acid sequence (31-692 aa) of mature human PCSK9 by a phage display technology, wherein the four polypeptides can be specifically combined with evolocumab and alirocumab, the optimally combined two polypeptides respectively correspond to two amino acid sequences S153-D212 and A363-D422 of PCSK9 protein, the lengths of the optimally combined two polypeptides are 60 amino acids and are respectively named as PC3 and PS6, the combined advantages of the optimally combined polypeptides are PC3, PS6 is suboptimal, the suboptimal combined polypeptides respectively correspond to two amino acid sequences F122-V182 and A393-G452 of PCSK9 protein, the lengths of the optimally combined polypeptides are respectively 61 and 60 amino acids and are respectively named as PS2 and PC7, and the combined advantages are equivalent. The relationship between these four polypeptides is that PC3 overlaps PS2 by 30 amino acids, and PS6 overlaps PC7 by 30 amino acids, and the specific amino acid sequence is shown below, and the underlining indicates the overlapping amino acid sequence:

①PC3:

SIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEED(SEQ ID NO：1)；

②PS6:

APGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKD(SEQ ID NO：2)；

③PS2:

FLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEV(SEQ ID NO：3)；

④PC7：

AGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHGAG(SEQ ID NO：4)。

According to the second aspect of the invention, the display diagram of the PCSK9 protein space structure of the dominant sequence obtained by screening is provided, through analysis of PCSK9 crystal space structures (ID:2PMW and 3BPS) disclosed by a protein database RCSB PDB, the adjacent regions of the two sections of amino acid sequences of PC3 and PS6 on the protein space structure are found, and the regions are positioned on the surface of the protein, and by combining the affinity screening result of a phage display technology, the PC3 and the PS6 can be judged to be important conformational epitopes of the PCSK9 protein, and the overlapping amino acid sequences of the four sections of polypeptides obtained by screening play a more critical role.

The third aspect of the invention provides four recombinant polypeptides capable of reacting PCSK9 dominant conformation epitopes, which are formed by analyzing the spatial structure of PCSK9 protein, recombining a dominant sequence obtained by affinity screening and a flexible polypeptide joint by using a gene recombination technology, and the obtained four recombinant polypeptides can directly react the dominant conformation epitopes of the PCSK9 protein. Four recombinant peptides are named SNRP1, SNRP2, LNRP1 and LNRP2 respectively, and can be used for the following specific components:

①SNRP1：

SIPWNLERITPPRYRADEYQPPDGGSLVEVGGGGSGGGGSGGGGSAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTH(SEQ ID NO：5)；

underlined is (Gly)₄Ser)₃The flexible connecting peptide comprises an overlapping amino acid sequence of PC3 and PS2 before the connecting peptide, and an overlapping amino acid sequence of PS6 and PS7 and an amino acid sequence from V423 to H449 in PCSK9 after the connecting peptide.

②SNRP2：

SIPWNLERITPPRYRADEYQPPDGGSLVEVGGGGSGGGGSGGGGSAGIAAMMLSAEPELTLAELRQRLIHFSAKD(SEQ ID NO：6)；

Underlined is (Gly)₄Ser)₃The flexible connecting peptide has the overlapping amino acid sequence of PC3 and PS2 before the connecting peptide and the overlapping amino acid sequence of PS6 and PS7 after the connecting peptide.

③LNRP1：

SIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGGGGSGGG GSGGGGSAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTH(SEQ ID NO：7)；

Underlined is (Gly)₄Ser)₃The flexible connecting peptide comprises a PC3 amino acid sequence in front of the connecting peptide, and a PS6 amino acid sequence and V423-H449 amino acid sequence in PCSK9 in back of the connecting peptide.

④LNRP2：

SIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGGGGSGGG GSGGGGSAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKD(SEQ ID NO：8)；

Underlined is (Gly)₄Ser)₃The flexible connecting peptide has PC3 amino acid sequence before connecting peptide and PS6 amino acid sequence after connecting peptide.

The fourth aspect of the invention provides an M13phage display vector, which is added with multiple cloning sites and (Gly)₄Ser)₃The deoxynucleotide sequence corresponding to the joint can better display the conformation of the target protein without influencing the titer of the phage and the infected bacteria, and the vector is named as pCANTAB 5E-Pro.

The fifth aspect of the invention provides four M13 phages displaying PCSK9 dominant conformational epitopes, namely M13Phage-SNRP1(SEQ ID NO: 9), M13Phage-SNRP2(SEQ ID NO: 10), M13Phage-LNRP1(SEQ ID NO: 11) and M13Phage-LNRP2(SEQ ID NO: 12), wherein the four phages are formed by packaging pCANTAB5E-Pro vector and super-helper Hyperphage M13K07 delta pIII in Escherichia coli TG1, and each PIII protein of the phages has fusion display target polypeptides, thereby greatly improving the titer of the phages.

