Recombinant dominant conformational epitope peptide lnrp2 of human pcsk9 protein and application thereof

Abstract

The application discloses a recombinant dominant conformational epitope peptide LNRP2 of human PCSK9 protein and application thereof. The application applies gene recombination technology to construct a nucleic acid fragment library covering a mature human PCSK9 gene reading frame based on an amino acid sequence and a protein structure of the mature human PCSK9 protein, expresses each nucleic acid fragment on the N terminal of PIII protein on the surface of M13 bacteriophage, applies bacteriophage display technology to perform epitope affinity screening on the PCSK9 library through specific antibodies, screens out a dominant sequence of the human PCSK9 protein combined with the antibodies, and recombines the dominant sequence to construct a dominant conformational epitope of the PCSK9 protein. The epitope can be used for detecting serum monoclonal antibody drugs in a clinical medication process, and developing interfering peptides, monoclonal antibodies and vaccine drugs in new drug research and development.

Description

Technical Field

[0001] This application relates to the recombinant dominant conformational epitope peptide LNRP2 of human PCSK9 protein and its applications, belonging to the field of biomedical technology. Background Technology

[0002] Cardiovascular disease is one of the leading causes of death, and low-density lipoprotein cholesterol (LDL-C) has been proven to be an independent and effective risk factor for its development. Lowering LDL-C levels can effectively reduce the incidence of adverse cardiovascular events. Currently, the main cholesterol-lowering drugs used clinically are statins. However, the increasing incidence of adverse events such as elevated transaminases, myalgia, and rhabdomyolysis after medication has created many uncertainties in clinical drug selection and patient tolerance.

[0003] The proprotein convertase subtilisin / kexin9 (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 disease in recent years. This enzyme is mainly expressed in the liver. After secretion, it directly binds to the low-density lipoprotein receptor (LDL-R), leading to post-translational downregulation of LDL-R on the surface of hepatocytes and an increase in circulating LDL-C levels. Therefore, blocking or inhibiting the expression or function of PCSK9 can effectively reduce circulating LDL-C levels. Current research directions targeting the PCSK9 protein mainly include monoclonal antibodies, small molecule inhibitors, interfering peptides, small interfering RNA, antisense oligonucleotides, gene editing, antibody-mimicking protein drugs, and vaccines. Among these, monoclonal antibody drugs have made the most progress. Currently, the monoclonal antibody drugs evolocumab (developed by Amgen) and alirocumab (co-developed by Sanofi and Regeneron) have been approved for marketing in Europe, the United States, and China. Global clinical trials have shown that both monoclonal antibody drugs can reduce LDL-C levels by 40% to 60% in various patient populations, exhibiting very similar and potent LDL-C-lowering effects. However, the high cost, limited duration of action, and high immunogenicity (leading to the production of drug-resistant antibodies) of monoclonal antibodies limit their widespread clinical application. Compared to monoclonal antibodies, vaccines have advantages such as smaller dosage, lower preparation costs, lower economic burden, and longer duration of action. Therefore, conducting PCSK9 vaccine research is a promising option for the long-term treatment and prevention of cardiovascular diseases such as hyperlipidemia and atherosclerosis. Identifying effective epitopes of the protein is the primary consideration in vaccine development.

[0004] The human PCSK9 gene is located on chromosome 1p32.3, contains 12 exons, and encodes a 692-amino acid glycoprotein. The PCSK9 protein initially forms a 75 kDa zymogen during endoplasmic reticulum synthesis, containing an N-terminal signal peptide, a predomain (residues 31-152), a catalytic domain (residues 153-452), and a cysteine-rich C-terminal domain (residues 453-692). Subsequently, in the endoplasmic reticulum, the PCSK9 zymogen removes the signal peptide sequence and undergoes intramolecular autocatalytic cleavage between amino acid residues 152 and 153, forming a 14 kDa predomain fragment and a 63 kDa mature fragment. The predomain remains tightly bound to the catalytic domain non-covalently, forming a complex that blocks the enzyme's substrate binding site. Therefore, PCSK9 lacks protease activity; the predomain simultaneously acts as a folding chaperone and an inhibitor of catalytic activity. Finally, it is transported from the endoplasmic reticulum to the Golgi apparatus, where it undergoes a series of modifications, including acetylation, and is eventually secreted into the bloodstream to exert its effects. Summary of the Invention

