CN116333149A

CN116333149A - Single-domain antibody combined with PCSK9 antigen and preparation method thereof

Info

Publication number: CN116333149A
Application number: CN202310380811.8A
Authority: CN
Inventors: 李新洋; 王青青; 舒羽; 陈娟; 霍瑞; 赵相杰; 陆居旭; 洪军; 于可豪
Original assignee: Henan University of Urban Construction
Current assignee: Henan University of Urban Construction
Priority date: 2023-04-11
Filing date: 2023-04-11
Publication date: 2023-06-27

Abstract

The invention relates to the technical field of antibodies of biomedical species, in particular to a single-domain antibody combined with PCSK9 antigen and a preparation method thereof. According to the invention, peripheral blood mononuclear cells are separated, total RNA is extracted, and a high-quality PCSK9 immune single-domain antibody library is obtained through reverse transcription and nested PCR library construction. The PCSK9 antigen is coated on an ELISA plate, a phage display technology is used for screening PCSK9 immune single-domain antibody library, and the screened single-domain antibody is converted into an escherichia coli expression system for mass expression, so that a monoclonal single-domain antibody strain of PCSK9 with high affinity can be obtained in a relatively short time.

Description

Single-domain antibody combined with PCSK9 antigen and preparation method thereof

Technical Field

The invention relates to the technical field of antibodies of biomedical species, in particular to a single-domain antibody combined with PCSK9 antigen and a preparation method thereof.

Background

Currently, drugs for reducing cholesterol in the market mainly include statins (statins), cholesterol absorption inhibitors, probucol, and the like. Although statins are excellent in the treatment of cardiovascular diseases, the disadvantages that result are gradually discovered as they are widely used. First, statin treated patients still have a high residual risk of cardiovascular events, with a risk of occurrence up to 22.4% within 2 years; second, a large number of patients cannot tolerate statin, especially familial hypercholesterolemia, and even with the highest dose of most effective statin treatment, the goal of lowering low-density lipoprotein cholesterol (low-density lipoprotein cholesterol, LDL-c) concentration is not achieved; most importantly, statins have various side effects, such as causing patient blood glucose abnormalities, muscle toxicity, memory and cognitive impairment, etc., with up to 20% of the incidence of side effects, serious side effects leading to rhabdomyolysis and acute renal failure, and a significant portion of patients terminate treatment because they cannot tolerate muscle pain from the side effects.

Proprotein convertase subtilisin 9 (Proprotein convertase subtilisin/kexin type 9, pcsk 9), a novel proprotein convertase belonging to the subtilisin subfamily, is one of the important influencing factors for autosomal dominant familial hypercholesterolemia. It was found that PCSK9 has a certain correlation with inflammatory responses in addition to affecting plasma cholesterol levels and modulating neuronal apoptosis. Current research on PCSK9 is mainly focused on the regulatory functions of liver lipid metabolism. Previous studies have shown that PCSK9 can regulate liver lipid metabolism by promoting the degradation of low density lipoprotein receptor (low-density lipoprotein receptor, LDL-R) of hepatocytes, thereby affecting the level of low density lipoprotein cholesterol (low-density lipoprotein cholesterol, LDL-c) in plasma. However, PCSK9 has two mutation types, a gain-of-function mutation and a loss-of-function mutation. Group experiments show that several mutations of PCSK9 "gain function" often occur in individuals with chromosomal dominant hypercholesterolemia, whereas mutations of PCSK9 "loss of function" are associated with a decrease in plasma cholesterol, and the risk of coronary heart disease in individuals with PCSK9 loss of function mutations is significantly reduced. In 2005, hobbs et al reported on Dallas Heart Study that LDL-c levels in individuals carrying PCSK9 nonsense mutant genes would be 28% lower than in average. Copenhagen Heart Study functional deletion of the PCSK9 gene was found to reduce LDL-c levels by 11-15% and coronary heart disease prevalence by 6-46%. Zimbabwe et al reported that deletion mutations in PCSK9 reduced LDL-c levels by 27% in African females. PCSK9 inhibitors offer a completely new therapeutic modality against LDL-c, considered as the greatest advancement in the field of lipid lowering after statins. The appearance of PCSK9 inhibitors brings good news to patients with serious side effects when the statin is taken and patients with the statin treatment failing to reach the LDL-c target level, such as hereditary hypercholesterolemia patients.

The PCSK9 inhibitor can inhibit LDL-R recovery and NF- κB channel, so as to reduce the risk of thrombus, inflammation, vascular endothelial cell activation and other acute coronary syndromes. Potential research projects in the field of PCSK9 inhibitors include inhibitor protein antibodies, siRNA, antisense oligonucleotides, small molecule inhibitors and the like. The monoclonal antibody medicament is a main field of research on the PCSK9 inhibitor at present because of the characteristics of strong targeting, high specificity, low toxic and side effects and the like. Studies at animal level showed that LDL-R expression levels in mouse liver were significantly increased and LDL-c concentration in blood was reduced by 30% after addition of neutralizing anti-PCSK 9 antibody. PCSK9 monoclonal antibodies also show significant effects in primates, and the effect of lowering LDL-c levels can be maintained for more than several weeks. Up to now, no obvious toxic and side effects of anti-PCSK 9 protein monoclonal antibodies are found, and only slight side effects such as local injection reaction, diarrhea and headache are reported. Praluent (Alirocumab) of Sainofil, repatha (evolocumab) of Ind and IBI-306 (tafoleimab) of Xindada are currently the only three approved humanized PCSK9 antibodies in the global market.