In the following examples of the present invention, the experimental instruments used are shown in Table 1 and the experimental reagents are shown in Table 2.

TABLE 1 Experimental apparatus

TABLE 2 test reagents

In the following examples, the phage display vector pCANTAB5E-Pro was modified from pCANTAB5E, E.coli TG1 was purchased from Shanghai Binyinan Biotechnology, Inc., and helper phage M13KO7 was purchased from New England Biolabs (NEB).

Example 1 construction of a PCSK9 polypeptide fragment phage display library

1. Experimental methods

(1) Preparation of PCSK9 reading frame sequence

Human PCSK9 gene information is obtained from NCBI website, open reading frame sequence is read from mature mRNA sequence (NM-174936.4), 498 th base C is mutated into T to eliminate Kpn I enzyme cutting site in the sequence, which is convenient for selection of enzyme cutting site in subsequent cloning, but does not change translated amino acid sequence. After the sequence is designed, the whole gene synthesis is carried out, and the sequence is sequenced to confirm the correctness of the sequence.

(2) Primer design

Combining the PCSK9 whole-gene open reading frame sequence, each structural domain division and each functional region difference, removing a signal peptide sequence and a stop codon, wherein the rest gene sequence is a base sequence for coding mature human PCSK9 protein, the length is 1986bp, the rest gene sequence is divided into 11 segments of designed primers, corresponding amplified segments are named as PC 1-PC 11, wherein the PC1 and PC2 segments are 183bp long and are N-terminal prodomain parts of the PCSK9, the rest are all base segments with the length of 180bp, the PC 3-PC 7 comprise sequences which are catalytic structural domains of the PCSK9, and the sequences in the PC 8-PC 11 are C-terminal structural domains of the PCSK 9; meanwhile, segments PS 1-PS 10 covered with intervals of PC 1-PC 11 are designed, namely each PS segment is composed of half of the base sequence of each adjacent PC segment, and the specific primer sequence is shown in Table 3:

TABLE 3 primer sequences

Restriction enzyme recognition sites Sfi I and Kpn I are underlined in the primers, F in the name of the primers is an upstream primer, R is a downstream primer, and PC 1-PC 11 and PS 1-PS 10 are 21 base fragments correspondingly designed and amplified.

(3) Construction and identification of recombinant phage display vectors

Amplifying the 21 fragments by using a plasmid containing a PCSK9 gene reading frame sequence as a template by adopting a conventional molecular biology method, purifying, enzyme-cutting and re-purifying the product, then connecting the product with a pCANTAB5E-Pro plasmid after enzyme-cutting and purification, transforming the connection product into escherichia coli DH5 alpha, carrying out PCR identification on the obtained monoclonal colony, and sending the identified positive clone to sequencing to confirm the correctness of the recombinant plasmid.

(4) Construction of phage display libraries

Respectively transforming 21 recombinant phage vectors which are correctly identified into escherichia coli TG1, culturing by using monoclonal bacteria, adding M13KO7 helper phage to prepare 21 recombinant phage, and mixing after adjusting the titer of each phage to be consistent to obtain a phage display library.

2. Results of the experiment

(1) Identification of PCSK9 reading frame sequence

Cloning the base sequence of the PCSK9 reading frame synthesized by the whole gene to a T vector, and sequencing, wherein the base sequence is shown as SEQ ID NO: 13, and:

ATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGAGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTATCGGGCGGATGAATACCAGCCCCCCGACGGAGGCAGCCTGGTGGAGGTGTATCTCCTAGACACCAGCATACAGAGTGACCACCGGGAAATCGAGGGCAGGGTCATGGTCACCGACTTCGAGAATGTGCCCGAGGAGGACGGGACCCGCTTCCACAGACAGGCCAGCAAGTGTGACAGTCATGGCACCCACCTGGCAGGGGTGGTCAGCGGCCGGGATGCCGGCGTGGCCAAGGGTGCCAGCATGCGCAGCCTGCGCGTGCTCAACTGCCAAGGGAAGGGCACGGTTAGCGGCACCCTCATAGGCCTGGAGTTTATTCGGAAAAGCCAGCTGGTCCAGCCTGTGGGGCCACTGGTGGTGCTGCTGCCCCTGGCGGGTGGGTACAGCCGCGTCCTCAACGCCGCCTGCCAGCGCCTGGCGAGGGCTGGGGTCGTGCTGGTCACCGCTGCCGGCAACTTCCGGGACGATGCCTGCCTCTACTCCCCAGCCTCAGCTCCCGAGGTCATCACAGTTGGGGCCACCAATGCCCAAGACCAGCCGGTGACCCTGGGGACTTTGGGGACCAACTTTGGCCGCTGTGTGGACCTCTTTGCCCCAGGGGAGGACATCATTGGTGCCTCCAGCGACTGCAGCACCTGCTTTGTGTCACAGAGTGGGACATCACAGGCTGCTGCCCACGTGGCTGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCACCCTGGCCGAGTTGAGGCAGAGACTGATCCACTTCTCTGCCAAAGATGTCATCAATGAGGCCTGGTTCCCTGAGGACCAGCGGGTACTGACCCCCAACCTGGTGGCCGCCCTGCCCCCCAGCACCCATGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCCACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGGGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGA。

(2) Identification result of recombinant phage display vector

The constructed monoclonal bacteria containing 21 recombinant phage display vectors are identified by PCR, and the result of agarose gel electrophoresis compared with a DNA Marker shows that the target fragments identified and amplified are all about 180bp, the initially identified positive single colony is cultured and then sequenced, and the sequencing result is consistent with the design.

(3) Phage display library results

After titer determination for the preparation of 21 recombinant phages, each phage titer was adjusted to 10¹¹Mixing after the CFU size is up to the titer of 10¹¹CFU size phage display library.

Example 2 affinity screening of PCSK9 polypeptide fragment phage display libraries

In the embodiment, the affinity screening design is mainly carried out through two phage display libraries, one is phage overload design aiming at quickly finding out the optimal binding sequence in 21 PCSK9 short peptides, and the other is monoclonal antibody overload design aiming at giving sufficient binding opportunity to the 21 PCSK9 short peptides so as to investigate the good and bad competition relationship of the binding of different polypeptide fragments, and the optimal conformation epitope of PCSK9 is obtained through analysis.

1. Experimental methods

(1) Affinity screening of phage display libraries for phage overload design

Diluting two monoclonal antibody molecules of evolocumab and alirocumab to 10 mu g/mL by using a pH9.6 carbonate buffer solution, adding 100 mu L of the diluted monoclonal antibody molecules to each hole to coat the diluted monoclonal antibody molecules on an enzyme-labeled strip plate, sealing the enzyme-labeled strip plate with a protein-free sealing solution overnight, washing the enzyme-labeled strip plate by TBST, adding 100 mu L of 10 mu L of the diluted monoclonal antibody molecules to each hole, and washing the enzyme-labeled strip plate by using the protein-free sealing solution¹¹And (3) carrying out shake reaction on the CFU phage library at 37 ℃ for 3 hours, washing by TBST, adding 100 mu L of escherichia coli TG1 in log phase into each hole, carrying out shake reaction at 37 ℃ for 1 hour, collecting bacterial liquid in the holes, taking 1 mu L of a coating plate, adding M13KO7 auxiliary phage into the rest of the bacterial liquid, carrying out amplification culture overnight, centrifuging, and collecting supernatant, namely the screened phage library. This was repeated for 4 rounds, and the monoclonal colonies in each round of plates were randomly picked out for 21 sequencing runs.

(2) Affinity screening of monoclonal antibody over-loaded design phage display library

The method is the same as the step (1), monoclonal antibody is diluted to 20 mu g/mL and coated on an enzyme label plate, and 100 mu L10 is added into each hole¹⁰Affinity screening of CFU phage library was performed, and 4 rounds were repeated to screen each roundThe individual colonies of the individual monoclonal 21 colonies were randomly picked for sequencing in each plate coated.

2. Results of the experiment

(1) Affinity screening result of phage display library designed by phage overload

The affinity screening process was designed for 2 more wells, each well was screened independently, detected independently, the sequencing results were compared and the results were summarized and counted for the corresponding peptide fragments shown in table 4:

TABLE 4 affinity screen results for phage display libraries designed for phage overload

In Table 4, PC 1-PC 11 and PS 1-PS 10 are peptide segments of 21 PCSK9, P1-P4 represent four-wheel screening processes, A1 and A2 represent complex holes coated by monoclonal antibody alirocumab, E1 and E2 represent complex holes coated by monoclonal antibody evolocumab, four-wheel affinity screening results are obtained after converting table data into percentages, and as shown in FIGS. 1A-D, the results show that under the condition of large enough phage amount, the optimal binding sequence PC3 and the suboptimal binding sequence PS6 are quickly found.