[0005] The purpose of this invention is to use phage display technology to screen for dominant polypeptide sequences that can bind to marketed monoclonal drugs, and then use gene recombination technology to recombine the dominant polypeptide sequences to obtain dominant conformational epitopes of human PCSK9 protein. These dominant epitopes can be used for the detection of monoclonal antibody drugs in blood, screening of monoclonal antibody drugs, and the development of new PCSK9 interfering peptide drugs and vaccines.

[0006] To achieve the above objectives, the present invention provides a natural dominant conformational epitope of human PCSK9 protein, which is any one of the polypeptide sequences shown in SEQ ID NO: 1 to 4, wherein the polypeptide sequence can specifically bind to anti-PCSK9 monoclonal antibody drugs.

[0007] Preferably, the naturally dominant conformational epitope is the polypeptide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] Preferably, the polypeptide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 have overlapping amino acid sequences: SIPWNLERITPPRYRADEYQPPDGGSLVEV; and the polypeptide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 have overlapping amino acid sequences: AGIAAMMLSAEPELTLAELRQRLIHFSAKD.

[0009] Preferably, the anti-PCSK9 monoclonal antibody drug includes evolocumab and alirocumab monoclonal antibodies.

[0010] The present invention also provides a recombinant dominant conformational epitope of human PCSK9 protein, which is any one of the recombinant sequences shown in SEQ ID NO: 5-8. The recombinant sequence is formed by recombination of at least two of the polypeptide sequences shown in SEQ ID NO: 1-4 and a flexible polypeptide linker, or by recombination of overlapping sequences of the polypeptide sequences shown in SEQ ID NO: 1-4 and a flexible polypeptide linker.

[0011] Preferably, the recombinant dominant conformational epitope is formed by recombination of the polypeptide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and a flexible polypeptide linker.

[0012] Preferably, the recombinant dominant conformational epitope further includes the V423~H449 amino acid sequence of the PCSK9 protein: VINEAWFPEDQRVLTPNLVAALPPSTH.

[0013] Preferably, the flexible polypeptide linker is a (Gly4Ser)3 flexible linker peptide with the amino acid sequence: GGGGSGGGGSGGGGS.

[0014] The present invention also provides an M13 phage display vector pCANTAB5E-Pro, which is a pCANTAB5E vector containing a multiple cloning site and a deoxynucleotide sequence corresponding to the (Gly4Ser)3 linker, wherein the multiple cloning site is GGCCCAGCCGGCC and GGTACC.

[0015] The present invention also provides an M13 recombinant phage comprising any one of the nucleotide sequences shown in SEQ ID NO: 9-12, wherein the nucleotide sequences shown in SEQ ID NO: 9-12 are used to encode the recombinant dominant conformational epitope of the above-mentioned human PCSK9 protein.

[0016] This invention also provides the application of recombinant dominant conformational epitopes of human PCSK9 protein, excluding applications as diagnostic and therapeutic methods.

[0017] Preferably, the application includes its use in the preparation of anti-PCSK9 monoclonal antibody drugs or vaccines, novel PCSK9 interfering peptide drugs or vaccines, and reagents or kits for detecting anti-PCSK9 monoclonal antibody drugs.