Antibody drugs are one of the main directions of the development of new drugs at present, and have been widely used in the fields of diagnosis, prevention and treatment of infectious diseases and bioscience research. Up to now, 100 remaining antibody drugs have been successfully marketed, 4 of the top 10 drugs sold worldwide in 2021 are antibody drugs. After natural deletion of heavy chain antibodies of light chain and constant region I (CH 1) regions was found in alpaca blood from Hamers et al in 1993, the variable region segments of heavy chain antibodies, also called single domain antibodies (Nb), gradually replaced other small antibodies, and gradually became a hotspot for the development of novel antibody drugs. Nb is usually only about 15KDa, about one tenth of the size of conventional antibodies, with disulfide bonds inside, a large number of hydrophilic residues on the surface, and a strong resistance to heat and pH; nb lacks Fc-segment and light chain properties that enable it to recognize cryptic or small epitopes that traditional antibodies cannot recognize, and avoids complement reactions; in addition, the single domain antibody has the advantages of high stability, low toxicity, strong solubility, easy target screening, easy direct expression in prokaryotic microorganism, good economy and the like. Sequence homology analysis showed that the VHH germline gene sequence of alpaca Nb was highly homologous to human VH3, but CDR1 and CDR3 were slightly longer than human, CDR3 protruding outward in tertiary structure, thus presumably higher specificity and affinity for antigen binding. In view of the above advantages, nb is being developed as a monoclonal antibody in disease diagnosis and treatment, and is widely used in development of inhibitors of enzymes, and biological inhibitors of tumors, infections, and inflammations. However, the small volume of single domain antibodies provides many advantages for their therapeutic function, but small molecule proteins are extremely easily eliminated in vivo. The Nb is modified into target enzyme, transmembrane protein or bivalent through genetic engineering, so that the activity and stability of the antibody can be effectively improved, and the research purpose can be achieved. In studies on inhibition of viral replication, it was found that bivalent single domain antibodies were at least 60-fold more effective than monovalent single domain antibodies and had a longer duration of action in animals, effectively delaying the death time of animals. The prospect of antibody drugs is huge, but domestic antibody drugs are still in an early stage. Therefore, the development of the domestic low-cost PCSK9 antibody inhibitor meets the urgent requirements of Chinese citizens on antibody medicines and has profound and positive significance.

The development of PCSK9 single-domain antibodies in the prior art is concentrated on murine traditional antibodies, the traditional antibodies are difficult to express in a large amount or humanized, the time and the cost are long, the effective antibody yield is low, the development of PCSK9 antibody inhibitors is severely limited, and particularly, domestic antibody medicines are just in a starting stage and can not meet the requirements of CVD patients.

Disclosure of Invention

The invention aims to provide a single domain antibody combined with PCSK9 antigen and a preparation method thereof, so as to solve the problems in the background technology.

In order to achieve the above purpose, the present invention provides the following technical solutions:

a single domain antibody that binds PCSK9 antigen, the single domain antibody comprising a heavy chain antibody variable region (VHH) consisting of a Framework Region (FR) and a Complementarity Determining Region (CDR), wherein the Complementarity Determining Region (CDR) comprises complementarity determining region 1 (CDR 1) having the sequence GRTFSDYA (SEQ ID NO: 1); complementarity determining region 2 (CDR 2) having the sequence IGWSGGQT (SEQ ID NO: 2); complementarity determining region 3 (CDR 3) having the sequence AASFLVIPGTVKTRYDS (SEQ ID NO: 3).

The amino acid sequence of the heavy chain antibody variable region (VHH) is shown in SEQ ID NO: 4.

The polynucleotide sequence comprises a sequence encoding the sequence of SEQ ID NO:1 to SEQ ID NO:3, and a nucleotide sequence of the complementarity determining region shown in (3).

The polynucleotide sequence comprises a sequence encoding the sequence of SEQ ID NO:4, and a heavy chain antibody variable region (VHH).

The polynucleotide sequence is shown in SEQ ID NO: shown at 5.

An expression vector comprising a polynucleotide sequence.

A host cell comprising an expression vector capable of expressing a single domain antibody that specifically binds to PCSK9 antigen.

The pharmaceutical composition comprises a single domain antibody, and a pharmaceutically acceptable carrier, diluent or excipient.

The method comprises the following steps: transforming an expression vector comprising a polynucleotide sequence into an expression host cell, culturing, performing substantial expression and purification of the single domain antibody;

preferably, the expression vector is a pMECS plasmid and the host cell is the strain e.coli HB 2151;

preferably, the expression vector is a ppiczα plasmid and the host cell is a yeast X33 strain;

preferably, the expression vector is a pcdna3.4 plasmid and the host cell is a HEK293F cell line.

