CN119192381A - A single domain antibody capable of targeting and binding to PCSK9 and its preparation method and application - Google Patents
A single domain antibody capable of targeting and binding to PCSK9 and its preparation method and application Download PDFInfo
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- CN119192381A CN119192381A CN202311569102.0A CN202311569102A CN119192381A CN 119192381 A CN119192381 A CN 119192381A CN 202311569102 A CN202311569102 A CN 202311569102A CN 119192381 A CN119192381 A CN 119192381A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/567—Framework region [FR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明提出一种能靶向结合PCSK9的单域抗体及其制备方法与应用。该单域抗体是来源于驼类的重链抗体可变区，其由框架区(FR区)和互补决定区(CDR区)组成，其中包括三个CDR区如SEQ ID NO：1至SEQ ID NO：3所示，以及四个FR区如SEQ ID NO：6至SEQ ID NO：9所示。该单域抗体制备方法是利用真核表达的PCSK9抗原免疫羊驼，分离单个核细胞，提取总RNA，经过逆转录和巢式PCR建库，获得PCSK9免疫单域抗体文库；再利用噬菌体展示技术进行筛选，并转化至大肠杆菌表达系统中进行表达，获得PCSK9的单克隆单域抗体株。本发明单域抗体与重组PCSK9蛋白的相互作用良好，能够用于研制抗PCSK9蛋白的抗体类药物，还可用于免疫学检测样本中的PCSK9水平。
The present invention proposes a single-domain antibody that can target and bind to PCSK9, and a preparation method and application thereof. The single-domain antibody is a heavy chain antibody variable region derived from camels, which is composed of a framework region (FR region) and a complementary determining region (CDR region), including 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 is to immunize alpacas with eukaryotically expressed PCSK9 antigen, separate mononuclear cells, extract total RNA, and obtain a PCSK9 immune single-domain antibody library through reverse transcription and nested PCR library construction; then screen using phage display technology, and transform into an Escherichia coli expression system for expression to obtain a monoclonal single-domain antibody strain of PCSK9. The single-domain antibody of the present invention has a good interaction 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.
Description
Technical Field
The invention relates to the field of antibody medicines in biological medicines, in particular to a single-domain antibody capable of targeting and combining PCSK9 (precursor subtilisin converting enzyme 9), and a preparation method and application of the single-domain antibody.
Background
At present, medicines for reducing cholesterol are mainly used for treating cardiovascular and cerebrovascular diseases in the market. The drugs for lowering cholesterol mainly include statins and the like. Although statins are excellent in the treatment of cardiovascular diseases, the disadvantages that occur are gradually discovered as they are widely used. Firstly, statin treatment patients still have a high residual risk of cardiovascular events, and secondly, a large number of patients cannot tolerate statin, especially familial hypercholesterolemia, and even with the maximum dose of the most effective statin treatment, the goal of lowering the low-density lipoprotein cholesterol concentration cannot be achieved. Most importantly, statins have various side effects, such as causing patient blood glucose abnormalities, muscle toxicity, memory and cognitive impairment, serious side effects leading to rhabdomyolysis and acute renal failure, and a significant proportion of patients terminate treatment due to intolerable side effect-related muscle pain.
The precursor subtilisin-converting enzyme 9 (PCSK 9) is a novel preprotein-converting enzyme belonging to the subtilisin subfamily 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 (LDL-R) of hepatocytes, thereby affecting the level of 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 in PCSK9 "gain of function" often occur in individuals with chromosomal dominant hypercholesterolemia, whereas mutations in PCSK9 "loss of function" are associated with reduced plasma cholesterol, with a significant reduction in the risk of coronary heart disease in individuals with PCSK9 loss of function mutations. In 2005, hobbs et al reported that LDL-c levels in individuals carrying PCSK9 nonsense mutant genes were 28% lower than in general, in 2006, hobbs et al also published the effect of PCSK9 gene mutations on coronary heart disease, based on an atherosclerosis risk survey, which was followed up to 15 years, and found that the incidence of coronary heart disease in populations lacking 1 or2 PCSK9 functional genes was significantly lower than in general. Copenhagen Heart Study it was found that functional deletion of the PCSK9 gene reduced 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 Sanofei, repatha (evolocumab) of Ind and IBI-306 (tafolecimab) 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 a heavy chain antibody naturally lacking the light chain and constant region I (CH 1) regions was found in alpaca blood from Hamers et al in 1993, the variable region of the heavy chain antibody is also called a Single-domain-antibody (sdAb) gradually replaces other small antibodies, and becomes a hotspot for the development of novel antibody drugs. sdabs are typically only about 16 kilodaltons in size, about one tenth of conventional antibodies in which disulfide bonds are present, and a large number of hydrophilic residues are present on the surface, which is more resistant to heat and pH. The sdAb lacks of Fc segment and light chain, so that the sdAb can recognize hidden epitope or small epitope which cannot be recognized by traditional antibodies, and complement reaction is avoided, and in addition, the single domain antibody has the advantages of high stability, low toxicity, strong solubility, easiness in target screening and direct expression in prokaryotic microorganisms, good economy and the like. Sequence homology analysis showed that the sdAb germline gene sequence of alpaca sdabs was highly homologous to human VH3, but CDR1 and CDR3 were slightly longer than human, CDR3 protruding outward in tertiary structure, thus presumably higher antigen binding specificity and affinity. In view of the above advantages, sdabs are being developed as monoclonal antibodies in disease diagnosis and treatment, and are widely used in the development of inhibitors of enzymes, and biological inhibitors of tumors, infections, and inflammation. 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 sdAb 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 aim of research is fulfilled. 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
Based on the technical problems, the invention provides a single-domain antibody capable of targeting PCSK9 binding, and a preparation method and application of the single-domain antibody.