[0018] Technical principle of the invention:

[0019] This invention utilizes phage display technology, a technique awarded the 2018 Nobel Prize in Chemistry, to insert genes containing different peptide segments of the mature human PCSK9 reading frame into the phage coat protein PⅢ gene, displaying exogenous peptides on the surface of phage particles. This cleverly combines the genotype and phenotype of the exogenous peptide, directly associating the exogenous protein displayed on the phage surface with its encoding gene. Through phage propagation and amplification, the activity and sequence of the exogenous protein can be directly identified. Screening with specific antibodies yields the target molecule that specifically binds. Furthermore, the repeated process of "adsorption," "elution," and "amplification" enriches the phage containing the target protein by tens of thousands to hundreds of millions of times, thus obtaining the dominant binding sequence within the PCSK9 sequence. Further sequence analysis ultimately yields the dominant conformational epitopes of PCSK9.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention utilizes phage display affinity screening technology to cleverly combine the genotype and phenotype of exogenous peptides. The exogenous protein displayed on the surface of the phage is directly associated with its encoding gene. Through phage propagation and amplification, the activity identification and sequence analysis of the exogenous protein can be directly performed, providing the most direct evidence for the superior target. This overcomes the shortcomings of current target screening for drugs acting on PCSK9 protein, which mostly rely on computer simulation and human speculation and cannot directly prove the superiority of the target. 2. This invention uses existing clinically proven and marketed monoclonal antibody drugs as bait to target the optimal selection of numerous epitopes of the PCSK9 protein, thereby obtaining the dominant conformational epitopes of PCSK9 and providing direct evidence for the selection of targets in subsequent new drug research. Attached Figure Description

[0021] Figure 1A ~D shows the results of four rounds of affinity screening for the phage overload design phage display library; Figure 2A ~D shows the results of four rounds of affinity screening for the monoclonal antibody overload-designed phage display library; Figure 3A , 3C 3E and 3G show the spatial structural locations of four recombination-advantageous conformational epitopes in PCSK9. Figure 3B , 3D 3F and 3H show the locations of four recombinant dominant conformational epitopes observed on the surface of the PCSK9 protein. Detailed Implementation

[0022] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0023] The first aspect of this invention involves using phage display technology to screen two peptides and two peptides that can optimally bind to the currently used anti-PCSK9 monoclonal antibody drugs evolocumab and alirocumab from the full-length amino acid sequence (31-692aa) of mature human PCSK9. All four peptides can specifically bind to evolocumab and alirocumab monoclonal antibodies. The two peptides with optimal binding correspond to the S153-D212 and A363-D422 amino acid sequences of the PCSK9 protein, respectively, both with a length of 60 amino acids, and are named PC3 and PS6, respectively. The binding advantage of PC3 is optimal, and that of PS6 is second optimal. The two peptides with second optimal binding correspond to the F122-V182 and A393-G452 amino acid sequences of the PCSK9 protein, respectively, with lengths of 61 and 60 amino acids, and are named PS2 and PC7, respectively. Their binding advantages are comparable. The four polypeptide segments are related as follows: PC3 overlaps with PS2 by 30 amino acids, and PS6 overlaps with PC7 by 30 amino acids. The specific amino acid sequences are shown below, with underlined sequences indicating overlapping amino acids: ①PC3: SIPWNLERITPPRYRADEYQPPDGGSLVEV YLLDTSIQSDHREIEGRVMVTDFENVPEED(SEQ IDNO:1); ② PS6: APGEDIIGASSDCSTCFVSQSGTSQAAAHV AGIAAMMLSAEPELTLAELRQRLIHFSAKD (SEQ ID NO: 2); ③ PS2: FLVKMSGDLLELALKLPHVDYIEEDSSVFAQ SIPWNLERITPPRYRADEYQPPDGGSLVEV (SEQ ID NO: 3); ④ PC7: AGIAAMMLSAEPELTLAELRQRLIHFSAKD VINEAWFPEDQRVLTPNLVAALPPSTHGAG (SEQ ID NO: 4).

