Single-domain antibody capable of being combined with PCSK9 in targeted manner as well as preparation method and application of single-domain antibody

Abstract

The invention provides a single-domain antibody capable of being combined with PCSK9 in a targeted manner as well as a preparation method and application of the single-domain antibody. The single-domain antibody is derived from a heavy-chain antibody variable region of a camel, consists of a framework region (FR region) and a complementary determining region (CDR region), and comprises three CDR regions as shown in SEQ ID NO: 1 to SEQ ID NO: 3 and four FR regions as shown in SEQ ID NO: 6 to SEQ ID NO: 9. The preparation method of the single-domain antibody comprises the following steps: immunizing alpaca by using an eukaryotically expressed PCSK9 antigen, separating mononuclear cells, extracting total RNA (Ribonucleic Acid), and carrying out reverse transcription and nested PCR (Polymerase Chain Reaction) library establishment to obtain a PCSK9 immunized single-domain antibody library; and screening by using a phage display technology, and transforming into an escherichia coli expression system for expression to obtain the PCSK9 monoclonal single-domain antibody strain. The interaction between the single-domain antibody and the recombinant PCSK9 protein is good, and the single-domain antibody can be used for developing antibody drugs for resisting the PCSK9 protein and also can be used for immunologically detecting the PCSK9 level in a sample.

Description

Technical Field

[0001] The present invention relates to the field of antibody drugs in biomedicine, and specifically to a single-domain antibody capable of targeting and binding to PCSK9 (pro-subtilisin / kexin convertase 9), as well as a preparation method and application of the single-domain antibody. Background Art

[0002] At present, the main drugs used on the market to treat cardiovascular and cerebrovascular diseases are cholesterol-lowering drugs. The main drugs used to lower cholesterol are statins. Although statins have performed well in the treatment of cardiovascular diseases, with their widespread use, the disadvantages they produce are gradually being discovered. First, patients treated with statins still have a high residual risk of cardiovascular events; second, a large number of patients cannot tolerate statins, especially patients with familial hypercholesterolemia. Even if they receive the maximum dose of the most effective statin treatment, they still cannot achieve the goal of lowering low-density lipoprotein cholesterol concentrations. Most importantly, statins have a variety of side effects, such as causing abnormal blood sugar, muscle toxicity, memory and cognitive impairment in patients. Severe side effects can lead to rhabdomyolysis and acute renal failure. A considerable number of patients terminated treatment because they could not tolerate the muscle pain caused by the side effects.

[0003] Pro-subtilisin / kexin convertase 9 (PCSK9) is a new proprotein convertase belonging to the subtilisin subfamily and is one of the important influencing factors of autosomal dominant familial hypercholesterolemia. Studies have found that in addition to affecting plasma cholesterol levels and regulating the apoptosis of nerve cells, PCSK9 is also related to inflammatory responses. At present, the research on PCSK9 mainly focuses on its regulatory function on liver lipid metabolism. Previous studies have shown that PCSK9 can regulate liver lipid metabolism by promoting the degradation of low-density lipoprotein receptors (LDL-R) in hepatocytes, thereby affecting the level of low-density lipoprotein cholesterol (LDL-c) in plasma. However, there are two types of mutations in PCSK9, gain-of-function mutations and loss-of-function mutations. Population trials have shown that several PCSK9 "gain-of-function" mutations often occur in individuals with chromosomal dominant hypercholesterolemia, while PCSK9 "loss-of-function" mutations are associated with reduced plasma cholesterol, and individuals with PCSK9 loss-of-function mutations have a significantly reduced risk of coronary heart disease. In 2005, Hobbs et al. reported in the Dallas Heart Study that LDL-c levels in individuals carrying nonsense mutations of PCSK9 were 28% lower than those of the general population; in 2006, Hobbs et al. published the effect of PCSK9 gene mutations on coronary heart disease. The results were based on a survey of atherosclerosis risk. They conducted a 15-year follow-up observation and found that the incidence of coronary heart disease in people who lacked 1 or 2 functional PCSK9 genes was significantly lower than that of the general population. The Copenhagen Heart Study found that functional loss of the PCSK9 gene would reduce LDL-c levels by 11-15% and the prevalence of coronary heart disease by 6-46%. Zimbabwe et al. reported that PCSK9 deletion mutations could reduce LDL-c levels in African women by 27%. PCSK9 inhibitors provide a new treatment model to combat LDL-c and are considered the greatest progress in lipid-lowering after statins. The emergence of PCSK9 inhibitors has brought good news to patients who experience severe side effects when taking statins and patients who cannot achieve LDL-c target levels with statin treatment, such as patients with hereditary hypercholesterolemia.

[0004] In addition to preventing LDL-R recycling, PCSK9 inhibitors can also inhibit NF-κB channels, thereby reducing the risk of acute coronary syndromes such as thrombosis, inflammation, and endothelial cell activation. At present, potential research projects in the field of PCSK9 inhibitors include inhibitory protein antibodies, siRNA, antisense oligonucleotides, and small molecule inhibitors. Monoclonal antibody drugs are currently the main area of ​​PCSK9 inhibitor research due to their strong targeting, high specificity, and low toxicity and side effects. Studies at the animal level have shown that after adding neutralizing anti-PCSK9 antibodies, the expression level of LDL-R in mouse livers increased significantly, and the LDL-c concentration in the blood decreased by 30%. For primates, PCSK9 monoclonal antibodies also showed significant effects, and the effect of reducing LDL-c levels can be maintained for more than several weeks. So far, no obvious toxic side effects have been found in anti-PCSK9 protein monoclonal antibody drugs, and only minor side effects such as local injection reactions, diarrhea, and headaches have been reported. Sanofi's Praluent (Alirocumab), Amgen's Repatha (evolocumab) and Innovent's IBI-306 (tafolecimab) are the only three humanized PCSK9 antibodies approved in the global market.

[0005] Antibody drugs are one of the main directions of new drug research and development at present, and have been widely used in the diagnosis, prevention and treatment of infectious diseases and biological science research. So far, more than 100 antibody drugs have been successfully launched on the market, and 4 of the top 10 drugs in global sales in 2021 are antibody drugs. Since Hamers et al. discovered heavy chain antibodies that naturally lack light chains and constant region I (CH1) in alpaca blood in 1993, the variable region segment of heavy chain antibodies, also known as single-domain antibodies (sdAb), has gradually replaced other small antibodies and become a hot spot for the development of new antibody drugs. sdAb is usually only about 16 kilodaltons, about one-tenth the size of traditional antibodies. It has disulfide bonds inside and a large number of hydrophilic residues on the surface, and has strong resistance to heat and pH. The lack of Fc segments and light chains in sdAb enables it to recognize hidden epitopes or small epitopes that traditional antibodies cannot recognize, and avoids complement reactions; in addition, single-domain antibodies also have many advantages such as high stability, low toxicity, strong solubility, easy target screening, easy direct expression in prokaryotic microorganisms, and good economy. Sequence homology analysis shows that the sdAb germline gene sequence of alpaca sdAb is highly homologous to human VH3, but CDR1 and CDR3 are slightly longer than those of humans, and CDR3 protrudes outward in the tertiary structure, so it is speculated that it has higher antigen binding specificity and affinity. In view of the above advantages, sdAb is gradually being developed as a monoclonal antibody drug for disease diagnosis and treatment, and is widely used in the development of enzyme inhibitors, tumors, infections, and inflammation. However, although the small size of single-domain antibodies provides many advantages for their therapeutic functions, small molecule proteins are easily eliminated in the body. Transforming sdAb into target enzymes, transmembrane proteins or bivalent through genetic engineering can effectively improve antibody activity and stability, etc., to achieve research purposes. In the study of inhibiting viral replication, it was found that the effectiveness of bivalent single-domain antibodies was at least 60 times that of monovalent single-domain antibodies, and the duration of action in animals was longer, effectively delaying the death of animals. The prospects for antibody drugs are huge, but domestic antibody drugs are still in the early stages. Therefore, the development of domestically produced low-cost PCSK9 antibody inhibitors to meet the urgent needs of our people for antibody drugs has far-reaching and positive significance.