(2) Affinity screening result of monoclonal antibody overload design phage display library

Similarly, the affinity screening process was performed with 2-fold-well design, each well was screened independently, detected independently, the sequencing results were compared, the results were summarized and counted for the corresponding peptide fragments, and the results are shown in table 5:

TABLE 5 affinity screening results of monoclonal antibody overload design phage display library

In the table, PC 1-PC 11 and PS 1-PS 10 are peptide segments of 21 PCSK9, P1-P4 represent four-wheel screening processes, A3 and A4 represent complex pores coated by monoclonal antibody alirocumab, E3 and E4 represent complex pores coated by monoclonal antibody evolocumab, four-wheel affinity screening results obtained by converting table data into percentages are shown in FIGS. 2A-D, the results show that under the condition of the dominant quantity of monoclonal antibody, the phage display peptide segments display a violent competition relationship, and finally, not only the PC3 and PS6 are proved to be optimal binding sequences and suboptimal binding sequences, but also two suboptimal binding sequences are found, and the suboptimal binding sequences have an overlapping relationship, and further, the existence of the epitope in the PC3-PS6 conformation is proved.

Example 3 preparation and identification of recombinant phages displaying four PCSK9 conformational epitopes

In this example, by analyzing the result of affinity screening in example 2, and combining with the spatial structure of PCSK9 protein, the nucleotide sequence of the dominant sequence obtained in the first aspect is recombined by using genetic recombination technology, and the nucleotide sequence of flexible linker peptide (Gly4Ser)3 is added between the dominant fragments, so that the dominant conformational epitope of PCSK9 can be better displayed.

1. Experimental methods

(1) Primer design

Gene recombination is carried out on the dominant base sequence by adopting an overlap PCR method to obtain four recombination sequences of SNRP1, SNRP2, LNRP1 and LNRP2, and the primer sequences are shown in Table 6:

TABLE 6 primer sequences

In Table 6, the restriction enzyme recognition sites Sfi I and Kpn I are underlined, the overlap region is underlined, and the flexible linker peptide (Gly)₄Ser)₃Base sequence of (1), list introductionIn the name of the compound, F is an upstream primer, and R is a downstream primer. The open reading frame sequence of PCSK9 is used as a template, a SNRP1 recombination sequence is obtained by amplification of primers SFKPC3-F, LapSUR1, LapSDF2 and LSR, an SNRP2 recombination sequence is obtained by amplification of primers SFKPC3-F, LapLUR2, LapSDF2 and SFKPC6-R, an LNRP1 recombination sequence is obtained by amplification of primers SFKPC3-F, LapSUR1, LapLDF1 and LSR, and an LNRP2 recombination sequence is obtained by amplification of primers SFKPC3-F, LapSUR1, LapLDF1 and SFKPC 6-R.

(3) Construction and identification of recombinant phage display vectors

Amplifying by adopting a conventional molecular biology method to obtain four recombinant sequences of SNRP1, SNRP2, LNRP1 and LNRP2, purifying products, cloning into a T vector, identifying positive clones by PCR, sequencing, amplifying four recombinant sequences of SNRP1, SNRP2, LNRP1 and LNRP2 by taking identified correct monoclonal bacteria as a template, purifying, enzyme-cutting and re-purifying the amplified products, connecting the amplified products with a pCANTAB5E-Pro plasmid after enzyme-cutting purification, converting the connected products into escherichia coli DH5 alpha, randomly picking out monoclonal colonies, carrying out PCR identification, and identifying the positive clones to sequence to confirm the correctness of the recombinant plasmids.

(4) Preparation and identification of four PCSK9 conformational epitope recombinant phages

Coli TG1 was transformed with the correctly identified 4 recombinant phage vectors, and after culture with monoclonals, 4 recombinant phages were prepared by adding M13KO7 helper phage and titer was determined.

2. Results of the experiment

(1) Preparation and identification of four recombinant sequences

Cloning four recombinant base sequences prepared by overlap PCR amplification to a T vector, and finally sequencing to identify the integrity of the sequences, wherein 4 base sequences obtained by sequencing are as follows, and underlines indicate base sequences of flexible connecting peptides:

SNRP1(SEQ ID NO：9)：

AGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTATCGGGCGGATGAATACCAGCCCCCCGACGGAGGCAGCCTGGTGGAGGTGGGTGGCGGCGGAAGTGGCGGTGGCGGAAGCGGCGGTGGTGGATCTGCTGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCACCCTGGCCGAGTTGAGGCAGAGACTGATCCACTTCTCTGCCAAAGATGTCATCAATGAGGCCTGGTTCCCTGAGGACCAGCGGGTACTGACCCCCAACCTGGTGGCCGCCCTGCCCCCCAGCACCCAT；