[0024] The second aspect of this invention provides a visualization of the spatial structure of the PCSK9 protein by the screened dominant sequences. This is achieved by analyzing the PCSK9 crystal spatial structures (ID: 2PMW and 3BPS) published in the RCSB PDB protein database. It was found that the two amino acid sequences PC3 and PS6 have adjacent regions in the protein spatial structure, and these regions are located on the protein surface. Combined with the affinity screening results of phage display technology, it can be determined that PC3 and PS6 are important conformational epitopes of the PCSK9 protein, and they play a more critical role in the overlapping amino acid sequences of the four selected polypeptides.

[0025] The third aspect of this invention provides four recombinant peptides capable of reflecting the dominant conformational epitopes of PCSK9. These peptides are obtained by analyzing the spatial structure of the PCSK9 protein and using gene recombination technology to recombine the dominant sequences obtained through affinity screening with flexible peptide linkers. The resulting four recombinant peptides directly reflect the dominant conformational epitopes of the PCSK9 protein. The four recombinant peptides are named SNRP1, SNRP2, LNRP1, and LNRP2, and their specific composition is as follows: ①SNRP1: SIPWNLERITPPRYRADEYQPPDGGSLVEV GGGGSGGGGSGGGGS AGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTH (SEQ ID NO: 5); The underlined part is the (Gly4Ser)3 flexible linker peptide. Before the linker peptide is the overlapping amino acid sequence of PC3 and PS2, and after the linker peptide is the overlapping amino acid sequence of PS6 and PS7 and the V423~H449 amino acid sequence of PCSK9.

[0026] ② SNRP2: SIPWNLERITPPRYRADEYQPPDGGSLVEV GGGGSGGGGSGGGGS AGIAAMMLSAEPELTLAELRQRLIHFSAKD (SEQ ID NO: 6); The underlined part is the (Gly4Ser)3 flexible linker peptide. Before the linker peptide is the overlapping amino acid sequence of PC3 and PS2, and after the linker peptide is the overlapping amino acid sequence of PS6 and PS7.

[0027] ③ LNRP1: SIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEED GGGGSGGG GSGGGGS APGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTH (SEQ ID NO: 7); The underlined part is the (Gly4Ser)3 flexible linker peptide. The part before the linker peptide is the PC3 amino acid sequence, and the part after the linker peptide contains the PS6 amino acid sequence and the V423~H449 amino acid sequence of PCSK9.

[0028] ④ LNRP2: SIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEED GGGGSGGG GSGGGGS APGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKD (SEQ IDNO: 8); The underlined part is the (Gly4Ser)3 flexible linker peptide, preceded by the PC3 amino acid sequence and followed by the PS6 amino acid sequence.

[0029] The fourth aspect of this invention is to provide an M13 phage display vector, which is based on the pCANTAB5E vector with the addition of a multiple cloning site and a deoxynucleotide sequence corresponding to the (Gly4Ser)3 linker, which can better display the conformation of the target protein without affecting the phage titer and the infected cells. This vector is named pCANTAB5E-Pro.

[0030] The fifth aspect of this invention provides four M13 phages displaying the PCSK9 dominant conformational epitope, 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). These four phages are packaged in Escherichia coli TG1 using the pCANTAB5E-Pro vector and the super helper phage Hyperphage M13 K07ΔpIII. Moreover, each PIII protein of the phage is fused with the target polypeptide, which greatly improves the phage titer.

[0031] In the following embodiments of the present invention, the experimental instruments used are shown in Table 1 and the experimental reagents are shown in Table 2.

[0032] Table 1 Experimental Apparatus

[0033] Table 2 Experimental Reagents

[0034] In the following examples, the phage display vector pCANTAB5E-Pro was modified from pCANTAB5E, Escherichia coli TG1 was purchased from Shanghai Beyotime Biotechnology Co., Ltd., and the helper phage M13KO7 was purchased from New England Biolabs (NEB).

