CN116106548A - Immunological reagent for detecting human PCSK9 protein level - Google Patents

Abstract

The invention belongs to the technical field of immunodetection, and discloses an immunological reagent for detecting the level of human PCSK9 protein, which comprises a coated antibody, a detection antibody and an antigen standard, wherein the coated antibody is a commercial antibody Repatha (American Anin Co., CAS# 1256937-27-5); the detection antibody is a single domain antibody HRP-VHH-C8 marked by horseradish peroxidase (HRP); VHH-C8 comprises three complementarity determining regions, SEQ ID NOs: 1 to SEQ ID NO:3, an amino acid sequence shown in 3; the antigen is commercial recombinant human PCSK9 protein (Cat#29698-H08H in Beijing Yiqiao Shenzhou). The single domain antibody VHH-C8 has obvious difference with the epitope of the coated antibody Repatha combined with PCSK9 protein; and can well interact with PCSK9 protein in serum, so that the method can be used for immunologically detecting the level of PCSK9 protein in serum and other samples.

Description

Immunological reagent for detecting human PCSK9 protein level

Technical Field

The invention belongs to the technical field of immunodetection, and discloses an immunological reagent for detecting the level of human PCSK9 protein.

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. This suggests that we should prevent and discover and treat cardiovascular and cerebrovascular diseases (especially hyperlipidemia level) early.

A novel preprotein converting enzyme of subtilisin converting enzyme precursor 9 (Proprotein convertase subtilisin/kexin type 9, PCSK9) belongs to the subfamily of subtilisins and is one of the important influencing factors of 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, and that PCSK9 antibody inhibitors offer a novel therapeutic modality against LDL-c in addition to statins, considered as the greatest advancement in the lipid lowering field after statins. However, currently, clinically, the four lipid tests only include Total Cholesterol (TC), high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c) and Triglycerides (TG), and do not include PCSK9 levels, which results in a physician's inability to determine whether high LDL-c levels in patients are caused by high PCSK9 levels, i.e., there is an unmet clinical need.

If the high level of LDL-c in the patient is caused by high levels of PCSK9 (including functionally acquired mutated PCSK 9), treatment with a PCSK9 inhibitor is possible; if the patient's PCSK9 level is normal, there is no need to blindly treat with PCSK9 inhibitors. After all, PCSK9 antibody inhibitors are not acceptable to many patients, either at an expensive price or by the mode of administration (subcutaneous injection).

The PCSK9 immunodetection reagent in the application can monitor the PCSK9 level in human serum, help doctors to judge whether the LDL-c high level of patients is caused by the high level of PCSK9 or not in time, guide doctors to reasonably take medicines for the patients, and bring good news for hereditary hypercholesterolemia patients. The reasonable medication is based on the medical theory, and the safe, effective, economical and proper medication is selected, so that the maximum effectiveness of the medication is emphasized, and the economic bearing capacity of the masses is considered. The world health organization proposes five reasonable medication standards: 1. the prescribed medication should be appropriate. 2. At the appropriate time, the supply of the medicament is guaranteed at a price payable by the public. 3. Correct prescription. 4. The medication is taken in the correct dosage, correct usage and administration time. 5. Ensuring the quality safety and effectiveness of the medicine. The PCSK9 immunological detection reagent adopts independently developed single-domain antibodies both of coated antibodies and detection antibodies, can be expressed by escherichia coli or yeast at low cost, has low production cost, is easy to further develop and popularize, and is easy to be accepted by common patients in future.

Antibody medicine is one of the main directions of the development of new medicines at present, and has been widely applied in the fields of diagnosis, prevention and treatment of various 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 the light chain and constant region I (CH 1) regions were found in camel blood from Hamers et al in 1993, the variable region segments of heavy chain antibodies, also known as 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.