The technical scheme adopted by the invention is as follows:
A single domain antibody capable of targeting PCSK9 comprises a heavy chain variable region comprising a framework region and a complementarity determining region comprising complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3, the sequence of complementarity determining region 1 being shown in sequence 1, the sequence of complementarity determining region 2 being shown in sequence 2, and the sequence of complementarity determining region 3 being shown in sequence 3.
The framework regions comprise a framework region 1, a framework region 2, a framework region 3 and a framework region 4, the sequence of the framework region 1 is shown as a sequence 4, the sequence of the framework region 2 is shown as a sequence 5, the sequence of the framework region 3 is shown as a sequence 6, and the sequence of the framework region 4 is shown as a sequence 7.
The sequence of the heavy chain variable region is shown in SEQ ID NO. 8.
An expression vector comprising a polynucleotide sequence as set forth in sequence 9.
A process for preparing the single-domain antibody able to target PCSK9 includes such steps as choosing the expression carrier containing polynucleotide sequence as shown in sequence 9 and host cell, transferring the expression carrier to host cell, culturing, and expressing the single-domain antibody able to target PCSK 9.
The expression vector adopts pMECS plasmid, and the host cell adopts escherichia coli HB2151 subtype strain;
Or the expression vector adopts pPICZa plasmid, and the host cell adopts yeast X33 subtype strain;
or the expression vector is selected from pCDNA3.1 plasmid, and the host cell is selected from HEK293E cell strain.
Further, the preparation method comprises the following steps:
Immunizing alpaca with eukaryotic expressed PCSK9 antigen, separating peripheral blood mononuclear cell to extract total RNA, reverse transcription and nest PCR library establishment to obtain PCSK9 immune single domain antibody library, coating PCSK9 antigen onto ELISA plate, phage display to screen PCSK9 immune single domain antibody library, and converting the screened single domain antibody into host cell for expression to obtain PCSK9 monoclonal single domain antibody strain.
More specifically, the preparation method comprises the following steps:
step one, construction of PCSK9 single-domain antibody phage display library
(11) PCSK9 immunized alpaca
Mixing PCSK9 with equal volume of Freund adjuvant, injecting into alpaca neck subcutaneous 3-5 points, immunizing once per month, and total immunizing 4 times, wherein 10mL of alpaca peripheral blood is taken in EDTA anticoagulant tube during each immunization, continuously and slowly shaking, and fully mixing;
(12) Blood lymphocyte sample separation
Separating lymphocytes from blood samples collected before and after each immunization;
(13) Total RNA extraction and cDNA Synthesis
Adding equal volume of isopropanol, mixing, standing at room temperature, centrifuging to remove supernatant, drying water, dissolving RNA with water without nuclease, and measuring 1 mu L of RNA for concentration and purity;
Taking RNA, and adopting a kit to synthesize cDNA;
(14) Phage display library construction
Amplifying the V region of the alpaca heavy chain antibody by using cDNA as a template and adopting Nest-PCR;
After the PCR reaction is finished, detecting a PCR product by agarose gel electrophoresis, cutting a target gene fragment of the first round of PCR at 700bp, recovering a target band, performing the second round of PCR, cutting the target gene fragment at 500bp, and recovering the target band, namely an sdAb fragment;
Double-enzyme cutting is carried out on the sdAb fragment and the carrier by using restriction endonucleases Not I and Pst I of NEB respectively, the enzyme cutting products of the carrier and the sdAb fragment are mixed, and the ligation is carried out overnight at 4 ℃ by using ligase of NEB to obtain a ligation product;
(15) Construction of phage display library
Purifying the connection product, taking 1 mu L of transformed TG competent cells, resuscitating for 2 hours at 37 ℃, carrying out gradient dilution to 10 1,102,103, respectively taking 300 mu L of coated plates, culturing at 37 ℃ overnight, and calculating the clone number;
the same transformation method is adopted for large quantity transformation until the clone number of the library reaches more than 10 7, all clones are eluted by the sterilized LB liquid culture medium, centrifugated, suspended by the sterilized LB liquid culture medium, added with glycerol with the same volume and frozen;
(16) Library diversity detection
Randomly picking 40 clones in the step (5) as templates to perform clone PCR reaction, detecting PCR products by using 2% agarose gel electrophoresis, verifying the recombination rate of the constructed PCSK9 single-domain antibody library, sequencing the recombinant PCSK9 single-domain antibody library, and analyzing the diversity of the PCSK9 single-domain antibody library;
(17) Phage amplification and rescue
Culturing the monoclonal library stored in the step (5) in a culture medium until the logarithmic phase, adding the auxiliary phage, standing at room temperature, centrifuging, suspending a sediment in the culture medium, inoculating the sediment in the culture medium, culturing overnight;
Step two, panning PCSK9 single domain antibody by phage display technology
(21) Affinity PCSK9 single domain antibody phage library panning
Taking PCSK9 antigen coated ELISA plate, incubating overnight, adding the rescued PCSK9 single-domain antibody phage the next day, incubating at room temperature, washing the hole by PBST, adding triethylamine, incubating at room temperature, and collecting phage, namely affinity washing the obtained PCSK9 single-domain antibody phage library;