The single domain antibody is used for preparing anti-PCSK 9 protein monoclonal antibodies or is used for immunologically detecting PCSK9 for the purpose of non-disease diagnosis and treatment.

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

according to the invention, peripheral blood mononuclear cells are separated, total RNA is extracted, and a high-quality PCSK9 immune single-domain antibody library is obtained through reverse transcription and nested PCR library construction. The PCSK9 antigen is coated on an ELISA plate, a phage display technology is used for screening PCSK9 immune single-domain antibody library, and the screened single-domain antibody is converted into an escherichia coli expression system for mass expression, so that a monoclonal single-domain antibody strain of PCSK9 with high affinity can be obtained in a relatively short time.

Drawings

FIG. 1 is a schematic diagram showing the measurement of the binding between PCSK9 and its single-domain antibody VHHH12 in example 4 of the present invention;

FIG. 2 is a schematic diagram showing the results of the assay for binding of PCSK9 to its single-domain antibody VHHH12 in example 4 of the present invention;

FIG. 3 is a schematic diagram showing the affinity assay results of Biacore T200 assay single domain antibodies VHHH12 and PCSK9 in example 4 of the present invention.

Detailed Description

The invention will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present invention. However, one skilled in the art will readily recognize that some of the features may be omitted, or replaced by other elements, materials, or methods in different situations.

Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.

According to the invention, firstly, the alpaca is immunized by using PCSK9 antigen expressed by CHO cells (Chinese hamster ovary cells), the peripheral blood cells (PBMC) of the alpaca after immunization are separated, a heavy chain variable region (VHH) library aiming at the PCSK9 antigen is amplified from the alpaca peripheral blood cells, and redundant background interference is deleted, so that the efficiency of obtaining effective antibodies is greatly improved. And secondly, the phage display technology is combined and used, so that the antibody affinity information can be intuitively obtained, and the single-domain antibody gene of the PCSK9 with high affinity can be obtained in a short time. In addition, the present invention provides a preparation scheme of the above PCSK9 single domain antibody, since pMECS (phage display vector) is an amber Terminator (TAG) between the HA TAG and M13 GIII gene, the conventional expression system cannot recognize the terminator effectively, thereby expressing the single domain antibody protein effectively. The invention optimizes a prokaryotic expression system, and carries out mass expression and purification on the PCSK9 single-domain antibody, and the single-domain antibody has high specificity and high affinity of targeting PCSK9 through ELISA and Biacore T200 system verification, which shows that the PCSK9 single-domain antibody obtained by the invention has continuous development value.

Immunizing alpaca with PCSK9 antigen expressed in CHO cells, collecting peripheral blood cells (PBMC) of the immunized alpaca, separating PCSK9 affinity lymphocyte from the peripheral blood cells, extracting total RNA, cloning a variable region (V region) of alpaca heavy chain antibody by using a Nest-PCR technology, inserting the variable region into phage plasmid to construct phage expression library, carrying out multiple rounds of screening on PCSK9 antigen by using phage display technology, carrying out mass expression purification on high affinity antibody obtained by screening in prokaryotic cells, and verifying the affinity and binding constant of the obtained single-domain antibody by ELISA and Biacore T200.

In one embodiment of the invention, the single domain antibody VHHH12, which specifically binds to PCSK9 antigen, has the amino acid sequence:

QVQLQESGGGLVQAGGSLRLSCAASGRTFSDYAVGWFRQAPGKEREFVAGIGWSGGQTTYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVCAASFLVIPGTVKTRYDSWGQGTQVTVPA(SEQ ID NO：4)。

wherein, the sequence of the framework region 1 is QVQLQESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 6), the sequence of the framework region 2 is VGWFRQAPGKEREFVAG (SEQ ID NO: 7), the sequence of the framework region 3 is TYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVC (SEQ ID NO: 8), the sequence of the framework region 4 is WGQGTQVTVPA (SEQ ID NO: 9), the sequence of the complementarity determining region 1 is GRTFSDYA (SEQ ID NO: 1), the sequence of the complementarity determining region 2 is IGWSGGQT (SEQ ID NO: 2), and the sequence of the complementarity determining region 3 is AASFLVIPGTVKTRYDS (SEQ ID NO: 3).

It is well known that the specific binding characteristics of antibodies are determined by the complementarity determining regions. Accordingly, one aspect of the present invention claims a single domain antibody that specifically binds to PCSK9 antigen comprising a heavy chain variable region (VHH) consisting of a Framework Region (FR) and a Complementarity Determining Region (CDR), wherein the Complementarity Determining Region (CDR) comprises complementarity determining region 1 (CDR 1) having the sequence GRTFSDYA (SEQ ID NO: 1); complementarity determining region 2 (CDR 2) having the sequence IGWSGGQT (SEQ ID NO: 2); complementarity determining region 3 (CDR 3) having the sequence AASFLVIPGTVKTRYDS (SEQ ID NO: 3). In a preferred embodiment, a single domain antibody that specifically binds PCSK9 antigen has the sequence of the heavy chain variable region (VHH) set forth in SEQ ID NO: 4.