[0035] Example 1: Construction of a phage display library of PCSK9 peptide fragments 1. Experimental Methods (1) Preparation of PCSK9 reading frame sequence 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). A mutation was designed to change the base C at position 498 to T to eliminate the KpnI restriction site within the sequence, facilitating the selection of restriction sites during subsequent cloning, without altering the translated amino acid sequence. After sequence design, the entire genome was synthesized, and sequencing was performed to confirm its accuracy.

[0036] (2) Primer design Based on the open reading frame sequence of the entire PCSK9 genome, the division of each domain, and the differences in each functional region, the signal peptide sequence and stop codon were removed. The remaining gene sequence is the base sequence encoding the mature human PCSK9 protein, with a length of 1986 bp. This sequence was divided into 11 segments, and primers were designed to amplify the corresponding fragments, named PC1~PC11. Among them, PC1 and PC2 are both 183 bp long and are the N-terminal pro-domain of PCSK9. The rest are 180 bp long fragments. The sequences in PC3~PC7 contain the catalytic domain of PCSK9, and the sequences in PC8~PC11 are the C-terminal domain of PCSK9. At the same time, fragments PS1~PS10 covering the intervals between PC1~PC11 were designed. That is, each PS fragment is composed of half of the base sequence of each adjacent PC fragment. The specific primer sequences are shown in Table 3. Table 3 Primer Sequences

[0037] The underlined primers represent the restriction enzyme recognition sites SfiⅠ and KpnⅠ. In the primer names, F is the upstream primer, R is the downstream primer, and PC1~PC11 and PS1~PS10 correspond to the 21-base fragments designed for amplification.

[0038] (3) Construction and identification of recombinant phage display vectors Using conventional molecular biology methods, the above 21 fragments were amplified using a plasmid containing the PCSK9 gene reading frame sequence as a template. The products were purified, digested with enzymes, and then purified again before being ligated with the enzyme-digested and purified pCANTAB5E-Pro plasmid. The ligation product was transformed into Escherichia coli DH5α, and the obtained single-clone colonies were identified by PCR. Positive clones were sent for sequencing to confirm the correctness of the recombinant plasmid.

[0039] (4) Construction of phage display library Twenty-one correctly identified recombinant phage vectors were transformed into Escherichia coli TG1, cultured with single-clone bacteria, and then M13KO7 helper phage was added to prepare 21 recombinant phages. After adjusting the titers of each phage to be consistent, they were mixed to form a phage display library.

[0040] 2. Experimental Results (1) Identification of PCSK9 reading frame sequence The fully synthesized PCSK9 reading frame sequence was cloned into a T vector and sequenced. The sequence is shown in SEQ ID NO: 13.

[0041] (2) Identification results of recombinant phage display vector The constructed monoclonal bacteria containing 21 recombinant phage display vectors were first identified by PCR. Agarose gel electrophoresis results comparing the DNA markers showed that the amplified target fragments were all around 180 bp. The preliminarily identified positive single colonies were cultured and then sent for sequencing. The sequencing results were consistent with the design.

[0042] (3) Phage display library results After titer determination of 21 recombinant phages, the titer of each phage was adjusted to 10. 11 After mixing CFU by size, a titer of 10 is obtained. 11 A CFU-sized phage display library.

[0043] Example 2: Affinity screening of PCSK9 peptide fragment phage display library This embodiment mainly employs two phage display library affinity screening designs: one is a phage overload design, which aims to quickly find the optimal binding sequence among 21 PCSK9 short peptides; the other is a monoclonal antibody overload design, which aims to provide sufficient binding opportunities for the 21 PCSK9 short peptides to examine the competitive relationship between different peptide fragments in binding, and to analyze and obtain the optimal conformational epitope of PCSK9.