[0006] The existing technology for the development of PCSK9 single-domain antibodies focuses on traditional mouse antibodies. Traditional antibodies are difficult to express in large quantities or to humanize them. They are time-consuming, costly, and have a low rate of obtaining effective antibodies, which seriously limits the development of PCSK9 antibody inhibitors. In particular, domestic antibody drugs are just in their infancy and cannot meet the needs of CVD patients at all. Summary of the invention

[0007] Based on the above technical problems, the present invention proposes a single-domain antibody that can target and bind to PCSK9, as well as a preparation method and application of the single-domain antibody.

[0008] The technical solution adopted by the present invention is:

[0009] A single-domain antibody capable of targeting and binding to PCSK9, the single-domain antibody comprises a heavy chain variable region, the heavy chain variable region comprises a framework region and a complementarity determining region; the complementarity determining region comprises complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3, the sequence of complementarity determining region 1 is shown in sequence 1, the sequence of complementarity determining region 2 is shown in sequence 2, and the sequence of complementarity determining region 3 is shown in sequence 3.

[0010] The above-mentioned framework region includes framework region 1, framework region 2, framework region 3 and framework region 4. The sequence of framework region 1 is shown in sequence 4, the sequence of framework region 2 is shown in sequence 5, the sequence of framework region 3 is shown in sequence 6, and the sequence of framework region 4 is shown in sequence 7.

[0011] The sequence of the above heavy chain variable region is shown in Sequence 8.

[0012] An expression vector comprising a polynucleotide sequence as shown in Sequence 9.

[0013] A method for preparing a single-domain antibody that can target and bind to PCSK9, comprising the following steps: selecting an expression vector and a host cell, wherein the polynucleotide sequence contained in the expression vector is as shown in Sequence 9; transforming the expression vector into the host cell, culturing, and expressing the single-domain antibody that can target and bind to PCSK9.

[0014] The above expression vector uses pMECS plasmid, and the host cell uses Escherichia coli HB2151 subtype strain;

[0015] Or the expression vector is selected from pPICZa plasmid, and the host cell is selected from yeast subtype X33 strain;

[0016] Alternatively, the expression vector may be pCDNA3.1 plasmid, and the host cell may be HEK293E cell strain.

[0017] Furthermore, the above preparation method comprises the following steps:

[0018] Alpacas were immunized with eukaryotically expressed PCSK9 antigen, and total RNA was extracted by isolating peripheral blood mononuclear cells. The PCSK9 immune single domain antibody library was obtained through reverse transcription and nested PCR library construction. The PCSK9 antigen was coated on an ELISA plate, and the PCSK9 immune single domain antibody library was screened using phage display technology. The screened single domain antibodies were then transformed into host cells for expression to obtain a monoclonal single domain antibody strain of PCSK9.

[0019] More specifically, the steps of the above preparation method are as follows:

[0020] Step 1: Construction of PCSK9 single domain antibody phage display library

[0021] (11) PCSK9 immunization of alpacas

[0022] Blood was collected from the ear vein of the alpaca before immunization; PCSK9 was mixed with an equal volume of Freund's adjuvant and injected into 3-5 points of the alpaca's neck subcutaneously. Immunization was performed once a month, for a total of 4 times; during each immunization, 10 mL of alpaca peripheral blood was collected in an EDTA anticoagulant tube, and continuously and slowly shaken to mix thoroughly;

[0023] (12) Blood lymphocyte sample separation

[0024] Lymphocytes were isolated from blood samples collected before immunization and after each immunization;

[0025] (13) Total RNA extraction and cDNA synthesis

[0026] Take the frozen lymphocytes, add Trizol, let stand at room temperature, add chloroform, shake, let stand at room temperature, wait for the solution to separate, centrifuge, and collect the upper aqueous phase; add an equal volume of isopropanol, mix well, let stand at room temperature, wait for the nucleic acid to precipitate, centrifuge to remove the supernatant, control the water, dissolve the RNA in nuclease-free water, and take 1 μL for concentration and purity determination;

[0027] RNA was obtained and cDNA was synthesized using a kit;

[0028] (14) Construction of phage display library

[0029] Nest-PCR was used to amplify the V region of the alpaca heavy chain antibody using cDNA as a template;

[0030] After the PCR reaction was completed, the PCR products were detected by agarose gel electrophoresis. The target gene fragment of the first round of PCR was at 700 bp, and the target band was recovered by gel cutting. The second round of PCR was performed, and the target gene fragment was at 500 bp. The target band, i.e., the sdAb fragment, was recovered by gel cutting.

[0031] The sdAb fragment and the vector were double-digested with restriction endonucleases Not I and Pst I from NEB, respectively; the digestion products of the vector and the sdAb fragment were mixed, and ligated with NEB ligase overnight at 4°C to obtain a ligation product;

[0032] (15) Construction of phage display library

[0033] After the ligation product was purified, 1 μL was taken to transform TG competent cells, revived at 37°C for 2 h, and gradiently diluted to 10 1 , 102 , 10 3 , 300 μL were taken to spread on the plate, cultured at 37°C overnight, and the number of clones was calculated;

[0034] Use the same transformation method as above to transform a large number of clones until the number of clones in the library reaches 10 7 All clones were eluted with sterilized LB liquid medium, centrifuged, the precipitate was suspended with sterilized LB liquid medium, an equal volume of glycerol was added, and frozen;

[0035] (16) Library diversity detection

[0036] Randomly select 40 clones from step (5) as templates to perform cloning PCR reaction; use 2% agarose gel electrophoresis to detect PCR products to verify the recombination rate of the constructed PCSK9 single domain antibody library; then sequence them to analyze the diversity of the PCSK9 single domain antibody library;

[0037] (17) Phage amplification and rescue

[0038] A helper phage is used to amplify and rescue the phage display library of PCSK9 single domain antibodies; the monoclonal library preserved in step (5) is inoculated into a culture medium and cultured to a logarithmic growth phase, a helper phage is added, the mixture is allowed to stand at room temperature, centrifuged, the precipitate is suspended with a culture medium, inoculated into a culture medium, and cultured overnight; the next day, the mixture is centrifuged, the supernatant is collected, PEG is added to precipitate the phage, the mixture is allowed to stand on ice, centrifuged, and the precipitate is a PCSK9 single domain antibody phage library;