In one embodiment of the invention, the nucleotide sequence encoding PCSK9 single domain antibody VHHH12 is:

5’-caggtgcagctgcaggagtctggaggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggacgcacgttcagcgactatgccgtgggctggttccgccaggctcccgggaaggagcgtgagtttgtagctggtataggctggagtggcggtcaaacaacctatgcagactccgtgaagggccgattcaccatctccagagacaacgccaaagacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttatgtctgtgcagcttcatttttggttatccccggcaccgtgaaaactcggtatgacagctggggccagggaacccaggtcaccgttccagcg-3’(SEQ ID NO：5)。

however, the specific binding characteristics of an antibody are determined by the complementarity determining regions in view of the degeneracy of the encoding gene. Accordingly, one aspect of the invention claims a polynucleotide sequence encoding PCSK9 single domain antibody VHHH12, comprising a sequence encoding the above-described SEQ ID NO:1 to SEQ ID NO:3, and a nucleotide sequence of the complementarity determining region shown in (3). Such polynucleotide sequences, due to the degeneracy of the encoding gene, may vary in base sequence as long as they are capable of encoding the sequence of SEQ ID NO:1 to SEQ ID NO:3, and a complementary determining region shown in FIG. 3. In a preferred embodiment, the polynucleotide sequence is as set forth in SEQ ID NO: shown at 5.

In one embodiment of the invention, an expression vector is provided comprising a polynucleotide sequence of the invention. Those skilled in the art will recognize that pET series vectors, pCYT, pMECS, pMG e, pPICZα, pUSE series, pCDNA series, etc. vectors may be used as expression vectors for the polynucleotide sequences of the present invention within the spirit of the present invention. In a preferred embodiment, the expression vector is the phage display vector pMECS (the present laboratory deposit).

In one embodiment of the invention, a host cell is provided comprising an expression vector of the invention capable of expressing a single domain antibody that specifically binds to PCSK9 antigen. Those skilled in the art will recognize that a variety of cell expression systems such as E.coli HB2151, lactic acid bacteria NZ9000, yeast X33, plant cells, insect cells or mammalian cells HEK293F, etc., can be used as host cells for the expression vectors of the invention. In a preferred embodiment, the host cell is E.coli strain HB2151 (deposited in this laboratory).

In one embodiment of the invention, a pharmaceutical composition is provided comprising a single domain antibody of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

The pharmaceutical compositions of the invention may be prepared by methods well known in the art (e.g., remington: the Science and Practice of Pharmacy,19th ed. (1995), a. Gennaro et al, mack Publishing co.) and comprise a single domain antibody as disclosed herein and one or more pharmaceutically acceptable carriers, diluents or excipients.

In one embodiment of the invention, there is provided a method of preparing a single domain antibody of the invention comprising: an expression vector comprising the polynucleotide sequence of the present invention is transformed into an expression host cell, cultured, and subjected to the above-described massive expression and purification of the single domain antibody. In a preferred embodiment, the expression vector is the phage display vector pMECS plasmid and the host cell is E.coli strain HB 2151.

The single domain antibody of the invention can be used for preparing anti-PCSK 9 protein monoclonal antibodies and can also be used for immunological detection of PCSK9. Thus, in one embodiment of the invention, there is provided the use of a single domain antibody of the invention in the manufacture of an anti-PCSK 9 protein mab or in the immunological detection of PCSK9 for non-diagnostic therapeutic purposes.

The invention utilizes eukaryotic expressed PCSK9 antigen to immunize alpaca, extracts total RNA by separating peripheral blood mononuclear cells, and obtains a high-quality PCSK9 immune single-domain antibody library through reverse transcription and nested PCR library construction. The PCSK9 antigen is coated on an ELISA plate, a phage display technology is used for screening PCSK9 immune single-domain antibody library, and the screened single-domain antibody is converted into an escherichia coli expression system for mass expression, so that a monoclonal single-domain antibody strain of PCSK9 with high affinity can be obtained in a relatively short time.

The following detailed description of the present invention is provided by way of example only, and should not be construed as limiting the scope of the invention.

EXAMPLE 1 construction of PCSK9 Single-domain antibody phage display library

(1) PCSK9 immunized alpaca

mu.L of PCSK9 (50. Mu.g) was mixed with an equal volume of Freund's adjuvant to 1mL, injected subcutaneously into the neck of alpaca at 3-5 sites, and collected from the limbic vein of alpaca prior to immunization. Once a month, total immunization was injected 4 times; at each immunization, 10mL of alpaca peripheral blood was taken. During blood collection, alpaca heads are fixed to one side, skin of an animal blood collection part is shaved firstly, 75% alcohol is disinfected, blood collection is carried out after drying, jugular vein groove is pressed by fingers, blood collection is carried out after blood vessels are angry, needle insertion and blood collection are carried out at the blood collection part, 10mL of blood is collected in an EDTA anticoagulation tube, and the blood collection device is immediately, continuously and slowly shaken, fully mixed, placed on ice and transported back to a laboratory.