[0044] 1. Experimental Methods (1) Affinity screening of phage display libraries based on phage overload design First, the two monoclonal antibodies, evolocumab and alirocumab, were diluted to 10 μg / mL with pH 9.6 carbonate buffer. 100 μL was added to each well to coat an ELISA plate. The plate was blocked overnight with protein-free blocking buffer, washed with TBST, and then 100 μL of 10... 11 CFU phage library was incubated at 37°C with shaking for 3 hours. After washing with TBST, 100 μL of logarithmic-phase E. coli TG1 was added to each well, and the incubation was continued at 37°C with shaking for 1 hour. The bacterial culture in the wells was collected, and 1 μL was plated. The remainder was expanded by adding M13KO7 helper phage and cultured overnight. After centrifugation, the supernatant was collected as the selected phage library. This process was repeated 4 times. From each round, 21 single colonies were randomly selected from the plates and sent for sequencing.

[0045] (2) Affinity screening of phage display libraries designed with monoclonal antibody overload The method is the same as step (1) above. The monoclonal antibody is diluted to 20 μg / mL and coated onto the microplate. 100 μL of 10 μg / mL is added to each well. 10Affinity screening was performed on CFU phage libraries, and this process was repeated four times. From each plate of screening in each round, 21 single-clone colonies were randomly selected and sent for sequencing.

[0046] 2. Experimental Results (1) Affinity screening results of phage display library based on phage overload design The affinity screening process employed a 2-well design, with each well performing independent screening and detection. Sequence alignment was performed on the sequencing results, and the corresponding peptides displayed in the alignment results were summarized and counted. The results are shown in Table 4. Table 4. Affinity screening results of phage display libraries designed for phage overload

[0047] Table 4 shows 21 PCSK9 peptides, with PC1-PC11 and PS1-PS10 representing four rounds of screening. A1 and A2 represent replicates coated with the monoclonal antibody alirocumab, and E1 and E2 represent replicates coated with the monoclonal antibody evolocumab. The results of the four rounds of affinity screening were obtained by converting the data in the table to percentages. Figure 1A As shown in -D, the results indicate that when the phage quantity is large enough, the optimal binding sequence PC3 and the second-best binding sequence PS6 were quickly found.

[0048] (2) Affinity screening results of monoclonal antibody overload-designed phage display libraries Similarly, a 2-well design was used for affinity screening, with each well performing independent screening and detection. Sequence alignment was performed on the sequencing results, and the corresponding peptides shown in the alignment results were counted. The results are shown in Table 5. Table 5. Affinity screening results of phage display libraries designed with monoclonal antibody overload.

[0049] In the table, PC1~PC11 and PS1~PS10 represent 21 PCSK9 peptides. P1~P4 indicate four rounds of screening. A3 and A4 represent replicates coated with the monoclonal antibody alirocumab, and E3 and E4 represent replicates coated with the monoclonal antibody evolocumab. The results of the four rounds of affinity screening are obtained by converting the data in the table to percentages. Figure 2A As shown in ~D, the results indicate that when the number of monoclonal antibodies is dominant, there is intense competition among the various peptides displayed by the phage. This not only confirms that PC3 and PS6 are the optimal and second-optimal binding sequences, but also identifies two second-optimal binding sequences that overlap with the optimal sequence, further confirming the existence of PC3-PS6 as a conformational epitope.

[0050] Example 3 demonstrates the preparation and identification of four PCSK9 conformational epitope recombinant phages. This embodiment analyzes the affinity screening results in Example 2 and combines them with the spatial structure of the PCSK9 protein. The base fragments of the dominant sequence obtained in the first aspect are recombined using gene recombination technology. The base sequence of the flexible linker peptide (Gly4Ser)3 is added between the dominant fragments so as to better display the dominant conformational epitopes of PCSK9.