While two PCSK9 antibody inhibitors, evolocumab and alirocumab, respectively, have been approved for global use, the development of traditional antibody drugs against PCSK9 has entered the "bottleneck" phase since 2015, with some limitations and disadvantages, in several aspects: (1) Both antibodies are traditional IgG monoclonal antibodies, generally need mammalian cell expression, and have high cost; (2) Only a single catalytic region epitope of PCSK9 is combined, the curative effect is limited, and the highest lipid-lowering efficiency is about 50%; (3) The selling price is high, and the selling price of Evolokumab and Alirocumb is up to $5850/year; (4) low temperature storage is required, increasing the cost of the drug.

In conclusion, the single domain antibody has obvious advantages in various aspects such as expression cost, transformation and the like compared with the traditional antibody, is suitable to be used as a substitute of the traditional antibody, and is used for developing an immunological reagent for detecting the level of human PCSK 9.

Disclosure of Invention

The invention aims to provide an immunological reagent for detecting the level of human PCSK9 protein.

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

an immunological reagent for detecting the level of human PCSK9 protein, the immunological reagent comprising a coated antibody, a detection antibody and an antigen standard, wherein the coated antibody is a commercial antibody repath (CAS #1256937-27-5, an ann corporation) and the antigen standard is a commercial recombinant human PCSK9 protein (Cat #29698-H08H, a king-senso), and the detection antibody is a horseradish peroxidase (HRP) labeled single domain antibody HRP-VHH-C8;

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 amino acid sequence GSTFSGYA (SEQ ID NO: 1); complementarity determining region 2 (CDR 2) having the amino acid sequence IEREIPG (SEQ ID NO: 2); complementarity determining region 3 (CDR 3) having the amino acid sequence EDTAVYYCAAGLKYPAQKHYDYDY (SEQ ID NO: 3).

The amino acid sequence of the heavy chain antibody variable region (VHH) in the single domain antibody HRP-VHH-C8 is shown as (SEQ ID NO: 4).

Preferably, the polynucleotide sequence of the single domain antibody HRP-VHH-C8 encodes the polynucleotide sequences of the complementarity determining regions shown in (SEQ ID NO: 1) to (SEQ ID NO: 3).

Preferably, the polynucleotide sequence of the single domain antibody HRP-VHH-C8 encodes the polynucleotide sequence of the heavy chain antibody variable region (VHH) shown in the (SEQ ID NO: 4).

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

A method for preparing an immunological reagent for detecting the level of human PCSK9 protein, the method comprising transforming an expression vector of a polynucleotide sequence into an expression host cell, culturing, and performing mass expression and purification of the single domain antibody HRP-VHH-C8;

the expression vector comprises the polynucleotide sequence of any one or more of claims 3-5;

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 plasmid of the pCDNA series and the host cell is a HEK293 cell line.

The host cell comprises an expression vector capable of expressing a single domain antibody that specifically binds to PCSK9 protein.

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

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.

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

the immunological reagent for detecting the level of the human PCSK9 protein has the advantages that firstly, the single-domain antibody HRP-VHH-C8 is small in size and easy to reconstruct, is very suitable for constructing bispecific and multispecific antibodies, has better effect by applying the nano antibody with smaller molecular weight and simpler structure compared with the traditional monoclonal antibody medicament, and has more advantages in the aspects of operation, absorption, synthesis, price and the like; secondly, the single domain antibody VHH-C8 of the invention has obvious difference with the epitope of the coated antibody Repatha binding to PCSK 9; and can well interact with PCSK9 protein in serum, so that the method can be used for immunologically detecting the level of PCSK9 in samples such as serum.

Drawings

FIG. 1 is a schematic diagram of the detection of PCSK9 levels in human samples using two antibodies based on ELISA in an embodiment of the invention;

FIG. 2 is a schematic diagram showing the verification of VHH-C8 and Repatha binding to different epitopes of recombinant human PCSK9 protein based on SPR technology in an embodiment of the invention;

FIG. 3 shows the determination of affinity constants of the single domain antibody VHH-C8 and human PCSK9 using a Biacore T200 instrument based on SPR technique in an embodiment of the 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 M13GIII 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 VHH-C8 has high specificity and high affinity of targeting PCSK9 through the verification of a Biacore T200 system, which shows that the PCSK9 single-domain antibody VHH-C8 obtained by the invention has development value as a diagnostic antibody.