(2) Blood lymphocyte sample separation

Lymphocytes were isolated from blood samples collected before and after each immunization as follows:

i. 7mL of lymphocyte separation liquid Ficoll is added into a 15mL centrifuge tube;

adding equal volume PBS (1X) or physiological saline into fresh whole blood added with anticoagulant (EDTA), diluting the blood, and fully mixing;

slowly adding an equal volume (7 mL) of diluted blood into a centrifuge tube of lymphocyte separation liquid by using a 1mL pipettor carefully, and keeping the mixed liquid above the liquid level of the lymphocyte separation liquid (namely, the two liquids are not mixed and a clear interface is kept), and centrifuging for 20min at 3000 g;

carefully transferring the supernatant (plasma sample) into a 1.5mL cell cryopreservation tube by using a 1mL pipetting gun, writing animal numbers and plasma word patterns, putting into a small cloth bag with ropes, and storing in a liquid nitrogen tank;

carefully separating the leukocyte layer into a 15mL centrifuge tube using a 1mL pipette; fill PBS (1X) to 15mL; washing white blood cells with PBS (1X), centrifuging (3000 g centrifuging for 20 min), carefully pouring off the supernatant, not stirring the cell pellet at the bottom of the tube, recovering white blood cells in the remaining 0.1-0.2mL PBS;

adding 5 times volume of RNA later, lightly mixing and dissolving cell blocks, dividing into 2 parts to 1.5mL of cell freezing tube, and storing in a liquid nitrogen tank.

(3) Total RNA extraction and cDNA Synthesis

Taking a part of frozen lymphocytes, adding 1mL of Trizol, standing at room temperature for 10min, adding 0.2mL of chloroform, shaking vigorously, standing at room temperature, layering (about 10 min) the solution, centrifuging at 12,000rpm, collecting an upper water phase, adding equal volume of isopropanol, mixing uniformly, standing at room temperature for 15min, standing at room temperature, removing the supernatant by high-speed centrifugation, adding 1mL of 75% ethanol (prepared by DEPC water) into RNA precipitate for washing, removing the supernatant by high-speed centrifugation, drying, dissolving RNA with water without nuclease, and taking 1 mu L of water for concentration and purity measurement respectively;

taking a proper amount (7-20 mug) of RNA and adopting SuperScript ^TM III First-Strand Synthesis SuperMix (Invitrogen) kit for cDNA synthesis, reverse transcription primer with Oligo dT, cDNA synthesized was frozen at-20deg.C.

(4) Phage display library construction

And (3) PCR amplification: the V region (VHH) of alpaca heavy chain antibody was amplified using Nest-PCR using the cDNA synthesized as described above as a template, and the names and sequences of the Nest-PCR primers are shown in Table 1.

TABLE 1 primer information for amplification of alpaca VHH fragments

The PCR reaction system is as follows:

first round: cDNA 2. Mu.L; 2 XMaster Mix 12.5. Mu.L; CALL 001.5. Mu.L; CALL 002.5. Mu.L; CALL005 0.5 μL; CALL 006.5 μl; the water was made up to 25 μl.2 XMaster Mix (available from KAPAbiosystems)

Reaction conditions: 95 ℃ for 5min;94 ℃ for 1min;57 ℃ for 1min; 1min at 72℃for each cycle; 7min at 72 ℃; amplification was performed for 35 cycles.

A second wheel: template (first round product) 40ng;2 x master Mix 25 μl; VHH-For (10. Mu.M) 1. Mu.L; VHH-Back (10. Mu.M) 1. Mu.L; the water was made up to 50 μl.

Reaction conditions: 95 ℃ for 5min;94 ℃ for 45s; 45s at 60 ℃; 45s at 72℃per cycle; amplification was performed for 25 cycles at 72℃for 7 min.

After the PCR reaction is finished, detecting the PCR product by using 1.5% agarose gel electrophoresis, cutting the target gene fragment of the first round of PCR at 700bp, recovering the target band, carrying out the second round of PCR, cutting the target gene fragment at 500bp, and recovering the target band, namely the VHH fragment.

The VHH fragment and vector (pMECS plasmid, stored in this laboratory) were double digested with NEB restriction enzymes Not I and Pst I, respectively, and the reaction system was as follows:

carrier enzyme cutting system: 20 μg of carrier; pst I10. Mu.L; not I20. Mu.L; cutsmart (10 Xbuffer, available from NEB Co.) 50. Mu.L; H2O was added to 500. Mu.L.

Fragment enzyme digestion system: 5 μg of VHH fragment; pstI 7. Mu.L; notI 14. Mu.L; cutsmart (10 Xbuffer) 50. Mu.L; H2O was added to 500. Mu.L.

Enzyme cutting overnight at 37 ℃, agarose gel electrophoresis, and gel cutting and recovery; the vector and the cleavage products of the VHH fragment were mixed and ligated overnight at 16℃with NEB ligase.