[0039] Step 2: Panning PCSK9 single domain antibodies using phage display technology

[0040] (21) Affinity PCSK9 single domain antibody phage library panning

[0041] Take the PCSK9 antigen to coat the ELISA plate and incubate overnight; the next day, add the rescued PCSK9 single domain antibody phage and incubate at room temperature; wash the wells with PBST, add triethylamine, incubate at room temperature, and the collected phage is the PCSK9 single domain antibody phage library obtained by affinity panning;

[0042] (22) Amplification and rescue of phage after screening

[0043] The amplification and rescue methods are the same as step (17);

[0044] (23) ELISA to evaluate the enrichment of specific antibodies

[0045] (24) Identification of PCSK9-specific single domain antibody positive clones

[0046] (25) Sequence analysis of positive clones

[0047] The DNA of the positive clone obtained in step (24) was extracted and the inserted fragment was verified by PCR, and the clone verified as positive by PCR was sequenced and analyzed; the sequencing results showed that two nucleotide sequences were obtained, and their amino acid sequences were analyzed. One of the sequences had a typical single-domain antibody structure, including a framework region and a complementary determining region. The nucleotide and amino acid sequences of this single-domain antibody monoclonal clone are as follows:

[0048] PCSK9 single domain antibody protein sdAb-C12, whose amino acid sequence is shown in sequence 8. Among them, the sequence of framework region 1 is shown in sequence 4, the sequence of framework region 2 is shown in sequence 5, the sequence of framework region 3 is shown in sequence 6, and the sequence of framework region 4 is shown in sequence 7; the sequence of complementary determining region 1 is shown in sequence 1, the sequence of complementary determining region 2 is shown in sequence 2, and the sequence of complementary determining region 3 is shown in sequence 3;

[0049] The nucleotide sequence encoding the PCSK9 single domain antibody protein sdAb-C12 is shown in SEQ ID NO: 9;

[0050] Step 3: Induced expression and purification of anti-PCSK9 single domain antibody sdAb-C12

[0051] (31) Construction of PCSK9 single domain antibody expression bacteria

[0052] First, the PCSK9 single-domain antibody monoclonal clone was transferred to the culture medium and cultured at 37°C overnight. The next day, the plasmid was extracted, and after agarose gel electrophoresis and concentration determination, the plasmid containing the PCSK9 single-domain antibody sequence was transformed into the expression bacteria HB2151, coated on the plate, and cultured at 37°C overnight.

[0053] (32) Inducible expression of anti-PCSK9 single domain antibody sdAb-C12

[0054] Pick 5 clones from the plate for clone PCR to verify whether the plasmid has been transferred into the expression strain; pick the positive clones and culture them at 37℃ until OD 600 The pH value was 0.6-0.8, and IPTG was added to induce expression; the bacterial solution was centrifuged, the bacterial precipitate was collected, the precipitate was resuspended with lysis buffer, the bacterial cells were ultrasonically disrupted, and the supernatant of the disrupted cells was collected by centrifugation;

[0055] (33) Purification of anti-PCSK9 single domain antibody sdAb-C12

[0056] PCSK9 single domain antibody sdAb-C12 was obtained by Ni column affinity purification.

[0057] In the above step (14), the names and sequences of the Nest-PCR primers are as follows:

[0058] In the first round, the primer name is CALL001, and its sequence is shown in SEQ ID NO: 10; the primer name is CALL002, and its sequence is shown in SEQ ID NO: 11;

[0059] In the second round, the primer is named sdAb-Back, and its sequence is shown in SEQ ID NO:12; the primer is named sdAb-For, and its sequence is shown in SEQ ID NO:13.

[0060] The use of the above-mentioned single-domain antibody capable of targeting and binding to PCSK9 in the preparation of anti-PCSK9 protein monoclonal antibody drugs; or the use of the single-domain antibody in immunological detection of PCSK9 for non-disease diagnosis and treatment purposes.

[0061] The beneficial technical effects of the present invention are:

[0062] The present invention obtains a single-domain antibody that can target and bind to precursor subtilisin convertase 9 (PCSK9) by limiting the complementary determining region sequence, etc. The single-domain antibody interacts well with the PCSK9 antigen and has the value of continued development. The single-domain antibody of the present invention also has many advantages such as high stability, low toxicity, strong solubility, easy target screening, easy direct expression in prokaryotic microorganisms, and good economy. The single-domain antibody of the present invention interacts well with the recombinant PCSK9 protein, can be used to develop antibody drugs against PCSK9 protein, and can also be used for immunological detection of PCSK9 levels in samples.

[0063] The single-domain antibody preparation method of the present invention utilizes eukaryotically expressed PCSK9 antigen to immunize alpacas, separates peripheral blood mononuclear cells, extracts total RNA, and constructs libraries through reverse transcription and nested PCR to obtain a high-quality PCSK9 immune single-domain antibody library. The PCSK9 antigen is coated on an ELISA plate, the PCSK9 immune single-domain antibody library is screened using phage display technology, and the screened single-domain antibodies are then transformed into an Escherichia coli expression system for mass expression, thereby obtaining a monoclonal single-domain antibody strain of PCSK9 with high affinity in a relatively short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The situation after the purification of PCSK9 single domain antibody was detected by SDS-PAGE protein gel electrophoresis in the embodiment of the present invention;

[0065] Figure 2 This is a schematic diagram of the binding principle of a single domain antibody and an antigen verified by ELISA in an embodiment of the present invention;

[0066] Figure 3 The affinity measurement results of the single domain antibody sdAb-C12 obtained in the examples of the present invention;

[0067] Figure 4This is the data processing result for analyzing the affinity constant of anti-PCSK9 single domain antibody sdAb-C12. DETAILED DESCRIPTION

[0068] The present invention provides a single-domain antibody capable of targeting and binding PCSK9, comprising a heavy chain variable region, wherein the heavy chain variable region comprises a framework region and a complementary determining region. The complementary determining region comprises complementary determining region 1, complementary determining region 2 and complementary determining region 3, wherein the sequence of complementary determining region 1 is shown in sequence 1, the sequence of complementary determining region 2 is shown in sequence 2, and the sequence of complementary determining region 3 is shown in sequence 3.

[0069] Sequence 1 (CDR1): GSTFKRYA (SEQ ID NO: 1); Sequence 2 (CDR2): IERDLVQPSPPFGGLT (SEQ ID NO: 2); Sequence 3 (CDR3): AAGLKYPAPNQLDYDY (SEQ ID NO: 3).

[0070] The above-mentioned framework region includes framework region 1, framework region 2, framework region 3 and framework region 4. The sequence of framework region 1 is shown in sequence 4, the sequence of framework region 2 is shown in sequence 5, the sequence of framework region 3 is shown in sequence 6, and the sequence of framework region 4 is shown in sequence 7.

[0071] Sequence 4 (FR1): LQESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 6); Sequence 5 (FR2): MAWFRQAPGKEREFVAA (SEQ ID NO: 7); Sequence 6 (FR3): YYAASVKGRFTISRDNAKNTVDLQMNSLKPEDAAVYYC (SEQ ID NO: 8); Sequence 7 (FR4): WGQGTQVTVSS (SEQ ID NO: 9).