Immunizing alpaca with PCSK9 antigen expressed in CHO cells, collecting peripheral blood cells (PBMC) of the immunized alpaca, separating PCSK9 affinity lymphocytes from the peripheral blood cells, extracting total RNA, cloning a variable region (V region) of a alpaca heavy chain antibody by adopting a nested PCR technology, inserting the variable region into phage plasmids to construct a phage expression library, carrying out multiple rounds of screening on the PCSK9 antigen by a phage display technology, carrying out mass expression purification on the screened high affinity antibody 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 VHH-C8, which specifically binds to PCSK9 antigen, has the amino acid sequence:

QVQLQESGGGLVQAGGSLRVSCVASGSTFSGYAMAWFRQAPGKERE FVAAIEREIPGHPAWSGLTYYADSKKGRFTISRDNAKNTVYLQMNSLKSEDTA VYYCAAGLKYPAQKHYDYDYWGQGTQVTVPA(SEQ ID NO：4)。

wherein, the sequence of the framework region 1 is QVQLQESGGGLVQAGGSLRVSCVAS (SEQ ID NO: 6), the sequence of the framework region 2 is MAWFRQAPGKEREFVAA (SEQ ID NO: 7), the sequence of the framework region 3 is HPAWSGLTYYADSKKGRFTISRDNAKNTVYLQMNSLKS (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 GSTFSGYA (SEQ ID NO: 1), the sequence of the complementarity determining region 2 is IEREIPG (SEQ ID NO: 2), and the sequence of the complementarity determining region 3 is EDTAVYYCAAGLKYPAQKHYDYDY (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 GSTFSGYA (SEQ ID NO: 1); complementarity determining region 2 (CDR 2) having the sequence IEREIPG (SEQ ID NO: 2); complementarity determining region 3 (CDR 3) having the sequence EDTAVYYCAAGLKYPAQKHYDYDY (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 VHH-C8 is: 5'-caggtgcagctgcaggagtctggaggaggattggtgcaggctgggggctctctgagagtctcctgtgtagct tctggaagcaccttcagtggttatgccatggcctggttccgccaggctccagggaaggagcgtgagtttgtagctgctattgagcgtgagattccaggacatcctgcctggagtggtttgacatactatgcagactccaagaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaagtctgaggacacggccgtttattactgtgcagcaggattgaaatatccggcccagaaacactatgactatgactactggggccaggggacccaggtcaccgttccagcgg-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 VHH-C8 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, pPICZ series, 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 a phage display vector pMECS.

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 HB 2151.

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, immunodetection agent, 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, immunodetectors 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 can be used for immunologically detecting PCSK9 and can also be used for preparing anti-PCSK 9 protein monoclonal antibodies. 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 RNAlater, lightly mixing and dissolving the cell mass, 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 LTrilzol, 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 12000rpm, 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 RNA 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; CALL0020.5 μl; 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 NotI and PstI, respectively, and the reaction system was as follows:

carrier enzyme cutting system: 20 μg of carrier; pstI 10 μl; notI 20. 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 96 clones in the step (5), taking the clones as templates, carrying out 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 96 monoclonal antibodies have 80 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, 3000g was centrifuged for 30min, the supernatant was collected, phage was precipitated by adding PEG-sodium chloride solution, left on ice for 30min, centrifuged for 3,000 min, and the precipitate was used as a PCSK9 single domain antibody phage library, which was suspended with PBS and the titer was determined to be 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 for 15min at 5000g, 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.