(5) Construction of phage display library

After purification of the ligation product by PCR Purification Kit (purchased from Beijing Tiangen biochemistry), 1. Mu.L of transformed TG competent cells were recovered at 37℃for 2h, and the mixture was diluted to 101, 102, 103 in a gradient, 300. Mu.L of coated plates were used, and cultured overnight at 37℃to calculate the number of clones, about 105 clones/plate.

The same transformation method is adopted, and a large amount of transformation is carried out until the clone number of the library reaches more than 107. All clones were eluted with sterilized LB liquid medium, centrifuged at 5,000g for 5min, the pellet was suspended in 2mL of sterilized LB liquid medium, and an equal volume of 30% glycerol was added for frozen storage at-80 ℃.

(6) Library diversity detection

Randomly picking 30 clones in the step (5) as templates, performing clone PCR reaction, detecting PCR products by 1.5% agarose gel electrophoresis, and verifying the recombination rate of the constructed PCSK9 single-domain antibody library. And then sequencing the PCSK9 single-domain antibody library, analyzing the diversity of the PCSK9 single-domain antibody library, and sequencing results show that 15 monoclonal antibodies have 13 amino acid sequences, thus indicating that the constructed library has better diversity.

(7) Phage amplification and rescue

Phage libraries of PCSK9 single domain antibodies were amplified and rescued using helper phage. And (3) inoculating the monoclonal library stored in the step (5) into 100mL of culture medium for culturing to a logarithmic growth phase, adding auxiliary phage M13 (stored in the laboratory) with MOI (multiplicity of infection ) of 20, standing at room temperature for 30min, centrifuging at a low speed, suspending the precipitate with culture medium, inoculating into 300mL of culture medium, and culturing overnight. The following day, centrifugation at 3,000g for 30min, collecting supernatant, adding PEG to precipitate phage, standing on ice for 30min, centrifuging at 3,000 for 30min, precipitating to obtain PCSK9 single domain antibody phage library, suspending the precipitate with PBS, and measuring the titer of 2.9X104 pfu/mL.

EXAMPLE 2 panning of PCSK9 Single-Domain antibodies Using phage display technology

(1) Affinity PCSK9 single domain antibody phage library panning

100ng of PCSK9 antigen was coated onto ELISA plates and incubated overnight at 4 ℃. The next day, adding the rescued PCSK9 single-domain antibody phage, and incubating for 2 hours at room temperature; PBST washing holes for 10 times, adding 100 mu L of triethylamine, incubating for 30min at room temperature, and collecting phage, namely affinity washing the obtained PCSK9 single-domain antibody phage library; mu.L of TG 1-infected E.coli cells were plated for determination of the number of clones after screening, and the remaining phages after screening were used for amplification.

(2) Amplification and rescue of phages after screening

Amplification and rescue procedure the PBS suspension obtained in step (7) of example 1, i.e.amplified phage from the first round of screening, was stored at 4℃and used for the next round of screening; the same screening steps are carried out, the antigen amount is gradually decreased, and 3-4 rounds of screening are carried out.

(3) ELISA evaluation of the enrichment degree of specific antibodies

ELISA plates were coated with 100ng of PCSK9 antigen, 4℃overnight; the following day was blocked with 2% BSA at room temperature for 1h; the amplified phage after each round of panning is added into the experimental group, the wild type phage with the same quantity is added into the control group, and the incubation is carried out for 2 hours at room temperature; PBST was washed 10 times to remove unbound phage; adding an HRP-labeled anti-M13 antibody, and incubating for 1h at room temperature; adding a color development liquid, carrying out light-proof reaction for 10-30min, measuring a light absorption value, gradually increasing the light absorption value along with the washing times, and stabilizing the light absorption value during washing from the third round to the fourth round, wherein the enrichment of specific antibodies is shown.

(4) Identification of PCSK 9-specific Single-Domain antibody Positive clones

ELISA plates were coated with 100ng PCSK9 antigen and incubated overnight at 4 ℃; taking phage coated plates obtained in the last round of screening, randomly picking 38 monoclonals in 1mL of culture medium, culturing at 37 ℃ until the logarithmic phase, and adding 1mM IPTG to induce overnight; the next day, centrifugally collecting bacteria, crushing, centrifuging at 5,000g for 15min, and collecting supernatant; simultaneously taking an ELISA plate, adding 2% BSA, and sealing for 1h at room temperature; adding monoclonal broken supernatant into each hole of an experimental group, adding blank TG1 broken supernatant into a control group, and incubating for 2h at room temperature; PBST is washed for 10 times, and a mouse anti-HA tag antibody is added for 1h at room temperature; PBST is washed 3-5 times, and AP marked anti-mouse IgG antibody is added for 1h at room temperature; adding a substrate, reacting for 5-20 min according to actual conditions, and reading a light absorption value on an enzyme label instrument; positive clones were judged when the absorbance to control well ratio was greater than 2.1 (Base line).