(22) Amplification and rescue of phages after screening
Amplification and rescue methods are the same as step (17);
(23) ELISA evaluation of the enrichment degree of specific antibodies
(24) Identification of PCSK 9-specific Single-Domain antibody Positive clones
(25) Positive clone sequence analysis
The DNA of the positive clone obtained in the step (24) is extracted to carry out PCR verification on the inserted fragment, the clone which is positive through the PCR verification is subjected to sequencing analysis, the sequencing result shows that two nucleotide sequences are obtained, the amino acid sequence of the nucleotide sequences is analyzed, one sequence has the structure of a typical single domain antibody and comprises a framework region and a complementarity determining region, and the nucleotide and amino acid sequence of the single domain antibody monoclonal is as follows:
The amino acid sequence of the PCSK9 single-domain antibody protein sdAb-C12 is shown in a sequence 8. Wherein the sequence of the framework region 1 is shown as a sequence 4, the sequence of the framework region 2 is shown as a sequence 5, the sequence of the framework region 3 is shown as a sequence 6, and the sequence of the framework region 4 is shown as a sequence 7, the sequence of the complementarity determining region 1 is shown as a sequence 1, the sequence of the complementarity determining region 2 is shown as a sequence 2, and the sequence of the complementarity determining region 3 is shown as a sequence 3;
The nucleotide sequence of the encoding PCSK9 single-domain antibody protein sdAb-C12 is shown as sequence 9;
step three, induced expression and purification of anti-PCSK 9 single domain antibody sdAb-C12
(31) Construction of PCSK9 single-domain antibody expression bacterium
Firstly, a PCSK9 single-domain antibody monoclonal transfer culture medium is cultured at 37 ℃ overnight, and after the next day, plasmid is extracted, agarose gel electrophoresis is carried out, and the concentration is measured, the plasmid containing the PCSK9 single-domain antibody sequence is transformed into an expression bacterium HB2151, a flat plate is coated, and the culture is carried out at 37 ℃ overnight;
(32) Inducible expression of anti-PCSK 9 single-domain antibody sdAb-C12
Selecting 5 clones from a flat plate, performing clone PCR to verify whether plasmid is transferred into an expression strain, selecting positive clones, culturing at 37 ℃ until OD 600 is 0.6-0.8, adding IPTG for induced expression, centrifuging bacterial liquid, collecting bacterial precipitate, re-suspending the precipitate with a lysis buffer, ultrasonically crushing the bacterial, centrifuging and collecting the crushed bacterial supernatant;
(33) Purification of anti-PCSK 9 Single-domain antibody sdAb-C12
PCSK9 single domain antibody sdAb-C12 was obtained by Ni column affinity purification.
In the step (14), the names and sequences of the Nest-PCR primers are as follows:
in the first round, the primer name is CALL001, the sequence of which is shown as sequence 10, the primer name is CALL002, the sequence of which is shown as sequence 11;
In the second round, the primer was named sdAb-Back, whose sequence is shown as sequence 12, and the primer was named sdAb-For, whose sequence is shown as sequence 13.
The application of the single-domain antibody capable of targeting and binding PCSK9 in preparing anti-PCSK 9 protein monoclonal antibody medicines or in immunological detection of PCSK9 for non-disease diagnosis and treatment purposes.
The beneficial technical effects of the invention are as follows:
The invention obtains the single domain antibody which can be targeted and combined with precursor subtilisin converting enzyme 9 (PCSK 9) by limiting the sequence of the complementary determining region and the like, has good interaction with PCSK9 antigen and has the value of continuous development. The single domain antibody of the invention has the advantages of high stability, low toxicity, strong solubility, easy target spot screening, easy direct expression in prokaryotic microorganism, good economy and the like. The single-domain antibody of the invention has good interaction with the recombinant PCSK9 protein, can be used for developing antibody medicines for resisting the PCSK9 protein, and can also be used for detecting the PCSK9 level in a sample by immunology.
The single-domain antibody preparation method 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.
Drawings
FIG. 1 shows the detection of PCSK9 single domain antibody after purification by SDS-PAGE protein gel electrophoresis in an embodiment of the invention;
FIG. 2 is a schematic diagram of ELISA for verifying binding of a single domain antibody to an antigen in an embodiment of the invention;
FIG. 3 shows the results of affinity assays for the single domain antibody sdAb-C12 obtained in the examples of the invention;
FIG. 4 is a data processing result when analyzing affinity constants of anti-PCSK 9 single-domain antibody sdAb-C12.
Detailed Description
The present invention provides a single domain antibody capable of targeting PCSK9 comprising a heavy chain variable region comprising a framework region and a complementarity determining region. The complementarity determining regions include complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3, the sequence of complementarity determining region 1 being shown in sequence 1, the sequence of complementarity determining region 2 being shown in sequence 2, the sequence of complementarity determining region 3 being shown in sequence 3.