[0072] The amino acid sequence of the single-domain antibody sdAb-C12 that specifically binds to the PCSK9 antigen is shown in Sequence 8.

[0073] Sequence 8:

[0074] LQESGGGLVQAGGSLRLSCAASGSTFKRYAMAWFRQAPGKEREFVAAIERDLVQPFPPSGGLTYYAASVKGRFTIS RDNAKNTVDLQMNSLKPEDAAVYYCAAGLKYPAPNQLDYDYWGQGTQVTVSS (SEQ ID NO: 4).

[0075] The specific binding properties of an antibody are determined by the complementarity determining region. Therefore, the present invention claims a single domain antibody that specifically binds to the PCSK9 antigen, which includes a heavy chain variable region (sdAb), and the heavy chain variable region (sdAb) is composed of a framework region (FR) and a complementarity determining region (CDR). The complementarity determining region (CDR) includes a complementarity determining region 1 (CDR1), whose sequence is GSTFKRYA (SEQ ID NO: 1); a complementarity determining region 2 (CDR2), whose sequence is IERDLVQPSPPFGGLT (SEQ ID NO: 2); and a complementarity determining region 3 (CDR3), whose sequence is AAGLKYPAPNQLDYDY (SEQ ID NO: 3). The sequence of the heavy chain variable region (sdAb) of a single domain antibody that specifically binds to the PCSK9 antigen is shown in SEQ ID NO: 4, i.e., sequence 8.

[0076] The nucleotide sequence encoding the PCSK9 single domain antibody sdAb-C12 is shown in SEQ ID NO:9.

[0077] 5'-

[0078] CTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTTCAAGAGGTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTTGTAGCCGCTATTGAGCGTGACCTTGTACAACCGTTTCCGCCGAGCGGTGGTTTGACATACTATGCA GCGTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGGTGGATCTGCAAATGAACAGCCTGAAACCTGAGGACGCGGCCGTTTATTACTGCGCAGCAGGATTGAAATATCCTGCCCCTAATCAGCTTGACTATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGCGGCC-3'(SEQ ID NO: 5).

[0079] Considering the degeneracy of the encoding gene, and considering that the specific binding characteristics of the antibody are determined by the complementary determining region. Therefore, the present invention claims a polynucleotide sequence encoding a PCSK9 single domain antibody sdAb-C12, including a nucleotide sequence encoding the complementary determining region shown in SEQ ID NO: 1 to SEQ ID NO: 3. Such a polynucleotide sequence, due to the degeneracy of the encoding gene, the base sequence can be changed, as long as it can encode the complementary determining region shown in SEQ ID NO: 1 to SEQ ID NO: 3. The preferred polynucleotide sequence is shown in SEQ ID NO: 5.

[0080] The present invention provides an expression vector containing the above polynucleotide sequence.

[0081] pET series vectors, pMECS, pPICZα, pFUSE series or pCDNA series vectors can all be used as expression vectors for the polynucleotide sequence of the present invention. Preferably, the expression vector is the phage display vector pMECS.

[0082] The present invention also provides a host cell, containing the above expression vector, capable of expressing a single domain antibody that specifically binds to the PCSK9 antigen. Many cell expression systems such as Escherichia coli HB2151, lactic acid bacteria NZ9000, yeast X33, plant cells, insect cells or mammalian cells HEK293F can be used as host cells for the expression vector of the present invention. Preferably, the host cell is the Escherichia coli HB2151 strain.

[0083] The present invention first immunizes alpacas with PCSK9 antigen expressed by CHO cells (Chinese hamster ovary cells), separates immunized alpacas peripheral blood cells (PBMC), amplifies heavy chain variable region (sdAb) library for PCSK9 antigen, deletes redundant background interference, and greatly improves the efficiency of obtaining effective antibodies. Secondly, the present invention combines the use of phage display technology, which can obtain antibody affinity information more intuitively, and obtains high-affinity PCSK9 single-domain antibody genes in a short time. In addition, the present invention provides a preparation scheme for the above-mentioned PCSK9 single-domain antibody. Since pMECS (phage display vector) is an amber terminator (TAG) between the HA tag and the M13 GIII gene, the ordinary expression system cannot effectively recognize the terminator, thereby effectively expressing the single-domain antibody protein. The present invention optimizes the prokaryotic expression system, expresses and purifies a large amount of PCSK9 single-domain antibodies, and the single-domain antibodies are verified by ELISA and Biacore T200 systems to have high specificity and high affinity for targeting PCSK9, indicating that the PCSK9 single-domain antibodies obtained by the present invention have continued development value.

[0084] Alpacas were immunized with PCSK9 antigen expressed in CHO cells, and peripheral blood cells (PBMC) of immunized alpacas were collected to separate PCSK9-affinity lymphocytes, extract total RNA, and clone the variable region (V region) of the alpaca heavy chain antibody using Nest-PCR technology, which was inserted into the phage plasmid to construct a phage expression library. Then, multiple rounds of screening of PCSK9 antigen were performed using phage display technology. Finally, the high-affinity antibodies obtained by screening were expressed and purified in large quantities in prokaryotic cells, and the affinity and binding constant of the obtained single-domain antibodies were verified by ELISA and Biacore T200.

[0085] The present invention provides a method for preparing a single-domain antibody that can target and bind to PCSK9, and the steps are as follows: selecting an expression vector and a host cell, wherein the polynucleotide sequence contained in the expression vector is as shown in Sequence 9; transforming the expression vector into the host cell, culturing, and expressing the single-domain antibody that can target and bind to PCSK9.

[0086] The above expression vector is selected from pMECS plasmid, and the host cell is selected from Escherichia coli HB2151 subtype strain. Alternatively, the expression vector is selected from pPICZa plasmid, and the host cell is selected from yeast X33 subtype strain. Alternatively, the expression vector is selected from pCDNA3.1 plasmid, and the host cell is selected from HEK293E cell strain.

[0087] Specifically, the preparation method of a single-domain antibody that can target and bind to PCSK9 is to immunize alpacas with eukaryotically expressed PCSK9 antigens, separate peripheral blood mononuclear cells, and extract total RNA. After reverse transcription and nested PCR library construction, a high-quality PCSK9 immune single-domain antibody library is obtained. The PCSK9 antigen is coated on an ELISA plate, and the PCSK9 immune single-domain antibody library is screened using phage display technology, and the screened single-domain antibodies are then transformed into an Escherichia coli expression system for mass expression, thereby being able to obtain a monoclonal single-domain antibody strain of PCSK9 with high affinity in a relatively short time.

[0088] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0089] A method for preparing a single domain antibody capable of targeting and binding to PCSK9, the specific steps are as follows:

[0090] Step 1: Construction of PCSK9 single domain antibody phage display library

[0091] (11) PCSK9 immunization of alpacas

[0092] Mix 500μL of PCSK9 (75μg) with an equal volume of Freund's adjuvant to 1mL and inject it at 3-5 points under the skin of the alpaca's neck. Collect blood from the alpaca's ear vein before immunization. Immunize once a month, for a total of 4 times; collect 10mL of peripheral blood from the alpaca during each immunization. When collecting blood, fix the alpaca's head to one side, shave the skin of the animal's blood collection site first, disinfect it with 75% alcohol, collect blood after it dries, press the jugular vein groove with your fingers, wait for the blood vessel to dilate, disinfect the needle at the blood collection site, collect blood, collect 10mL of blood in an EDTA anticoagulant tube, shake it continuously and slowly immediately, mix it thoroughly, put it on ice, and transport it back to the laboratory.