(5) Positive clone sequence analysis

Extracting the DNA of 30 positive clones obtained in the step (4), carrying out PCR verification on the inserted fragments, and carrying out sequencing analysis on the clones which are positive through the PCR verification. Sequencing results showed that two nucleotide sequences were obtained, the amino acid sequences of which were analyzed, one sequence having the structure of a typical single domain antibody, i.e., consisting of a framework region (FR 1, FR2, FR3 and FR 4) and a complementarity determining region (CDR 1, CDR2 and CDR 3). The monoclonal nucleotide and amino acid sequence of the single domain antibody is as follows:

the amino acid sequence of the PCSK9 single domain antibody protein VHHH12 is as follows: QVQLQESGGGLVQAGGSLRLSCAASGRTFSDYAVGWFRQAPGKEREFVAGIGWSGGQTTYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVCAASFLVIPGTVKTRYDSWGQGTQVTVPA (SEQ ID NO: 4). Wherein, the sequence of the framework region 1 is QVQLQESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 6), the sequence of the framework region 2 is VGWFRQAPGKEREFVAG (SEQ ID NO: 7), the sequence of the framework region 3 is TYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVC (SEQ ID NO: 8), the sequence of the framework region 4 is WGQGTQVTVPA (SEQ ID NO: 9), the sequence of the complementarity determining region 1 is GRTFSDYA (SEQ ID NO: 1), the sequence of the complementarity determining region 2 is IGWSGGQT (SEQ ID NO: 2), and the sequence of the complementarity determining region 3 is AASFLVIPGTVKTRYDS (SEQ ID NO: 3).

The nucleotide sequence encoding PCSK9 single domain antibody protein VHHH12 is:

5’-caggtgcagctgcaggagtctggaggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggac gcacgttcagcgactatgccgtgggctggttccgccaggctcccgggaaggagcgtgagtttgtagctggtataggctggagtggcggtcaaacaacctatgcagactccgtgaagggccgattcaccatctccagagacaacgccaaagacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttatgtctgtgcagcttcatttttggttatccccggcaccgtgaaaactcggtatgacagctggggccagggaacccaggtcaccgttccagcg-3’(SEQ ID NO：5)。

example 3 inducible expression and purification of the anti-PCSK 9 Single Domain antibody VHHH12

(1) Construction of PCSK9 single-domain antibody expression bacterium

Firstly, monoclonal transfer culture medium of PCSK9 single domain antibody is cultured overnight at 37 ℃; the next day, plasmids were extracted using Plasmid miniprep kit (purchased from OMEGA), agarose gel electrophoresis was performed and the concentration was determined, and plasmids containing PCSK9 single domain antibody sequences were transformed into expression bacteria HB2151, plated, and incubated overnight at 37 ℃.

(2) Inducible expression of anti-PCSK 9 single domain antibody VHHH12

The next day, 5 clones were picked from the plate for cloning PCR to verify if the plasmid was transferred into the expression strain; positive clones were picked and cultured at 37 ℃ until the OD600 was 0.6-0.8, and IPTG was added for induction expression. Centrifuging the bacterial liquid, collecting bacterial precipitate, re-suspending the precipitate with lysis buffer, ultrasonically crushing the bacterial, and centrifuging to collect the crushed bacterial supernatant.

(3) Purification of anti-PCSK 9 single domain antibody VHHH12

The PCSK9 single domain antibody is obtained through Ni column affinity purification. The Ni column is firstly washed by ultrapure water and then washed by a lysate; adding the crushed supernatant of the PCSK9 single-domain antibody expression bacterium into a Ni column at a flow rate of 1 mL/min; washing off the impurity protein by using 5 times of column volume of affinity A solution (20 mM imidazole), eluting the target protein by using equal volume of affinity B solution (250 mM imidazole), and collecting the eluent; finally, 15% concentration SDS-PAGE protein gel electrophoresis is used for detecting the expression and purification condition of the PCSK9 single domain antibody.

Example 4 affinity assay of PCSK9 with its Single Domain antibody VHHH12

(1) ELISA method for analysis of binding of PCSK9 Single Domain antibody VHHH12 (FIG. 1)

The experimental group is characterized in that 100ng of PCSK9 protein is used for coating an ELISA plate (a PBS blank control group is reserved), the No-coating control group is a control group without antigen coating, and the incubation is carried out at 4 ℃ overnight; the following day was blocked with 2% BSA at room temperature for 1h; adding purified PCSK9 single-domain antibodies into experimental groups respectively, adding PBS into blank groups, and incubating for 2h at room temperature; washing for 10 times by 1 XPBST, adding mouse anti-HA tag antibody, and standing for 1h at room temperature; washing with 1 XPBST for 3-5 times, adding AP labeled anti-mouse IgG antibody, and standing for 1 hr at room temperature; adding a substrate, reacting for 10-20min, and reading the absorbance value on an enzyme label instrument. ELISA detection results (figure 2) show that the VHHH12 single domain antibody has better binding property to PCSK9, and the binding force is far higher than that of a control group.