(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 VHH-C8 is as follows: QVQLQESGGGLVQAGGSLRVSCVASGSTFSGYAMAWFRQAPGKEREFVAAIE REIPGHPAWSGLTYYADSKKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYCA AGLKYPAQKHYDYDYWGQGTQVTVPA (SEQ ID NO: 4). Wherein, the sequence of framework region 1 is QVQLQESGGGLVQAGGSLRVSCVAS (SEQ ID NO: 6), the sequence of framework region 2 is MAWFRQAPGKEREFVAA (SEQ ID NO: 7), the sequence of framework region 3 is HPAWSGLTYYADSKKGRFTISRDNAKNTVYLQMNSLKS (SEQ ID NO: 8), the sequence of framework region 4 is WGQGTQVTVPA (SEQ ID NO: 9), the sequence of complementarity determining region 1 is GSTFSGYA (SEQ ID NO: 1), the sequence of complementarity determining region 2 is IEREIPG (SEQ ID NO: 2), and the sequence of complementarity determining region 3 is EDTAVYYCAAGLKYPAQKHYDYDY (SEQ ID NO: 3).

The nucleotide sequence encoding PCSK9 single domain antibody protein VHH-C8 is: 5'-Caggtgcagctgcaggagtctggaggaggattggtgcaggctgggggctctctgagagtctcctgtgtagcttctggaa gcaccttcagtggttatgccatggcctggttccgccaggctccagggaaggagcgtgagtttgtagctgctattgagcgtgagattccaggacatcctgcctggagtggtttgacatactatgcagactccaagaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaagtctgaggacacggccgtttattactgtgcagcaggattgaaatatccggcccagaaacactatgactatgactactggggccaggggacccaggtcaccgttccagcgg-3' (SEQ ID NO: 5).

EXAMPLE 3 inducible expression and purification of the anti-PCSK 9 Single-domain antibody VHH-C8

(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 beijing Tiangen biochemistry), agarose gel electrophoresis was performed and the concentration was determined, and then 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 VHH-C8

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 VHH-C8

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 identification of different epitopes of VHH-C8 and Repatha binding PCSK9

The epitope difference identification results are shown in FIG. 2, and are determined by using the affinity kinetics method, and comprise a plurality of stages: the protein A chip captures the coated antibody, the repath stage, the PCSK9 sample injection and binding to the chip saturation stage, the detection antibody VHH-C8 and two positive control antibody sample injection stage and the chip regeneration stage. In the first stage, after capture of repath, the response curve stabilizes around-180 RU; after PCSK9 injection, repatha began to bind to PCSK9 and finally stabilized at 0RU, demonstrating that the specific binding epitope of Repatha on PCSK9 was saturated (the graph was corrected using Biacore T200 self-contained software, i.e., baseline 0RU with the saturation level of Repatha versus PCSK 9), along with separate injections of VHH-C8 and two positive control antibodies (developed in this laboratory, verifying that they were different from the epitope of Repatha that bound PCSK 9), the reaction curve (black line) of VHH-C8 also further increased and stabilized at-50 RU, indicating that the specific binding epitope of VHH-C8 antibody was present on PCSK9 protein in addition to the specific binding epitope of Repatha, confirming that the epitopes of VHH-C8 antibody and Repatha bound PCSK9 were indeed different, and can be adapted for the development of a sandwich immunodetection reagent for diabody.

Example 5 affinity assay of PCSK9 with the Single Domain antibody VHH-C8

(1) Analysis of affinity constants of anti-PCSK 9 Single Domain antibody VHH-C8 with Biacore T200

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 VHH-C8 of the PCSK9 after gradient dilution is pumped by a machine (25 nM-12.5nM-6.25nM-3.125nM-1.56 nM) to flow through the chip surface in sequence at a rate of 25 mu L/min, combined for 120s and dissociated for 180s; after data were obtained, the results were processed and as shown in fig. 3, parameters of interaction of single domain antibody VHH-C8 with antigen PCSK9 were Kon (1/Ms) =1.613e+6 as binding constant, koff (1/s) =2.661E-3 as dissociation constant, rmax (RU) =6.5 maximum binding response value, KD (M) =1.649E-9 (i.e. about 1.65 nM), respectively, as affinity value of antigen-antibody interaction; the single domain antibody has good interaction with PCSK9 antigen and has the value of continuous development.

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.