SNRP2(SEQ ID NO：10)：

AGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTATCGGGCGGATGAATACCAGCCCCCCGACGGAGGCAGCCTGGTGGAGGTGGGTGGCGGCGGAAGTGGCGGTGGCGGAAGCGGCGGTGGTGGATCTGCTGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCACCCTGGCCGAGTTGAGGCAGAGACTGATCCACTTCTCTGCCAAAGAT；

LNRP1(SEQ ID NO：11)：

AGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTATCGGGCGGATGAATACCAGCCCCCCGACGGAGGCAGCCTGGTGGAGGTGTATCTCCTAGACACCAGCATACAGAGTGACCACCGGGAAATCGAGGGCAGGGTCATGGTCACCGACTTCGAGAATGTGCCCGAGGAGGACGGTGGCGGCGGAAGTGGCGGTGGCGGAAGCGGCGGTGGTG GATCTGCCCCAGGGGAGGACATCATTGGTGCCTCCAGCGACTGCAGCACCTGCTTTGTGTCACAGAGTGGGACATCACAGGCTGCTGCCCACGTGGCTGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCACCCTGGCCGAGTTGAGGCAGAGACTGATCCACTTCTCTGCCAAAGATGTCATCAATGAGGCCTGGTTCCCTGAGGACCAGCGGGTACTGACCCCCAACCTGGTGGCCGCCCTGCCCCCCAGCACCCAT；

LNRP2(SEQ ID NO：12)：

AGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTATCGGGCGGATGAATACCAGCCCCCCGACGGAGGCAGCCTGGTGGAGGTGTATCTCCTAGACACCAGCATACAGAGTGACCACCGGGAAATCGAGGGCAGGGTCATGGTCACCGACTTCGAGAATGTGCCCGAGGAGGACGGTGGCGGCGGAAGTGGCGGTGGCGGAAGCGGCGGTGGTG GATCTGCCCCAGGGGAGGACATCATTGGTGCCTCCAGCGACTGCAGCACCTGCTTTGTGTCACAGAGTGGGACATCACAGGCTGCTGCCCACGTGGCTGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCACCCTGGCCGAGTTGAGGCAGAGACTGATCCACTTCTCTGCCAAAGAT。

(2) Preparation and identification of four PCSK9 conformational epitope recombinant phages

The titer of 4 recombinant phages displaying PCSK9 dominant conformation epitopes prepared by determination can reach 10¹¹CFU (circulating fluid Unit) proves that the polypeptide of the 4 dominant epitopes is successfully displayed on the P III protein on the surface of phage particles, and the polypeptide of the 4 dominant epitopes is subjected to positioning analysis on the crystal structure of the PCSK9 protein, so that the existence of the dominant conformational epitope of PC3-PS6 is confirmed, and the results are shown in figures 3A-H, and show that the epitope sequence is mainly on the surface of the protein structure, and the results show thatWithout being covered by the prodomain binding site and the LDLR binding and site, it can be determined that the dominant conformational epitope of PCSK9, which is currently discovered, provides strong evidence for target selection for subsequent drug development.

The invention obtains the natural dominant sequence of human PCSK9 protein by screening through a phage display technology, carries out gene recombination on the dominant sequence, obtains the sequence information of four recombinant polypeptides capable of reacting PCSK9 dominant conformation epitope, can directly carry out protein expression or construct recombinant virus particles and the like, and is used for developing a subsequent specific antibody drug blood concentration detection kit, screening anti-PCSK 9 monoclonal antibody drugs, researching and developing cholesterol-lowering interfering peptide drugs, researching and developing PCSK9 vaccines for preventing and treating hypercholesterolemia and the like.

The above-described embodiments are intended to be preferred embodiments of the present invention only, and not to limit the invention in any way and in any way, it being noted that those skilled in the art will be able to make modifications and additions without departing from the scope of the invention, which shall be deemed to also encompass the scope of the invention.