Sequence 1 (CDR 1) GSTFKRYA (SEQ ID NO: 1), sequence 2 (CDR 2) IERDLVQPSPPFGGLT (SEQ ID NO: 2), sequence 3 (CDR 3) AAGLKYPAPNQLDYDY (SEQ ID NO: 3).
The framework regions comprise a framework region 1, a framework region 2, a framework region 3 and a framework region 4, the sequence of the framework region 1 is shown as a sequence 4, the sequence of the framework region 2 is shown as a sequence 5, the sequence of the framework region 3 is shown as a sequence 6, and the sequence of the framework region 4 is shown as a sequence 7.
Sequence 4 (FR 1) LQESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 6), sequence 5 (FR 2) MAWFRQAPGKEREFVAA (SEQ ID NO: 7), sequence 6 (FR 3) YYAASVKGRFTISRDNAKNTVDLQMNSLKPEDAAVYYC (SEQ ID NO: 8), sequence 7 (FR 4) WGQGTQVTVSS (SEQ ID NO: 9).
The amino acid sequence of the single domain antibody sdAb-C12 specifically binding to PCSK9 antigen is shown in sequence 8.
Sequence 8:
LQESGGGLVQAGGSLRLSCAASGSTFKRYAMAWFRQAPGKEREFVAAIERDLVQPFPPSGGLTYYAASVKGRFTIS RDNAKNTVDLQMNSLKPEDAAVYYCAAGLKYPAPNQLDYDYWGQGTQVTVSS(SEQ ID NO：4).
The specific binding characteristics of an antibody are determined by the complementarity determining regions. Accordingly, the present invention claims a single domain antibody that specifically binds PCSK9 antigen, comprising a heavy chain variable region (sdAb) consisting of a Framework Region (FR) and a Complementarity Determining Region (CDR). Wherein the Complementarity Determining Regions (CDRs) comprise complementarity determining region 1 (CDR 1) having sequence GSTFKRYA (SEQ ID NO: 1), complementarity determining region 2 (CDR 2) having sequence IERDLVQPSPPFGGLT (SEQ ID NO: 2), complementarity determining region 3 (CDR 3) having sequence AAGLKYPAPNQLDYDY (SEQ ID NO: 3). A single domain antibody specifically binding to PCSK9 antigen has a heavy chain variable region (sdAb) sequence shown in SEQ ID NO. 4, sequence 8.
The nucleotide sequence encoding PCSK9 single domain antibody sdAb-C12 is shown as sequence 9.
5’-
CTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTTCAAGAGGTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCCGCTATTGAGCGTGACCTTGTACAACCGTTTCCGCCGAGCGGTGGTTTGACATACTATGCAGCGTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGGTGGATCTGCAAATGAACAGCCTGAAACCTGAGGACGCGGCCGTTTATTACTGCGCAGCAGGATTGAAATATCCTGCCCCTAATCAGCTTGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCC-3'(SEQ ID NO：5).
The complementarity determining regions are determined by consideration of the degeneracy of the coding genes and, at the same time, by consideration of the specific binding characteristics of the antibody. Accordingly, the present invention claims a polynucleotide sequence encoding PCSK9 single domain antibody sdAb-C12, comprising a nucleotide sequence encoding the complementarity determining regions set forth in SEQ ID No. 1 to SEQ ID No. 3. Such polynucleotide sequences may vary in base sequence due to the degeneracy of the coding gene, as long as they are capable of encoding the complementarity determining regions shown in SEQ ID NO. 1 through SEQ ID NO. 3. Preferably, the polynucleotide sequence is shown in SEQ ID NO. 5.
The present invention provides an expression vector comprising the polynucleotide sequence described above.
PET series vectors, pMECS, pPICZα, pUSE series vectors, pCDNA series vectors, etc. can be used as expression vectors for the polynucleotide sequences of the present invention. Preferably the expression vector is a phage display vector pMECS.
The invention also provides a host cell containing the expression vector, which can express a single domain antibody specifically binding to PCSK9 antigen. A wide 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. Preferably, the host cell is E.coli strain HB 2151.
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 (sdAb) 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, so that the single domain antibody protein is expressed 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 cell, collecting peripheral blood cell (PBMC) of the alpaca after immunization, separating PCSK9 affinity lymphocyte from the alpaca, extracting total RNA, cloning variable region (V region) of alpaca heavy chain antibody by using Nest-PCR technology, inserting the alpaca heavy chain antibody into phage plasmid, and constructing phage expression library. The PCSK9 antigen was then screened multiple rounds by phage display technology. And finally, carrying out mass expression purification on the 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.
The invention provides a preparation method of a single-domain antibody capable of targeting and binding PCSK9, which comprises the steps of selecting an expression vector and a host cell, wherein a polynucleotide sequence contained in the expression vector is shown as a sequence 9, transforming the expression vector into the host cell, and culturing to express the single-domain antibody capable of targeting and binding PCSK 9.
The expression vector adopts pMECS plasmid, and the host cell adopts E.coli HB2151 subtype strain. Or the expression vector adopts pPICZa plasmid, and the host cell adopts yeast X33 subtype strain. Or the expression vector is selected from pCDNA3.1 plasmid, and the host cell is selected from HEK293E cell strain.