(2) Biacore T200 analysis of affinity constant of anti-PCSK 9 Single-domain antibody VHHH12

After chip activation, PCSK9 antigen is coupled to a CM5 chip special for Biacore machine, and the coupling reaction is stopped until the coupling reaction reaches the level of about 790 RU; subsequently, 150. Mu.l of 1M ethanolamine salt are added to wash off residual reactive carboxyl groups; then the single domain antibody VHHH12 (218 nM-109nM-54nM-27nM-13.5 nM) of the PCSK9 diluted in gradient is pumped through the chip surface in sequence at a rate of 25 μL/min, combined for 120s, dissociated for 230s; after data were obtained, the results were processed and as shown in fig. 3, parameters of interaction of single domain antibody VHHH12 with antigen PCSK9 were, respectively, kon (1/Ms) =2.221e+5 as binding constant, koff (1/s) =5.844E-4 as dissociation constant, rmax (RU) =31.76 as maximum binding response value, KD (M) = 2.631E-9 (i.e., about 2.6 nM), as affinity value of antigen-antibody interaction; the single domain antibody has good interaction with PCSK9 antigen and has the value of continuous development.

SEQ ID NO：1

GRTFSDYA

SEQ ID NO：2

IGWSGGQT

SEQ ID NO：3

AASFLVIPGTVKTRYDS

SEQ ID NO：4

QVQLQESGGGLVQAGGSLRLSCAASGRTFSDYAVGWFRQAPGKEREFVAGIGWSGGQTTYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVCAASFLVIPGTVKTRYDSWGQGTQVTVPA

SEQ ID NO：5

5’-caggtgcagctgcaggagtctggaggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggacgcacgttcagcgactatgccgtgggctggttccgccaggctcccgggaaggagcgtgagtttgtagctggtataggctggagtggcggtcaaacaacctatgcagactccgtgaagggccgattcaccatctccagagacaacgccaaagacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttatgtctgtgcagcttcatttttggttatccccggcaccgtgaaaactcggtatgacagctggggccagggaacccaggtcaccgttccagcg-3’

SEQ ID NO：6

QVQLQESGGGLVQAGGSLRLSCAAS

SEQ ID NO：7

VGWFRQAPGKEREFVAG

SEQ ID NO：8

TYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVC

SEQ ID NO：9

WGQGTQVTVPA

SEQ ID NO：10

GTCCTGGCTGCTCTTCTACAAGG

SEQ ID NO：11

GGTACGTGCTGTTGAACTGTTCC

SEQ ID NO：12

TGGTGGCAGGTCCCCAAGGT

SEQ ID NO：13

TTCTTGGTGGCAGTAGCCGCAGT

SEQ ID NO：14

GATGTGCAGCTGCAGGAGTCTGGRGGAGG

SEQ ID NO：15

CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.

Claims

1. A single domain antibody that binds PCSK9 antigen, characterized in that: the single domain antibody comprises a heavy chain antibody variable region (VHH) consisting of a Framework Region (FR) and a Complementarity Determining Region (CDR), wherein the Complementarity Determining Region (CDR) comprises complementarity determining region 1 (CDR 1) having the sequence GRTFSDYA (SEQ ID NO: 1); complementarity determining region 2 (CDR 2) having the sequence IGWSGGQT (SEQ ID NO: 2); complementarity determining region 3 (CDR 3) having the sequence AASFLVIPGTVKTRYDS (SEQ ID NO: 3).

2. The single domain antibody of claim 1, wherein: the amino acid sequence of the heavy chain antibody variable region (VHH) is shown in SEQ ID NO: 4.

3. A polynucleotide sequence encoding the single domain antibody of claim 1 or 2, characterized in that: the polynucleotide sequence comprises a sequence encoding the sequence of SEQ ID NO:1 to SEQ ID NO:3, and a nucleotide sequence of the complementarity determining region shown in (3).

4. A polynucleotide sequence according to claim 3, wherein: the polynucleotide sequence comprises a sequence encoding the sequence of SEQ ID NO:4, and a heavy chain antibody variable region (VHH).

5. The polynucleotide sequence according to claim 3 or 4, wherein: the polynucleotide sequence is shown in SEQ ID NO: shown at 5.

6. An expression vector, characterized in that: the expression vector contains the polynucleotide sequence of any one of claims 3-5.

7. A host cell, characterized in that: the host cell comprises the expression vector of claim 6, which is capable of expressing a single domain antibody that specifically binds to PCSK9 antigen.

8. A pharmaceutical composition characterized by: the pharmaceutical composition comprises the single domain antibody of claim 1 or 2, and a pharmaceutically acceptable carrier, diluent or excipient.

9. A method of making the single domain antibody of claim 1 or 2, characterized by: the method comprises the following steps: transforming an expression vector comprising the polynucleotide sequence of any one of claims 3-5 into an expression host cell, culturing, performing substantial expression and purification of the single domain antibody;

the expression vector is a pMECS plasmid, and the host cell is an Escherichia coli HB2151 strain;

the expression vector is a pPICZ alpha plasmid, and the host cell is a yeast X33 strain;

the expression vector is a pCDNA3.4 plasmid and the host cell is a HEK293F cell line.

10. The single domain antibody of claim 1 or 2, characterized in that: the single domain antibody is used for preparing anti-PCSK 9 protein monoclonal antibodies or is used for immunologically detecting PCSK9 for the purpose of non-disease diagnosis and treatment.