[0093] (12) Separation of blood lymphocyte samples

[0094] Lymphocytes were isolated from blood samples collected before and after each immunization using the following method:

[0095] i. Add 7 mL of lymphocyte separation medium Ficoll into a 15 mL centrifuge tube.

[0096] ii. Add an equal volume of PBS (1×) or normal saline to fresh whole blood to which anticoagulant (EDTA) has been added, dilute the blood, and mix thoroughly.

[0097] iii. Use a 1 mL pipette to carefully and slowly add an equal volume (7 mL) of diluted blood to the centrifuge tube of lymphocyte separation fluid, and make the mixed liquid above the surface of lymphocyte separation fluid (i.e., the two liquids should not be mixed, and a clear interface should be retained), and centrifuge at 3000 g for 20 min.

[0098] iv. Use a 1mL pipette to carefully transfer the supernatant (plasma sample) into a 1.5mL cell cryopreservation tube, write the animal number and the word plasma on it, put it into a small cloth bag with a rope, and store it in a liquid nitrogen tank.

[0099] v. Use a 1mL pipette to carefully separate the white blood cell layer into a 15mL centrifuge tube; fill up to 15mL with PBS (1×); wash the white blood cells with PBS (1×), centrifuge (3000g for 20min), carefully pour off the supernatant without disturbing the cell clumps at the bottom of the tube, and recover the white blood cells in the remaining 0.1-0.2mL PBS.

[0100] vi. Add 5 times the volume of RNA later, gently dissolve the cell clumps, divide into 2 portions into 1.5 mL cell cryopreservation tubes, and store in liquid nitrogen tank.

[0101] (13) Total RNA extraction and cDNA synthesis

[0102] Take a portion of frozen lymphocytes, add 1mL Trizol, let stand at room temperature for 10min, then add 0.2mL chloroform, shake vigorously, let stand at room temperature, wait for the solution to separate (about 10min), centrifuge at 12,000rpm, and collect the upper aqueous phase. Add an equal volume of isopropanol, mix well, let stand at room temperature for 15min, wait for nucleic acid precipitation, centrifuge at high speed to remove the supernatant, add 1mL of 75% ethanol (prepared with DEPC water) to wash the RNA precipitate, and centrifuge at high speed to remove the supernatant. After controlling the water, dissolve the RNA in nuclease-free water, and take 1μL for concentration and purity determination.

[0103] Take an appropriate amount (7-20 μg) of RNA and use SuperScript TM III First-Strand Synthesis SuperMix (Invitrogen) kit was used for cDNA synthesis, Oligo dT was used as the reverse transcription primer, and the synthesized cDNA was frozen at -20°C.

[0104] (14) Construction of phage display library

[0105] PCR amplification: Using the above-synthesized cDNA as a template, Nest-PCR was used to amplify the V region of the alpaca heavy chain antibody (sdAb). Table 1 shows the names and sequences of the Nest-PCR primers, i.e., the primer information and sequence information used for amplifying the alpaca sdAb fragment.

[0106] Table 1

[0107]

[0108] In Table 1, sequence SEQ ID NO: 10 corresponds to sequence 10, sequence SEQ ID NO: 11 corresponds to sequence 11; sequence SEQ ID NO: 12 corresponds to sequence 12, and sequence SEQ ID NO: 13 corresponds to sequence 13.

[0109] The PCR reaction system is as follows:

[0110] First round: cDNA 2μL; 2×Master Mix 12.5μL; CALL001 0.5μL; CALL002 0.5μL; water to 25μL. 2×Master Mix (purchased from KAPA Biosystems)

[0111] Reaction conditions: 95℃5min; 94℃1min; 57℃1min; 72℃1min per cycle; 72℃7min; amplification for 35 cycles.

[0112] Second round: Template (product of the first round) 40 ng; 2*Master Mix 25 μL; sdAb-For (10 μM) 1 μL; sdAb-Back (10 μM) 1 μL; make up to 50 μL with water.

[0113] Reaction conditions: 95°C for 5 min; 94°C for 45 s; 60°C for 45 s; 72°C for 45 s per cycle; 72°C for 7 min for 25 cycles.

[0114] After the PCR reaction was completed, the PCR product was detected by 1.5% agarose gel electrophoresis. The target gene fragment of the first round of PCR was at 700 bp, and the gel was cut to recover the target band. The second round of PCR was performed, and the target gene fragment was at 500 bp. The gel was cut to recover the target band, that is, the sdAb fragment.

[0115] The sdAb fragment and the vector (pMECS plasmid) were double-digested with restriction endonucleases Not I and Pst I from NEB, respectively. The reaction system was as follows:

[0116] Vector enzyme digestion system: vector 20 μg; Pst I 10 μL; Not I 20 μL; Cutsmart (10× buffer, purchased from NEB) 50 μL; add H 2 0 to 500 μL.

[0117] Fragment digestion system: sdAb fragment 5μg; PstI 7μL; NotI 14μL; Cutsmart (10× buffer) 50μL; add H 2 0 to 500 μL.

[0118] The enzyme digestion was carried out at 37°C overnight, and the agarose gel was electrophoresed and the gel was cut and recovered; the enzyme digestion products of the vector and sdAb fragment were mixed and connected with NEB's ligase at 4°C overnight.

[0119] (15) Construction of phage display library

[0120] After the ligation product was purified by PCR Purification Kit (purchased from Beijing Tiangen Biochemical), 1 μL was taken to transform TG competent cells, revived at 37°C for 2 h, and gradiently diluted to 10 1 , 10 2 , 10 3 , take 300 μL of each plate, culture at 37°C overnight, and calculate the number of clones, about 10 5 clones / plate.

[0121] Use the same transformation method as above to transform a large number of clones until the number of clones in the library reaches 10 7All clones were eluted with sterilized LB liquid medium, centrifuged at 5,000 g for 5 min, the precipitate was suspended with 2 mL of sterilized LB liquid medium, an equal volume of 30% glycerol was added, and frozen at -80°C.

[0122] (16) Library diversity detection

[0123] 40 clones from step (5) were randomly selected as templates for cloning PCR reaction, and the PCR products were detected by 2% agarose gel electrophoresis to verify the recombination rate of the constructed PCSK9 single domain antibody library. Then, the library was sequenced to analyze the diversity of the PCSK9 single domain antibody library. The sequencing results showed that the 40 monoclones had 35 kinds of amino acid sequences, indicating that the constructed library had good diversity.

[0124] (17) Phage amplification and rescue

[0125] The PCSK9 single domain antibody phage library was amplified and rescued using helper phage. The monoclonal library preserved in step (15) was inoculated into 100 mL of culture medium and cultured to the logarithmic growth phase. Helper phage M13 with an MOI (multiplicity of infection) of 20 was added. The mixture was allowed to stand for 30 min at room temperature. After low-speed centrifugation, the precipitate was suspended with culture medium and inoculated into 300 mL of culture medium and cultured overnight. The next day, the mixture was centrifuged at 3,000 g for 30 min, the supernatant was collected, PEG was added to precipitate the phage, the mixture was allowed to stand on ice for 30 min, and the precipitate was centrifuged at 3,000 g for 30 min. The precipitate was the PCSK9 single domain antibody phage library. After the precipitate was suspended with PBS, its titer was determined to be 2.9×10 12 pfu / mL.