Sequence listing

<110> Zhongshan Hospital affiliated to double-denier university

<120> dominant conformational epitope of human PCSK9 protein and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 60

<212> PRT

<213> human PCSK9 protein

<400> 1

Ser Ile Pro Trp Asn Leu Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala

1 5 10 15

Asp Glu Tyr Gln Pro Pro Asp Gly Gly Ser Leu Val Glu Val Tyr Leu

20 25 30

Leu Asp Thr Ser Ile Gln Ser Asp His Arg Glu Ile Glu Gly Arg Val

35 40 45

Met Val Thr Asp Phe Glu Asn Val Pro Glu Glu Asp

50 55 60

<210> 2

<211> 60

<212> PRT

<213> human PCSK9 protein

<400> 2

Ala Pro Gly Glu Asp Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys

1 5 10 15

Phe Val Ser Gln Ser Gly Thr Ser Gln Ala Ala Ala His Val Ala Gly

20 25 30

Ile Ala Ala Met Met Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu

35 40 45

Leu Arg Gln Arg Leu Ile His Phe Ser Ala Lys Asp

50 55 60

<210> 3

<211> 61

<212> PRT

<213> human PCSK9 protein

<400> 3

Phe Leu Val Lys Met Ser Gly Asp Leu Leu Glu Leu Ala Leu Lys Leu

1 5 10 15

Pro His Val Asp Tyr Ile Glu Glu Asp Ser Ser Val Phe Ala Gln Ser

20 25 30

Ile Pro Trp Asn Leu Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala Asp

35 40 45

Glu Tyr Gln Pro Pro Asp Gly Gly Ser Leu Val Glu Val

50 55 60

<210> 4

<211> 60

<212> PRT

<213> human PCSK9 protein

<400> 4

Ala Gly Ile Ala Ala Met Met Leu Ser Ala Glu Pro Glu Leu Thr Leu

1 5 10 15

Ala Glu Leu Arg Gln Arg Leu Ile His Phe Ser Ala Lys Asp Val Ile

20 25 30

Asn Glu Ala Trp Phe Pro Glu Asp Gln Arg Val Leu Thr Pro Asn Leu

35 40 45

Val Ala Ala Leu Pro Pro Ser Thr His Gly Ala Gly

50 55 60

<210> 5

<211> 102

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ser Ile Pro Trp Asn Leu Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala

1 5 10 15

Asp Glu Tyr Gln Pro Pro Asp Gly Gly Ser Leu Val Glu Val Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gly Ile

35 40 45

Ala Ala Met Met Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu

50 55 60

Arg Gln Arg Leu Ile His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala

65 70 75 80

Trp Phe Pro Glu Asp Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala

85 90 95

Leu Pro Pro Ser Thr His

100

<210> 6

<211> 75

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Ser Ile Pro Trp Asn Leu Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala

1 5 10 15

Asp Glu Tyr Gln Pro Pro Asp Gly Gly Ser Leu Val Glu Val Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gly Ile

35 40 45

Ala Ala Met Met Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu

50 55 60

Arg Gln Arg Leu Ile His Phe Ser Ala Lys Asp

65 70 75

<210> 7

<211> 162

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Ser Ile Pro Trp Asn Leu Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala

1 5 10 15

Asp Glu Tyr Gln Pro Pro Asp Gly Gly Ser Leu Val Glu Val Tyr Leu

20 25 30

Leu Asp Thr Ser Ile Gln Ser Asp His Arg Glu Ile Glu Gly Arg Val

35 40 45

Met Val Thr Asp Phe Glu Asn Val Pro Glu Glu Asp Gly Gly Gly Gly

50 55 60

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Gly Glu Asp

65 70 75 80

Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser

85 90 95

Gly Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met

100 105 110

Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu

115 120 125

Ile His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu

130 135 140

Asp Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser

145 150 155 160

Thr His

<210> 8

<211> 135

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Ser Ile Pro Trp Asn Leu Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala

1 5 10 15

Asp Glu Tyr Gln Pro Pro Asp Gly Gly Ser Leu Val Glu Val Tyr Leu

20 25 30

Leu Asp Thr Ser Ile Gln Ser Asp His Arg Glu Ile Glu Gly Arg Val

35 40 45

Met Val Thr Asp Phe Glu Asn Val Pro Glu Glu Asp Gly Gly Gly Gly

50 55 60

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Gly Glu Asp

65 70 75 80

Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser

85 90 95

Gly Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met

100 105 110

Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu

115 120 125

Ile His Phe Ser Ala Lys Asp

130 135

<210> 9

<211> 306

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agcatcccgt ggaacctgga gcggattacc cctccacggt atcgggcgga tgaataccag 60