Gene sequence table

SEQ ID NO：1

GSTFSGYA

SEQ ID NO：2

IEREIPG

SEQ ID NO：3

EDTAVYYCAAGLKYPAQKHYDYDY

SEQ ID NO：4

QVQLQESGGGLVQAGGSLRVSCVASGSTFSGYAMAWFRQAPGKEREFVAAIEREIPGHPAWSGLTYYADSKKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYCAAGLKYPAQKHYDYDYWGQGTQVTVPA

SEQ ID NO：5

‘5-caggtgcagctgcaggagtctggaggaggattggtgcaggctgggggctctctgagagtctcctgtgtagcttctggaagcaccttcagtggttatgccatggcctggttccgccaggctccagggaaggagcgtgagtttgtagctgctattgagcgtgagattccaggacatcctgcctggagtggtttgacatactatgcagactccaagaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaagtctgaggacacggccgtttattactgtgcagcaggattgaaatatccggcccagaaacactatgactatgactactggggccaggggacccaggtcaccgttccagcgg-3’

SEQ ID NO：6

QVQLQESGGGLVQAGGSLRVSCVAS

SEQ ID NO：7

MAWFRQAPGKEREFVAA

SEQ ID NO：8

HPAWSGLTYYADSKKGRFTISRDNAKNTVYLQMNSLKS

SEQ ID NO：9

WGQGTQVTVPA。

Claims (8)

1. An immunological reagent for detecting the level of human PCSK9 protein, the immunological reagent comprising a coated antibody, a detection antibody, and an antigen standard, wherein the coated antibody is a commercial antibody repath (CAS #1256937-27-5, ann corporation, usa), and the antigen standard is a commercial recombinant human PCSK9 protein (Cat #29698-H08H, beijing, seemingly characterized in that: the detection antibody is a single-domain antibody HRP-VHH-C8 marked by horseradish peroxidase (HRP);

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 amino acid sequence GSTFSGYA (SEQ ID NO: 1); complementarity determining region 2 (CDR 2) having the amino acid sequence IEREIPG (SEQ ID NO: 2); complementarity determining region 3 (CDR 3) having the amino acid sequence EDTAVYYCAAGLKYPAQKHYDYDY (SEQ ID NO: 3).

2. An immunological reagent for detecting the level of human PCSK9 protein of claim 1, wherein: the amino acid sequence of the heavy chain antibody variable region (VHH) in the single domain antibody HRP-VHH-C8 is shown as (SEQ ID NO: 4).

3. An immunological reagent for detecting the level of human PCSK9 protein according to claim 1 or 2, wherein: the polynucleotide sequence of the single domain antibody HRP-VHH-C8 codes for the polynucleotide sequences of the complementarity determining regions shown in (SEQ ID NO: 1) to (SEQ ID NO: 3).

4. An immunological reagent for detecting the level of human PCSK9 protein of claim 3, wherein: the polynucleotide sequence of the single domain antibody HRP-VHH-C8 codes for the polynucleotide sequence of the heavy chain antibody variable region (VHH) shown in the (SEQ ID NO: 4).

5. The polynucleotide sequence of claim 3 or 4, wherein said polynucleotide sequence is as set forth in SEQ ID NO: 5) As shown.

6. A method of preparing an immunological reagent for detecting the level of human PCSK9 protein according to claims 3-5, wherein: the preparation method comprises the steps of transforming an expression vector of a polynucleotide sequence into an expression host cell, culturing, and carrying out mass expression and purification of the single-domain antibody HRP-VHH-C8;

the expression vector comprises the polynucleotide sequence of any one or more of claims 3-5;

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 pCDNA series plasmid, and the host cell is HEK293 cell strain.

7. The method of preparing an immunological reagent for detecting the level of human PCSK9 protein of claim 6, wherein: the host cell comprises an expression vector capable of expressing a single domain antibody that specifically binds to PCSK9 protein.

8. An immunological reagent for detecting the level of human PCSK9 protein as claimed in claims 1-7, wherein: the single domain antibody is used for preparing anti-PCSK 9 protein monoclonal antibody medicines or is used for immunologically detecting PCSK9 for the purpose of non-disease diagnosis and treatment.

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