[0051] 1. Experimental Methods (1) Primer design Gene recombination of the dominant base sequence was performed using overlap PCR, yielding four recombinant sequences: SNRP1, SNRP2, LNRP1, and LNRP2. The primer sequences are shown in Table 6. Table 6 Primer Sequences

[0052] In Table 6, single underlines in the primer sequences represent restriction enzyme recognition sites SfiⅠ and KpnⅠ, while double underlines represent overlap segments, which are also the base sequences of the flexible linker peptide (Gly4Ser)3. In the primer names, F is the upstream primer and R is the downstream primer. Using the PCSK9 open reading frame sequence as a template, primers SFKPC3-F, LapSUR1, LapSDF2, and LSR amplified the SNRP1 recombinant sequence; primers SFKPC3-F, LapLUR2, LapSDF2, and SFKPC6-R amplified the SNRP2 recombinant sequence; primers SFKPC3-F, LapSUR1, LapLDF1, and LSR amplified the LNRP1 recombinant sequence; and primers SFKPC3-F, LapSUR1, LapLDF1, and SFKPC6-R amplified the LNRP2 recombinant sequence.

[0053] (3) Construction and identification of recombinant phage display vectors Four recombinant sequences, SNRP1, SNRP2, LNRP1, and LNRP2, were amplified using conventional molecular biology methods. After purification, the products were cloned into a T vector. Positive clones were identified by PCR and then sequenced. Using correctly identified monoclonal bacteria as templates, the four recombinant sequences SNRP1, SNRP2, LNRP1, and LNRP2 were amplified. The amplified products were then purified, digested with enzymes, and purified again before being ligated into the enzyme-digested and purified pCANTAB5E-Pro plasmid. The ligation product was transformed into Escherichia coli DH5α, and single colonies were randomly selected for PCR identification. Positive clones were then sequenced to confirm the correctness of the recombinant plasmid.

[0054] (4) Preparation and identification of four PCSK9 conformational epitope recombinant phages Four correctly identified recombinant phage vectors were transformed into Escherichia coli TG1, cultured with monoclonal bacteria, and then M13KO7 helper phage was added to prepare four recombinant phages, and their titers were determined.

[0055] 2. Experimental Results (1) Preparation and identification of four recombinant sequences Four recombinant base sequences prepared by overlap PCR amplification were cloned into the T vector, and their integrity was finally confirmed by sequencing. The four base sequences obtained by sequencing are as follows, with underlined sequences indicating flexible linker peptide base sequences: SNRP1 (SEQ ID NO: 9): AGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTATCGGGCGGATGAATACCAGCCCCCCGACGGAGGCAGCCTGGTGGAGGTG GGTGGCGGCGGAAGTGGCGGTGGCGGAAGCGGCGGTGGTGGATCT GCTGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCACCCTGGCCGAGTTGAGGCAGAGACTGATCCACTTCTCTGCCAAAGATGTCATCAATGAGGCCTGGTTCCCTGAGGACCAGCGGGTACTGACCCCCAACCTGGTGGCCGCCCTGCCCCCCAGCACCCAT; SNRP2 (SEQ ID NO: 10): AGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTATCGGGCGGATGAATACCAGCCCCCCGACGGAGGCAGCCTGGTGGAGGTG GGTGGCGGCGGAAGTGGCGGTGGCGGAAGCGGCGGTGGTGGATCT GCTGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCACCCTGGCCGAGTTGAGGCAGAGACTGATCCACTTCTCTGCCAAAGAT; LNRP1 (SEQ ID NO: 11): AGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTATCGGGCGGATGAATACCAGCCCCCCGACGGAGGCAGCCTGGTGGAGGTGTATCTCCTAGACACCAGCATACAGAGTGACCACCGGGAAATCGAGGGCAGGGTCATGGTCACCGACTTCGAGAATGTGCCCGAGGAGGAC GGTGGCGGCGGAAGTGGCGGTGGCGGAAGCGGCGGTGGTG GATCT GCCCCAGGGGAGGACATCATTGGTGCCTCCAGCGACTGCAGCACCTGCTTTGTGTCACAGAGTGGGACATCACAGGCTGCTGCCCACGTGGCTGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCACCCTGGCCGAGTTGAGGCAGAGACTGATCCACTTCTCTGCCAAAGATGTCATCAATGAGGCCTGGTTCCCTGAGGACCAGCGGGTACTGACCCCCAACCTGGTGGCCGCCCTGCCCCCCAGCACCCAT; LNRP2 (SEQ ID NO: 12): AGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTATCGGGCGGATGAATACCAGCCCCCCGACGGAGGCAGCCTGGTGGAGGTGTATCTCCTAGACACCAGCATACAGAGTGACCACCGGGAAATCGAGGGCAGGGTCATGGTCACCGACTTCGAGAATGTGCCCGAGGAGGAC GGTGGCGGCGGAAGTGGCGGTGGCGGAAGCGGCGGTGGTG GATCT GCCCCAGGGGAGGACATCATTGGTGCCTCCAGCGACTGCAGCACCTGCTTTGTGTCACAGAGTGGGACATCACAGGCTGCTGCCCACGTGGCTGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCACCCTGGCCGAGTTGAGGCAGAGACTGATCCACTTCTCTGCCAAAGAT.