[0126] Step 2: Panning PCSK9 single domain antibodies using phage display technology

[0127] (21) Affinity PCSK9 single domain antibody phage library panning

[0128] Take 50ng PCSK9 antigen to coat the ELISA plate and incubate overnight at 4°C. The next day, add the rescued PCSK9 single domain antibody phage and incubate at room temperature for 2h; wash the wells 10 times with PBST, add 100μL triethylamine, incubate at room temperature for 30min, and the collected phages are the PCSK9 single domain antibody phage library obtained by affinity panning. Take 10μL of infected TG1 Escherichia coli cells to coat the plate for determining the number of clones after screening, and the remaining screened phages are used for amplification.

[0129] (22) Amplification and rescue of phage after screening

[0130] The amplification and rescue methods are the same as step (17). The obtained PBS suspension is the amplified phage after the first round of screening, which is stored at 4°C and used for the next round of screening. According to the same screening steps as above, the amount of antigen is gradually reduced, and screening is carried out for 3-4 rounds.

[0131] (23) ELISA to evaluate the enrichment of specific antibodies

[0132] ELISA plates were coated with 100 ng of PCSK9 antigen at 4°C overnight; the next day, 5% BSA was added and blocked at room temperature for 1 hour. The experimental group added the amplified phages after each round of panning, and the control group added an equal amount of wild-type phages and incubated at room temperature for 2 hours. Washed 10 times with PBST to remove unbound phages. HRP-labeled anti-M13 antibody was added and incubated at room temperature for 1 hour; color development solution was added, and the reaction was protected from light for 15 minutes. The absorbance value was measured. The absorbance value gradually increased with the number of panning times and stabilized from the third to the fourth round of panning, indicating that specific antibodies were enriched.

[0133] (24) Identification of PCSK9-specific single domain antibody positive clones

[0134] ELISA plates were coated with 100 ng of PCSK9 antigen and incubated at 4°C overnight. Take the phage-coated plate obtained in the last round of screening, randomly pick 38 monoclones in 1 mL of culture medium, culture at 37°C to the logarithmic phase, and add 1 mM IPTG for induction overnight; the next day, collect the bacterial pellet by centrifugation, break it, centrifuge it at 5,000g for 15 minutes, and collect the supernatant. At the same time, take the ELISA plate, add 2% BSA and block it at room temperature for 1 hour. Add monoclonal supernatant to each well of the experimental group, and add blank TG1 supernatant to each well of the control group, incubate at room temperature for 2 hours. Wash 10 times with PBST, add mouse anti-HA tag antibody, and incubate at room temperature for 1 hour. Wash 3-5 times with PBST, add AP-labeled anti-mouse IgG antibody, and incubate at room temperature for 1 hour. Add substrate, react for 5-20 minutes according to the actual situation, and read the absorbance value on the microplate reader. When the absorbance value is greater than 2.1 (Base line) with the control well, it is judged as a positive clone.

[0135] (25) Sequence analysis of positive clones

[0136] The DNA of the 25 positive clones obtained in step (24) was extracted to perform PCR verification on the inserted fragments, and the clones verified as positive by PCR were sequenced and analyzed. The sequencing results showed that two nucleotide sequences were obtained, and their amino acid sequences were analyzed. One of the sequences had a typical single-domain antibody structure, that is, composed of a framework region (FR1, FR2, FR3 and FR4) and a complementary determining region (CDR1, CDR2 and CDR3). The nucleotide and amino acid sequences of this single-domain antibody monoclonal clone are as follows:

[0137] PCSK9 single domain antibody protein sdAb-C12, whose amino acid sequence is:

[0138] LQESGGGLVQAGGSLRLSCAASGSTFKRYAMAWFRQAPGKEREFVAAIEREIPGHPAWSGLTYYAASVKGRFTISR DNAKNTVDLQMNSLKPEDAAVYYCAAGLKYPAPNQLDYDYWGQGTQVTVSS (SEQ ID NO: 4). Among them, the sequence of framework region 1 is LQESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 6), the sequence of framework region 2 is MAWFRQAPGKEREFVAA (SEQ ID NO: 7), the sequence of framework region 3 is YYAASVKGRFTISRDNAKNTVDLQMNSLKPEDAAVYYC (SEQ ID NO: 8), and the sequence of framework region 4 is WGQGTQVTVSS (SEQ ID NO: 9); the sequence of complementarity determining region 1 is GSTFKRYA (SEQ ID NO: 1), the sequence of complementarity determining region 2 is IERDLVQPSPPFGGLT (SEQ ID NO: 2), and the sequence of complementarity determining region 3 is AAGLKYPAPNQLDYDY (SEQ ID NO: 3).

[0139] The nucleotide sequence encoding the PCSK9 single domain antibody protein sdAb-C12 is:

[0140] 5'-CTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGC ACCTTCAAGAGGTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCCGCTATTGAGCGTGACCTTGTACAACCGTTTCCGCCGAGCGGTGGTTTGACATACTATGCAGCGTCCGTGAAGGGCCGATTCACCATCTCCAGAGAC AATGCCAAGAACACGGTGGATCTGCAAATGAACAGCCTGAAACCTGAGGACGCGGCCGTTTATTACTGCGCAGCAGGATTGAAATATCCTGCCCCTAATCAGCTTGACTATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGCGGCC-3'(SEQ ID NO: 5).

[0141] Step 3: Induced expression and purification of anti-PCSK9 single domain antibody sdAb-C12

[0142] (31) Construction of PCSK9 single domain antibody expression bacteria

[0143] First, the PCSK9 single-domain antibody monoclonal was transferred to the culture medium and cultured overnight at 37°C. The next day, the plasmid was extracted using a Plasmid Extraction Kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.). After agarose gel electrophoresis and concentration determination, the plasmid containing the PCSK9 single-domain antibody sequence was transformed into the expression bacteria HB2151, coated on a plate, and cultured overnight at 37°C.

[0144] (32) Inducible expression of anti-PCSK9 single domain antibody sdAb-C12

[0145] The next day, five clones were selected from the plate for cloning PCR to verify whether the plasmid was transferred into the expression strain. Positive clones were selected and cultured at 37°C until OD 600 The concentration of 1:1 was 0.6-0.8, and IPTG was added to induce expression. The bacterial solution was centrifuged, the bacterial precipitate was collected, the precipitate was resuspended with lysis buffer, the bacterial cells were ultrasonically disrupted, and the supernatant of the disrupted bacterial cells was collected by centrifugation.