Specifically, the preparation method of the single domain antibody capable of targeting and binding PCSK9 utilizes eukaryotic expressed PCSK9 antigen to immunize alpaca, and total RNA is extracted by separating peripheral blood mononuclear cells. Through 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, 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 invention will be further described with reference to the drawings and the specific embodiments.
A preparation method of a single domain antibody capable of targeting and binding PCSK9 comprises the following specific steps:
step one, construction of PCSK9 single-domain antibody phage display library
(11) PCSK9 immunized alpaca
Mu.L of PCSK9 (75. 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, and 10mL of alpaca peripheral blood was taken for each immunization. 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.
(12) Blood lymphocyte sample separation
Lymphocytes were isolated from blood samples collected before and after each immunization as follows:
i. 7mL of lymphocyte isolate Ficoll was added to 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.
In a centrifuge tube of lymphocyte separation liquid, carefully and slowly adding an equal volume (7 mL) of diluted blood by using a 1mL pipettor, 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, placing into a small cloth bag with ropes, and storing in a liquid nitrogen tank.
Carefully separating the leukocyte layer into a 15mL centrifuge tube with a 1mL pipette, topping up the PBS (1X) to 15mL, washing the leukocytes 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, and recovering the leukocytes 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.
(13) Total RNA extraction and cDNA Synthesis
One frozen portion of lymphocytes was taken, 1mL of Trizol was added, after 10min at room temperature, 0.2mL of chloroform was added, vigorously shaken, and after 10min of solution was allowed to separate, after centrifugation at 12,000rpm, the upper aqueous phase was collected. Adding equal volume of isopropanol, mixing, standing at room temperature for 15min, centrifuging at high speed to remove supernatant, adding 1mL of 75% ethanol (prepared by DEPC water) into RNA precipitate, washing, and centrifuging at high speed to remove supernatant. After the water content was drained, RNA was dissolved in nuclease-free water, and 1. Mu.L of each was used for concentration and purity measurement.
And (3) taking a proper amount (7-20 mug) of RNA, adopting a SuperScript TM III First-STRAND SYNTHESIS SuperMix (Invitrogen) kit to carry out cDNA synthesis, using Oligo dT as a reverse transcription primer, and freezing the synthesized cDNA at-20 ℃.
(14) Phage display library construction
PCR amplification, namely, amplifying a V region (sdAb) of the alpaca heavy chain antibody by using the synthesized cDNA as a template and adopting Nest-PCR, wherein the names and sequences of the Nest-PCR primers are shown in Table 1, namely, primer information and sequence information used for amplifying alpaca sdAb fragments.
TABLE 1
In Table 1, SEQ ID NO 10 corresponds to SEQ ID NO 10, SEQ ID NO 11 corresponds to SEQ ID NO 11, SEQ ID NO 12 corresponds to SEQ ID NO 12, and SEQ ID NO 13 corresponds to SEQ ID NO 13.
The PCR reaction system is as follows:
In the first round, cDNA was 2. Mu.L, 2 XMaster Mix 12.5. Mu.L, CALL 001.5. Mu.L, CALL002 0.5. Mu.L, and water was made up to 25. Mu.L. 2 XMaster Mix (from KAPA Biosystems)
The reaction conditions were 95℃for 5min, 94℃for 1min, 57℃for 1min, 72℃for 1min per cycle, 72℃for 7min, and 35 cycles of amplification.
The second round was 40ng;2*Master Mix 25. Mu.L of template (first round product), 1. Mu.L of sdAb-For (10. Mu.M), 1. Mu.L of sdAb-Back (10. Mu.M), and water make up to 50. Mu.L.
The reaction conditions were 95℃for 5min, 94℃for 45s, 60℃for 45s, 72℃for 45s for each cycle, and 72℃for 7min for 25 cycles of amplification.
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 sdAb fragment.
The sdAb fragment and vector (pMECS plasmid) were double digested with NEB restriction enzymes Not I and Pst I, respectively, and the reaction was as follows:
Carrier cleavage System-20. Mu.g of carrier, 10. Mu.L of Pst I, 20. Mu.L of Not I, cutsmart (10 Xbuffer, available from NEB Co.) 50. Mu.L, and H 2 O to 500. Mu.L.
The fragment cleavage system was 5. Mu.g of sdAb fragment, 7. Mu.L of PstI, 14. Mu.L of NotI, cutsmart (10 Xbuffer) 50. Mu.L and H 2 O to 500. Mu.L.
The vector and the cleaved product of the sdAb fragment were mixed and ligated overnight at 4 ℃ with the ligase of NEB.
(15) Construction of phage display library
After PCR Purification Kit (purchased from Beijing Tiangen biochemistry) purification of the ligation products, 1. Mu.L of transformed TG competent cells were recovered at 37℃for 2h, diluted to 10 1,102,103 in a gradient, 300. Mu.L of coated plates each, cultured overnight at 37℃and the number of clones calculated to be about 10 5 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 10 7. 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-80℃cryopreservation.
(16) Library diversity detection
Randomly picking 40 clones in the step (5) as templates, performing clone PCR reaction, detecting PCR products by using 2% 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 40 monoclonal antibodies have 35 amino acid sequences, thus indicating that the constructed library has better diversity.
(17) 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 (15) into 100mL of culture medium for culturing to a logarithmic phase, adding helper phage M13 with MOI (multiplicity of infection ) of 20, standing at room temperature for 30min, centrifuging at a low speed, suspending the precipitate with the 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 to 2.9X10 12 pfu/mL.