ccccccgacg gaggcagcct ggtggaggtg ggtggcggcg gaagtggcgg tggcggaagc 120

ggcggtggtg gatctgctgg cattgcagcc atgatgctgt ctgccgagcc ggagctcacc 180

ctggccgagt tgaggcagag actgatccac ttctctgcca aagatgtcat caatgaggcc 240

tggttccctg aggaccagcg ggtactgacc cccaacctgg tggccgccct gccccccagc 300

acccat 306

<210> 10

<211> 225

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

agcatcccgt ggaacctgga gcggattacc cctccacggt atcgggcgga tgaataccag 60

ccccccgacg gaggcagcct ggtggaggtg ggtggcggcg gaagtggcgg tggcggaagc 120

ggcggtggtg gatctgctgg cattgcagcc atgatgctgt ctgccgagcc ggagctcacc 180

ctggccgagt tgaggcagag actgatccac ttctctgcca aagat 225

<210> 11

<211> 486

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

agcatcccgt ggaacctgga gcggattacc cctccacggt atcgggcgga tgaataccag 60

ccccccgacg gaggcagcct ggtggaggtg tatctcctag acaccagcat acagagtgac 120

caccgggaaa tcgagggcag ggtcatggtc accgacttcg agaatgtgcc cgaggaggac 180

ggtggcggcg gaagtggcgg tggcggaagc ggcggtggtg gatctgcccc aggggaggac 240

atcattggtg cctccagcga ctgcagcacc tgctttgtgt cacagagtgg gacatcacag 300

gctgctgccc acgtggctgg cattgcagcc atgatgctgt ctgccgagcc ggagctcacc 360

ctggccgagt tgaggcagag actgatccac ttctctgcca aagatgtcat caatgaggcc 420

tggttccctg aggaccagcg ggtactgacc cccaacctgg tggccgccct gccccccagc 480

acccat 486

<210> 12

<211> 405

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

agcatcccgt ggaacctgga gcggattacc cctccacggt atcgggcgga tgaataccag 60

ccccccgacg gaggcagcct ggtggaggtg tatctcctag acaccagcat acagagtgac 120

caccgggaaa tcgagggcag ggtcatggtc accgacttcg agaatgtgcc cgaggaggac 180

ggtggcggcg gaagtggcgg tggcggaagc ggcggtggtg gatctgcccc aggggaggac 240

atcattggtg cctccagcga ctgcagcacc tgctttgtgt cacagagtgg gacatcacag 300

gctgctgccc acgtggctgg cattgcagcc atgatgctgt ctgccgagcc ggagctcacc 360

ctggccgagt tgaggcagag actgatccac ttctctgcca aagat 405

<210> 13

<211> 2079

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgggcaccg tcagctccag gcggtcctgg tggccgctgc cactgctgct gctgctgctg 60