[0146] (33) Purification of anti-PCSK9 single domain antibody sdAb-C12

[0147] PCSK9 single domain antibody sdAb-C12 was obtained by Ni column affinity purification. The Ni column was first washed with ultrapure water and then with lysis buffer; the supernatant of the PCSK9 single domain antibody expression bacteria was added to the Ni column at a flow rate of 1 mL / min. The impurities were washed away with 5 column volumes of affinity A solution (20 mM imidazole), and then the target protein was eluted with an equal volume of affinity B solution (250 mM imidazole), and the eluate was collected. Finally, the purified PCSK9 single domain antibody was detected by SDS-PAGE protein gel electrophoresis at a concentration of 13%, as shown in FIG. Figure 1 The molecular weight standard is shown in the left lane of the figure, and the unit is kilodalton (KDa); lanes 1 and 2 are the band positions (~15KDa) of sdAb-C12 under reducing conditions and non-reducing conditions, respectively.

[0148] The above-mentioned single-domain antibody capable of targeting and binding to PCSK9 can be used in the preparation of anti-PCSK9 protein monoclonal antibody drugs; or the single-domain antibody can be used in immunological detection of PCSK9 for non-disease diagnosis and treatment purposes.

[0149] For example, a pharmaceutical composition may be provided, comprising the single domain antibody of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.

[0150] The affinity of PCSK9 and its single domain antibody sdAb-C12 was determined as follows:

[0151] (1) Analysis of the binding of PCSK9 single-domain antibody sdAb-C12 by ELISA

[0152] The experimental group was coated with 50ng PCSK9 protein (PBS blank control group was reserved), and the No-coating control group was a control group without antigen coating, and incubated at 25°C for 2 hours. Then 5% BSA was added to block at room temperature for 1 hour; the purified PCSK9 single domain antibody sdAb-C12 was added to the experimental group, and PBS was added to the blank group, and incubated at room temperature for 2 hours. Figure 2 As shown, wash 8 times with 1×PBST, add mouse anti-his tag antibody, and react at room temperature for 1 hour; wash 5 times with 1×PBST; add substrate 3,3',5,5'-tetramethylbenzidine (TMB), react for about 10 minutes, and read the absorbance at OD450nm on an ELISA reader. ELISA test results are shown in Figure 3 As shown, from Figure 3 The results showed that the sdAb-C12 single domain antibody had good binding to PCSK9, and the binding force was much higher than that of the control group.

[0153] (2) Biacore T200 analysis of affinity constants of anti-PCSK9 single domain antibody sdAb-C12

[0154] After activating the chip, the PCSK9 antigen was coupled to the CM5 chip used in the Biacore machine, and the coupling reaction was stopped after reaching a level of about 800RU. Then 200μl of 1M ethanolamine hydrochloric acid was added to wash away the residual active carboxyl groups; then the machine pumped the gradient diluted PCSK9 single domain antibody sdAb-C12 (250nM-125nM-62.5nM-31.25nM-15.6nM-7.8nM-3.9nM) sequentially through the chip surface at a rate of 30μL / min, binding for 120s and dissociation for 180s; after obtaining the data, the results were processed, and the results are as follows Figure 4 As shown. The parameters of the interaction between the single-domain antibody sdAb-C12 and the antigen PCSK9 are Kon (1 / Ms) = 1.594E+5 as the binding constant, Koff (1 / s) = 0.01063 as the dissociation constant, Rmax (RU) = 32.43 as the maximum binding response value, and KD (M) = 6.669E-8M as the affinity value of the antigen-antibody interaction; indicating that this single-domain antibody has a good interaction with the PCSK9 antigen and is worth further development.

[0155] Sequence 1: GSTFKRYA;

[0156] Sequence 2: IERDLVQPSPPFGGLT;

[0157] Sequence 3: AAGLKYPAPNQLDYDY;

[0158] Sequence 4: LQESGGGLVQAGGSLRLSCAAS;

[0159] Sequence 5: MAWFRQAPGKEREFVAA;

[0160] Sequence 6: YYAASVKGRFTISRDNAKNTVDLQMNSLKPEDAAVYYC;

[0161] Sequence 7: WGQGTQVTVSS;

[0162] Sequence 8:

[0163] LQESGGGLVQAGGSLRLSCAASGSTFKRYAMAWFRQAPGKEREFVAAIERDLVQPFPPSGGLTYYAASVKGRFTIS RDNAKNTVDLQMNSLKPEDAAVYYCAAGLKYPAPNQLDYDYWGQGTQVTVSS;

[0164] Sequence 9:

[0165] 5’-

[0166] CTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTTCAAGAGGTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCCGCTATTGAGCGTGACCTTGTACAACCGTTTCCGCCGAGCGGTGGTTTGACATACTATGCAGCGTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGGTGGATCTGCAAATGAACAGCCTGAAACCTGAGGACGCGGCCGTTTATTACTGCGCAGCAGGATTGAAATATCCTGCCCCTAATCAGCTTGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCC-3’;

[0167] Sequence 10: GTCCTGGCTGCTCTTCTACAAGG;

[0168] sequence11:GGTACGTGCTGTTGAACTGTTCC;

[0169] sequence12:GATGTGCAGCTGCAGGAGTCTGGRGGAGG;

[0170] sequence13:CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT。

Claims

1. A single domain antibody capable of targeting and binding to PCSK9, characterized in that: The single-domain antibody comprises a heavy chain variable region, which includes a framework region and a complementarity determining region; the complementarity determining region includes complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3, the sequence of complementarity determining region 1 is shown in sequence 1, the sequence of complementarity determining region 2 is shown in sequence 2, and the sequence of complementarity determining region 3 is shown in sequence 3.

2. A single domain antibody capable of targeting and binding to PCSK9 according to claim 1, characterized in that: The framework region includes framework region 1, framework region 2, framework region 3 and framework region 4. The sequence of framework region 1 is shown in sequence 4, the sequence of framework region 2 is shown in sequence 5, the sequence of framework region 3 is shown in sequence 6, and the sequence of framework region 4 is shown in sequence 7.

3. A single domain antibody capable of targeting and binding to PCSK9 according to claim 2, characterized in that: The sequence of the heavy chain variable region is shown in Sequence 8.

4. An expression vector, characterized in that: The polynucleotide sequence contained in the expression vector is shown in Sequence 9.

5. A method for preparing a single domain antibody capable of targeting and binding to PCSK9, characterized in that The steps are as follows: selecting an expression vector and a host cell, wherein the polynucleotide sequence contained in the expression vector is as shown in Sequence 9; transforming the expression vector into the host cell, culturing, and expressing a single domain antibody that can target and bind to PCSK9.

6. The method for preparing a single domain antibody capable of targeting and binding to PCSK9 according to claim 5, characterized in that: The expression vector is selected from pMECS plasmid, and the host cell is selected from Escherichia coli HB2151 subtype strain; Or the expression vector is pPICZa plasmid, and the host cell is yeast X33 subtype strain; Or the expression vector is selected from pCDNA3.1 plasmid, and the host cell is selected from HEK293E cell strain.

7. The method for preparing a single domain antibody capable of targeting and binding to PCSK9 according to claim 5, characterized in that The following steps are involved: Alpacas were immunized with eukaryotically expressed PCSK9 antigen, and total RNA was extracted by isolating peripheral blood mononuclear cells. The PCSK9 immune single domain antibody library was obtained through reverse transcription and nested PCR library construction. The PCSK9 antigen was coated on an ELISA plate, and the PCSK9 immune single domain antibody library was screened using phage display technology. The screened single domain antibodies were then transformed into host cells for expression to obtain a monoclonal single domain antibody strain of PCSK9.