Step two, panning PCSK9 single domain antibody by phage display technology
(21) Affinity PCSK9 single domain antibody phage library panning
50Ng PCSK9 antigen coated ELISA plate, 4℃overnight incubation. The next day, the rescued PCSK9 single-domain antibody phage is added, incubated at room temperature for 2h, PBST is washed 10 times, 100 mu L of triethylamine is added, the phage is incubated at room temperature for 30min, and the collected phage is the PCSK9 single-domain antibody phage library obtained by affinity panning. 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.
(22) Amplification and rescue of phages after screening
Amplification and rescue methods were the same as step (17), and the obtained PBS suspension, the phage after the first round of amplification 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.
(23) ELISA evaluation of the enrichment degree of specific antibodies
ELISA plates were coated with 100ng of PCSK9 antigen, 4℃overnight, and blocked for 1h the next day with 5% BSA at room temperature. The phages amplified after each round of panning were added to the experimental group, the control group was added with an equal amount of wild-type phages, and incubated for 2h at room temperature. PBST was washed 10 times to remove unbound phage. Adding HRP-labeled anti-M13 antibody, incubating at room temperature for 1 hr, adding color development liquid, reacting in dark for 15min, measuring light absorption value, gradually increasing with the times of elutriation, and stabilizing during the third round to fourth round of elutriation to obtain enriched specific antibody.
(24) Identification of PCSK 9-specific Single-Domain antibody Positive clones
ELISA plates were coated with 100ng PCSK9 antigen and incubated overnight at 4 ℃. The phage-coated plate obtained in the last round of screening is taken, 38 monoclonals are randomly picked in 1mL of culture medium, the culture is carried out at 37 ℃ until the logarithmic phase, 1mM IPTG is added for induction overnight, the bacterial settlement is collected by centrifugation the next day, after breaking, the supernatant is collected by centrifugation at 5,000g for 15 min. Simultaneously ELISA plates were blocked with 2% BSA at room temperature for 1h. The monoclonal disruption supernatant was added to each well of the experimental group, the blank TG1 disruption supernatant was added to the control group, and the mixture was incubated at room temperature for 2 hours. PBST was washed 10 times, and murine anti-HA-tagged antibody was added for 1h at room temperature. PBST was washed 3-5 times, and AP-labeled anti-mouse IgG antibody was added thereto at room temperature for 1h. And 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).
(25) Positive clone sequence analysis
The DNA of 25 positive clones obtained in the step (24) is extracted to carry out PCR verification on the inserted fragments, and the clones which are positive after the PCR verification are subjected to sequencing analysis. 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 sdAb-C12 is as follows:
LQESGGGLVQAGGSLRLSCAASGSTFKRYAMAWFRQAPGKEREFVAAIEREIPGHPAWSGLTYYAASVKGRFTISR DNAKNTVDLQMNSLKPEDAAVYYCAAGLKYPAPNQLDYDYWGQGTQVTVSS(SEQ ID NO：4). Wherein, 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), 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).
The nucleotide sequence encoding the PCSK9 single domain antibody protein sdAb-C12 is:
5'-CTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGC ACCTTCAAGAGGTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCCGCTATTGAGCGTGACCTTGTACAACCGTTTCCGCCGAGCGGTGGTTTGACATACTATGCAGCGTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGGTGGATCTGCAAATGAACAGCCTGAAACCTGAGGACGCGGCCGTTTATTACTGCGCAGCAGGATTGAAATATCCTGCCCCTAATCAGCTTGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCC-3'(SEQ ID NO：5).
step three, induced expression and purification of anti-PCSK 9 single domain antibody sdAb-C12
(31) Construction of PCSK9 single-domain antibody expression bacterium
Firstly, PCSK9 single-domain antibody monoclonal transfer culture medium is cultured at 37 ℃ overnight, and the next day, plasmid is extracted by using a Plasmid small extraction kit (purchased from Beijing Tiangen Biochemical technology Co.), after agarose gel electrophoresis and concentration measurement, the Plasmid containing PCSK9 single-domain antibody sequence is transformed into an expression bacterium HB2151, coated on a flat plate and cultured at 37 ℃ overnight.
(32) Inducible expression of anti-PCSK 9 single-domain antibody sdAb-C12
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, cultured at 37 ℃ to an OD 600 of 0.6-0.8, and induced to express by addition of IPTG. 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.
(33) Purification of anti-PCSK 9 Single-domain antibody sdAb-C12
PCSK9 single domain antibody sdAb-C12 was obtained by Ni column affinity purification. The Ni column is washed by ultrapure water and then by lysate, and the broken supernatant of the PCSK9 single domain antibody expression bacteria is added into the Ni column at the flow rate of 1 mL/min. The impurity protein was washed with 5 column volumes of affinity A (20 mM imidazole), the target protein was eluted with an equal volume of affinity B (250 mM imidazole), and the eluate was collected. Finally, the purified PCSK9 single domain antibody was detected by electrophoresis on a 13% SDS-PAGE protein gel as shown in FIG. 1. Molecular weight criteria are shown in the left lane of the figure in kilodaltons (kDa), lanes 1 and 2 are the band positions of sdAb-C12 under reducing and non-reducing conditions, respectively (15 kDa).