ctgctcctgg gtcccgcggg cgcccgtgcg caggaggacg aggacggcga ctacgaggag 120

ctggtgctag ccttgcgttc cgaggaggac ggcctggccg aagcacccga gcacggaacc 180

acagccacct tccaccgctg cgccaaggat ccgtggaggt tgcctggcac ctacgtggtg 240

gtgctgaagg aggagaccca cctctcgcag tcagagcgca ctgcccgccg cctgcaggcc 300

caggctgccc gccggggata cctcaccaag atcctgcatg tcttccatgg ccttcttcct 360

ggcttcctgg tgaagatgag tggcgacctg ctggagctgg ccttgaagtt gccccatgtc 420

gactacatcg aggaggactc ctctgtcttt gcccagagca tcccgtggaa cctggagcgg 480

attacccctc cacggtatcg ggcggatgaa taccagcccc ccgacggagg cagcctggtg 540

gaggtgtatc tcctagacac cagcatacag agtgaccacc gggaaatcga gggcagggtc 600

atggtcaccg acttcgagaa tgtgcccgag gaggacggga cccgcttcca cagacaggcc 660

agcaagtgtg acagtcatgg cacccacctg gcaggggtgg tcagcggccg ggatgccggc 720

gtggccaagg gtgccagcat gcgcagcctg cgcgtgctca actgccaagg gaagggcacg 780

gttagcggca ccctcatagg cctggagttt attcggaaaa gccagctggt ccagcctgtg 840

gggccactgg tggtgctgct gcccctggcg ggtgggtaca gccgcgtcct caacgccgcc 900

tgccagcgcc tggcgagggc tggggtcgtg ctggtcaccg ctgccggcaa cttccgggac 960

gatgcctgcc tctactcccc agcctcagct cccgaggtca tcacagttgg ggccaccaat 1020

gcccaagacc agccggtgac cctggggact ttggggacca actttggccg ctgtgtggac 1080

ctctttgccc caggggagga catcattggt gcctccagcg actgcagcac ctgctttgtg 1140

tcacagagtg ggacatcaca ggctgctgcc cacgtggctg gcattgcagc catgatgctg 1200

tctgccgagc cggagctcac cctggccgag ttgaggcaga gactgatcca cttctctgcc 1260

aaagatgtca tcaatgaggc ctggttccct gaggaccagc gggtactgac ccccaacctg 1320

gtggccgccc tgccccccag cacccatggg gcaggttggc agctgttttg caggactgta 1380

tggtcagcac actcggggcc tacacggatg gccacagccg tcgcccgctg cgccccagat 1440

gaggagctgc tgagctgctc cagtttctcc aggagtggga agcggcgggg cgagcgcatg 1500

gaggcccaag ggggcaagct ggtctgccgg gcccacaacg cttttggggg tgagggtgtc 1560

tacgccattg ccaggtgctg cctgctaccc caggccaact gcagcgtcca cacagctcca 1620

ccagctgagg ccagcatggg gacccgtgtc cactgccacc aacagggcca cgtcctcaca 1680

ggctgcagct cccactggga ggtggaggac cttggcaccc acaagccgcc tgtgctgagg 1740

ccacgaggtc agcccaacca gtgcgtgggc cacagggagg ccagcatcca cgcttcctgc 1800

tgccatgccc caggtctgga atgcaaagtc aaggagcatg gaatcccggc ccctcaggag 1860

caggtgaccg tggcctgcga ggagggctgg accctgactg gctgcagtgc cctccctggg 1920

acctcccacg tcctgggggc ctacgccgta gacaacacgt gtgtagtcag gagccgggac 1980

gtcagcacta caggcagcac cagcgaaggg gccgtgacag ccgttgccat ctgctgccgg 2040

agccggcacc tggcgcaggc ctcccaggag ctccagtga 2079

Claims

1. A naturally dominant conformational epitope of a human PCSK9 protein, which is SEQ ID NO: 1-4, wherein the polypeptide sequence can specifically bind to anti-PCSK 9 monoclonal antibody drugs.

2. The naturally dominant conformational epitope of a human PCSK9 protein of claim 1, which is SEQ ID NO: 1 or SEQ ID NO: 2.

3. The naturally dominant conformational epitope of a human PCSK9 protein of claim 1, wherein the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 3 has an overlapping amino acid sequence: SIPWNLERITPPRYRADEYQPPDGGSLVEV; the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 4 has an overlapping amino acid sequence: AGIAAMMLSAEPELTLAELRQRLIHFSAKD are provided.

4. The natural dominant conformational epitope of a human PCSK9 protein of any one of claims 1 to 3, wherein the anti-PCSK 9 monoclonal antibody drug comprises evolocumab and alirocumab.

5. A recombinant dominant conformational epitope of human PCSK9 protein, which is characterized in that the epitope is represented by SEQ ID NO: 5-8, wherein the recombination sequence is represented by SEQ ID NO: 1-4 and a flexible polypeptide linker, or a polypeptide sequence represented by SEQ ID NO: 1-4 and a flexible polypeptide linker.

6. The recombinant dominant conformational epitope of human PCSK9 protein of claim 5, wherein said recombinant dominant conformational epitope consists of the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2 and a flexible polypeptide linker.

7. The recombinant dominant conformational epitope of human PCSK9 protein of claim 6, further comprising the amino acid sequence V423 to H449 of PCSK9 protein: VINEAWFPEDQRVLTPNLVAALPPSTH are provided.

8. The recombinant dominant conformational epitope of a human PCSK9 protein of claim 7, wherein the flexible polypeptide linker is (Gly)₄Ser)₃A flexible linker peptide having the amino acid sequence: GGGGSGGGGSGGGGS.

9. An M13 phage display vector pCANTAB5E-Pro, which is characterized by comprising a multiple cloning site and (Gly)₄Ser)₃The pCANTAB5E carrier of deoxynucleotide sequence corresponding to joint, and the multiple cloning site is GGCCCAGCCGGCC and GGTACC.

10. An M13 recombinant bacteriophage comprising the amino acid sequence set forth in SEQ ID NO: 9-12, wherein the nucleotide sequence shown in SEQ ID NO: 9-12 for encoding a recombinant dominant conformational epitope of a human PCSK9 protein according to any of claims 5-8.

11. The use of the recombinant dominant conformational epitope of human PCSK9 protein of any of claims 5 to 8, which excludes use as a diagnostic or therapeutic method.

12. The use as claimed in claim 12, which comprises use in the manufacture of anti-PCSK 9 mab drugs or vaccines, PCSK9 interference peptide new drugs or vaccines and reagents or kits for the detection of anti-PCSK 9 mab drugs.