8. The method for preparing a single domain antibody capable of targeting and binding to PCSK9 according to claim 7, characterized in that The specific steps are as follows: Step 1: Construction of PCSK9 single domain antibody phage display library (11) PCSK9 immunization of alpacas Blood was collected from the ear vein of the alpaca before immunization; PCSK9 was mixed with an equal volume of Freund's adjuvant and injected into 3-5 points of the alpaca's neck subcutaneously. Immunization was performed once a month, for a total of 4 times; during each immunization, 10 mL of alpaca peripheral blood was collected in an EDTA anticoagulant tube, and continuously and slowly shaken to mix thoroughly; (12) Blood lymphocyte sample separation Lymphocytes were isolated from blood samples collected before immunization and after each immunization; (13) Total RNA extraction and cDNA synthesis Take the frozen lymphocytes, add Trizol, let stand at room temperature, add chloroform, shake, let stand at room temperature, wait for the solution to separate, centrifuge, and collect the upper aqueous phase; add an equal volume of isopropanol, mix well, let stand at room temperature, wait for the nucleic acid to precipitate, centrifuge to remove the supernatant, control the water, dissolve the RNA in nuclease-free water, and take 1 μL for concentration and purity determination; RNA was obtained and cDNA was synthesized using a kit; (14) Construction of phage display library Nest-PCR was used to amplify the V region of the alpaca heavy chain antibody using cDNA as a template; After the PCR reaction was completed, the PCR products were detected by agarose gel electrophoresis. The target gene fragment of the first round of PCR was at 700 bp, and the target band was recovered by gel cutting. The second round of PCR was performed, and the target gene fragment was at 500 bp. The target band, i.e., the sdAb fragment, was recovered by gel cutting. The sdAb fragment and the vector were double-digested with restriction endonucleases Not I and Pst I from NEB, respectively; the digestion products of the vector and the sdAb fragment were mixed, and ligated with NEB ligase overnight at 4°C to obtain a ligation product; (15) Construction of phage display library After the ligation product was purified, 1 μL was taken to transform TG competent cells, revived at 37°C for 2 h, and gradiently diluted to 10 1 , 10 2 , 10 3 , 300 μL were taken to spread on the plate, cultured at 37°C overnight, and the number of clones was calculated; Use the same transformation method as above to transform a large number of clones until the number of clones in the library reaches 10 7 All clones were eluted with sterilized LB liquid medium, centrifuged, the precipitate was suspended with sterilized LB liquid medium, an equal volume of glycerol was added, and frozen; (16) Library diversity detection Randomly select 40 clones from step (5) as templates to perform cloning PCR reaction; use 2% agarose gel electrophoresis to detect PCR products to verify the recombination rate of the constructed PCSK9 single domain antibody library; then sequence them to analyze the diversity of the PCSK9 single domain antibody library; (17) Phage amplification and rescue Amplify and rescue the phage display library of PCSK9 single domain antibody using helper phage; inoculate the monoclonal library preserved in step (5) into the culture medium and culture it to the logarithmic growth phase, add helper phage, stand at room temperature, centrifuge, suspend the precipitate with culture medium, inoculate it into the culture medium, and culture it overnight; The next day, the mixture was centrifuged, the supernatant was collected, PEG was added to precipitate the phage, the mixture was placed on ice, and centrifuged to precipitate the PCSK9 single domain antibody phage library; Step 2: Panning PCSK9 single domain antibodies using phage display technology (21) Affinity PCSK9 single domain antibody phage library panning Take the PCSK9 antigen to coat the ELISA plate and incubate overnight; the next day, add the rescued PCSK9 single domain antibody phage and incubate at room temperature; wash the wells with PBST, add triethylamine, incubate at room temperature, and the collected phage is the PCSK9 single domain antibody phage library obtained by affinity panning; (22) Amplification and rescue of phage after screening The amplification and rescue methods are the same as step (17); (23) ELISA to evaluate the enrichment of specific antibodies (24) Identification of PCSK9-specific single domain antibody positive clones (25) Sequence analysis of positive clones The DNA of the positive clone obtained in step (24) was extracted and the inserted fragment was verified by PCR, and the clone verified as positive by PCR was sequenced and analyzed; the sequencing results showed that two nucleotide sequences were obtained, and their amino acid sequences were analyzed. One of the sequences had a typical single-domain antibody structure, including a framework region and a complementary determining region. The nucleotide and amino acid sequences of this single-domain antibody monoclonal clone are as follows: PCSK9 single domain antibody protein sdAb-C12, whose amino acid sequence is shown in sequence 8. Among them, the sequence of framework region 1 is shown in sequence 4, the sequence of framework region 2 is shown in sequence 5, the sequence of framework region 3 is shown in sequence 6, and the sequence of framework region 4 is shown in sequence 7; the sequence of complementary determining region 1 is shown in sequence 1, the sequence of complementary determining region 2 is shown in sequence 2, and the sequence of complementary determining region 3 is shown in sequence 3; The nucleotide sequence encoding the PCSK9 single domain antibody protein sdAb-C12 is shown in SEQ ID NO: 9; Step 3: Induced expression and purification of anti-PCSK9 single domain antibody sdAb-C12 (31) Construction of PCSK9 single domain antibody expression bacteria First, the PCSK9 single-domain antibody monoclonal clone was transferred to the culture medium and cultured at 37°C overnight. The next day, the plasmid was extracted, and after agarose gel electrophoresis and concentration determination, the plasmid containing the PCSK9 single-domain antibody sequence was transformed into the expression bacteria HB2151, coated on the plate, and cultured at 37°C overnight. (32) Inducible expression of anti-PCSK9 single domain antibody sdAb-C12 Pick 5 clones from the plate for clone PCR to verify whether the plasmid has been transferred into the expression strain; pick the positive clones and culture them at 37℃ until OD 600 The pH value was 0.6-0.8, and IPTG was added to induce expression; the bacterial solution was centrifuged, the bacterial precipitate was collected, the precipitate was resuspended with lysis buffer, the bacterial cells were ultrasonically disrupted, and the supernatant of the disrupted cells was collected by centrifugation; (33) Purification of anti-PCSK9 single domain antibody sdAb-C12 PCSK9 single domain antibody sdAb-C12 was obtained by Ni column affinity purification.

9. The method for preparing a single domain antibody capable of targeting and binding to PCSK9 according to claim 8, characterized in that: In step (14), the names and sequences of the Nest-PCR primers are as follows: In the first round, the primer name is CALL001, and its sequence is shown in SEQ ID NO: 10; the primer name is CALL002, and its sequence is shown in SEQ ID NO: 11; In the second round, the primer is named sdAb-Back, and its sequence is shown in SEQ ID NO:12; the primer is named sdAb-For, and its sequence is shown in SEQ ID NO:

13.

10. Use of a single domain antibody capable of targeting and binding to PCSK9 as claimed in any one of claims 1 to 3 in the preparation of anti-PCSK9 protein monoclonal antibody drugs; or use of a single domain antibody in immunological detection of PCSK9 for non-disease diagnosis and treatment purposes.

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