The single domain antibody capable of targeting and binding PCSK9 can be applied to preparation of anti-PCSK 9 protein monoclonal antibodies, or can be applied to immunological detection of PCSK9 for non-disease diagnosis and treatment purposes.
A pharmaceutical composition comprising a single domain antibody of the invention, together with a pharmaceutically acceptable carrier, diluent or excipient, may be provided, for example.
Affinity assay for PCSK9 and its single domain antibody sdAb-C12, specifically as follows:
(1) ELISA method for analyzing binding of PCSK9 single domain antibody sdAb-C12
The experimental group was incubated with 50ng of PCSK9 protein coated ELISA plate (reserved PBS blank control group), no-coating control group as control group without antigen coating, at 25℃for 2 hours. The purified PCSK9 single domain antibody sdAb-C12 was then blocked with 5% BSA for 1 hour at room temperature, and the experimental groups, blank groups, were each incubated with PBS for 2 hours at room temperature. As shown in FIG. 2, the mouse anti-his-tag antibody was added by 1 XPBST washing 8 times and 1 XPBST washing 5 times at room temperature, and the substrate 3,3', 5' -Tetramethylbenzidine (TMB) was added and reacted for about 10 minutes, and the absorbance at OD450nm was read on an ELISA reader. ELISA detection results are shown in FIG. 3, and the sdAb-C12 single domain antibody has better binding property to PCSK9 from the view of FIG. 3, 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 sdAb-C12
After activation of the chip, PCSK9 antigen was coupled to CM5 chip for Biacore machine and the coupling reaction was stopped to a level of about 800 RU. Subsequently 200. Mu.l of 1M ethanolamine salt was added to wash out residual reactive carboxyl groups, then the machine sequentially pumps the single domain antibody sdAb-C12 (250 nM-125nM-62.5nM-31.25nM-15.6nM-7.8nM-3.9 nM) of the gradient diluted PCSK9 through the chip surface at a rate of 30. Mu.L/min, binding 120s, dissociating 180s, and after data were obtained, the results were processed and are shown in FIG. 4. Parameters of interaction of the single domain antibody sdAb-C12 with the antigen PCSK9 are respectively that Kon (1/Ms) =1.59te+5 is a binding constant, koff (1/s) = 0.01063 is a dissociation constant, rmax (RU) =32.43 is a maximum binding response value, KD (M) = 6.669E-8M is an affinity value of antigen-antibody interaction, which indicates that the interaction of the single domain antibody with PCSK9 antigen is good, and the method has a value of continuous development.
Sequence 1 GSTFKRYA;
sequence 2 IERDLVQPSPPPFGGLT;
sequence 3 AAGLKYPAPNQLDYDY;
sequence 4 LQESGGGLVQAGGSLRLSCAAS;
Sequence 5 MAWFRQAPGKEREFVAA;
Sequence 6 YYAASVKGRFTISRDNAKNTVDLQMNSLKPEDAVYYC;
sequence 7 WGQGTQVTVSS;
Sequence 8:
LQESGGGLVQAGGSLRLSCAASGSTFKRYAMAWFRQAPGKEREFVAAIERDLVQPFPPSGGLTYYAASVKGRFTIS RDNAKNTVDLQMNSLKPEDAAVYYCAAGLKYPAPNQLDYDYWGQGTQVTVSS;
Sequence 9:
5’-
CTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTTCAAGAGGTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCCGCTATTGAGCGTGACCTTGTACAACCGTTTCCGCCGAGCGGTGGTTTGACATACTATGCAGCGTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGGTGGATCTGCAAATGAACAGCCTGAAACCTGAGGACGCGGCCGTTTATTACTGCGCAGCAGGATTGAAATATCCTGCCCCTAATCAGCTTGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCC-3';
Sequence 10: GTCCTGGGCTGCTCTTCTACAAGG;
sequence 11 GGTACGTGCTGTTGAACTGTTCC;
sequence 12 GATGTGCAGCTGCAGGAGTCTGGRGGAGG;
Sequence 13 CTAGTGGGCCGCTGGAGACGGTGACCTGGGT.
Claims (10)
1. A single domain antibody capable of targeting PCSK9 comprises a heavy chain variable region, wherein the heavy chain variable region comprises a framework region and a complementarity determining region, the complementarity determining region comprises a complementarity determining region 1, a complementarity determining region 2 and a complementarity determining region 3, the sequence of the complementarity determining region 1 is shown in sequence 1, the sequence of the complementarity determining region 2 is shown in sequence 2, and the sequence of the complementarity determining region 3 is shown in sequence 3.
2. A single domain antibody capable of targeting PCSK9 binding according to claim 1, wherein the framework regions comprise framework region 1, framework region 2, framework region 3 and framework region 4, the sequence of framework region 1 is shown in SEQ ID NO. 4, the sequence of framework region 2 is shown in SEQ ID NO. 5, the sequence of framework region 3 is shown in SEQ ID NO. 6, and the sequence of framework region 4 is shown in SEQ ID NO. 7.
3. A single domain antibody capable of targeting PCSK9 binding according to claim 2, wherein the sequence of the heavy chain variable region is shown in SEQ ID NO. 8.
4. An expression vector is characterized in that the expression vector comprises a polynucleotide sequence shown as a sequence 9.