[0056] (2) Preparation and identification of four recombinant phages displaying PCSK9 conformational epitopes The titers of the four recombinant phages exhibiting the dominant conformational epitope of PCSK9 obtained by testing all reached 10. 11 CFU (Cellular Fusion) was used to demonstrate successful display of the PⅢ protein on the surface of phage particles. Furthermore, the localization analysis of the peptides containing these four dominant epitopes in the PCSK9 protein crystal structure confirmed the existence of the PC3-PS6 dominant conformational epitopes. The results are as follows: Figure 3A As shown in ~H, the results indicate that this epitope sequence is mainly located on the surface of the protein structure and is not covered by the pre-domain binding site and the LDLR binding site. It can be determined that this should be the currently discovered dominant conformational epitope of PCSK9, providing strong evidence for the selection of targets for subsequent drug development.

[0057] This invention uses phage display technology to screen for the natural dominant sequence of human PCSK9 protein and then performs gene recombination on the dominant sequence to obtain the sequence information of four recombinant polypeptides that can reflect the dominant conformational epitopes of PCSK9. These sequences can be directly expressed as proteins or used to construct recombinant viral particles for subsequent development of specific antibody drug blood concentration detection kits, screening of anti-PCSK9 monoclonal antibody drugs, research and development of cholesterol-lowering interfering peptide drugs, and research and development of PCSK9 vaccines for the prevention and treatment of hypercholesterolemia.

[0058] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant dominant conformational epitope peptide of human PCSK9 protein, characterized in that, The recombinant sequence is shown in SEQ ID NO: 8, which is formed by recombining the polypeptide sequences shown in SEQ ID NO: 1~2 with a flexible polypeptide linker.

2. The recombinant dominant conformational epitope peptide of human PCSK9 protein as described in claim 1, characterized in that, The flexible polypeptide linker is a (Gly4Ser)3 flexible linker peptide with the following amino acid sequence: GGGGSGGGGSGGGGS.

3. An M13 recombinant phage, characterized in that, It comprises a nucleotide sequence as shown in SEQ ID NO: 12, the nucleotide sequence being used to encode a recombinant dominant conformational epitope peptide of the human PCSK9 protein as described in claim 1.

4. The application of the recombinant dominant conformational epitope peptide of the human PCSK9 protein as described in claim 1 or 2, characterized in that, This excludes its use as a diagnostic or treatment method.

5. The application as described in claim 4, characterized in that, This includes applications in the preparation of anti-PCSK9 monoclonal antibody drugs or vaccines, novel PCSK9 interfering peptide drugs or vaccines, and reagents or kits for detecting anti-PCSK9 monoclonal antibody drugs.

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