5. A preparation method of a single-domain antibody capable of targeting and binding PCSK9 is characterized by comprising the steps of selecting an expression vector and a host cell, wherein the expression vector comprises a polynucleotide sequence shown as a sequence 9, transforming the expression vector into the host cell, and culturing to express the single-domain antibody capable of targeting and binding PCSK 9.
6. The method for preparing a single domain antibody capable of targeting PCSK9 binding according to claim 5, wherein the expression vector is pMECS plasmid, and the host cell is E.coli HB2151 subtype strain;
or the expression vector adopts pPICZa plasmid, and the host cell adopts 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 the single domain antibody capable of targeting and binding to PCSK9 according to claim 5, which is characterized by comprising the following steps:
Immunizing alpaca with eukaryotic expressed PCSK9 antigen, separating peripheral blood mononuclear cell to extract total RNA, reverse transcription and nest PCR library establishment to obtain PCSK9 immune single domain antibody library, coating PCSK9 antigen onto ELISA plate, phage display to screen PCSK9 immune single domain antibody library, and converting the screened single domain antibody into host cell for expression to obtain PCSK9 monoclonal single domain antibody strain.
8. The method for preparing the single domain antibody capable of targeting and binding to PCSK9 according to claim 7, which is characterized by comprising the following specific steps:
step one, construction of PCSK9 single-domain antibody phage display library
(11) PCSK9 immunized alpaca
Mixing PCSK9 with equal volume of Freund adjuvant, injecting into alpaca neck subcutaneous 3-5 points, immunizing once per month, and total immunizing 4 times, wherein 10mL of alpaca peripheral blood is taken in EDTA anticoagulant tube during each immunization, continuously and slowly shaking, and fully mixing;
(12) Blood lymphocyte sample separation
Separating lymphocytes from blood samples collected before and after each immunization;
(13) Total RNA extraction and cDNA Synthesis
Adding equal volume of isopropanol, mixing, standing at room temperature, centrifuging to remove supernatant, drying water, dissolving RNA with water without nuclease, and measuring 1 mu L of RNA for concentration and purity;
Taking RNA, and adopting a kit to synthesize cDNA;
(14) Phage display library construction
Amplifying the V region of the alpaca heavy chain antibody by using cDNA as a template and adopting Nest-PCR;
After the PCR reaction is finished, detecting a PCR product by agarose gel electrophoresis, cutting a target gene fragment of the first round of PCR at 700bp, recovering a target band, performing the second round of PCR, cutting the target gene fragment at 500bp, and recovering the target band, namely an sdAb fragment;
Double-enzyme cutting is carried out on the sdAb fragment and the carrier by using restriction endonucleases Not I and Pst I of NEB respectively, the enzyme cutting products of the carrier and the sdAb fragment are mixed, and the ligation is carried out overnight at 4 ℃ by using ligase of NEB to obtain a ligation product;
(15) Construction of phage display library
Purifying the connection product, taking 1 mu L of transformed TG competent cells, resuscitating for 2 hours at 37 ℃, carrying out gradient dilution to 10 1,102,103, respectively taking 300 mu L of coated plates, culturing at 37 ℃ overnight, and calculating the clone number;
the same transformation method is adopted for large quantity transformation until the clone number of the library reaches more than 10 7, all clones are eluted by the sterilized LB liquid culture medium, centrifugated, suspended by the sterilized LB liquid culture medium, added with glycerol with the same volume and frozen;
(16) Library diversity detection
Randomly picking 40 clones in the step (5) as templates to perform clone PCR reaction, detecting PCR products by using 2% agarose gel electrophoresis, verifying the recombination rate of the constructed PCSK9 single-domain antibody library, sequencing the recombinant PCSK9 single-domain antibody library, and analyzing the diversity of the PCSK9 single-domain antibody library;
(17) Phage amplification and rescue
Culturing the monoclonal library stored in the step (5) in a culture medium until the logarithmic phase, adding the auxiliary phage, standing at room temperature, centrifuging, suspending a sediment in the culture medium, inoculating the sediment in the culture medium, culturing overnight;
Step two, panning PCSK9 single domain antibody by phage display technology
(21) Affinity PCSK9 single domain antibody phage library panning
Taking PCSK9 antigen coated ELISA plate, incubating overnight, adding the rescued PCSK9 single-domain antibody phage the next day, incubating at room temperature, washing the hole by PBST, adding triethylamine, incubating at room temperature, and collecting phage, namely affinity washing the obtained PCSK9 single-domain antibody phage library;
(22) Amplification and rescue of phages after screening
Amplification and rescue methods are the same as step (17);
(23) ELISA evaluation of the enrichment degree of specific antibodies
(24) Identification of PCSK 9-specific Single-Domain antibody Positive clones
(25) Positive clone sequence analysis
The DNA of the positive clone obtained in the step (24) is extracted to carry out PCR verification on the inserted fragment, the clone which is positive through the PCR verification is subjected to sequencing analysis, the sequencing result shows that two nucleotide sequences are obtained, the amino acid sequence of the nucleotide sequences is analyzed, one sequence has the structure of a typical single domain antibody and comprises a framework region and a complementarity determining region, and the nucleotide and amino acid sequence of the single domain antibody monoclonal is as follows:
The amino acid sequence of the PCSK9 single-domain antibody protein sdAb-C12 is shown in a sequence 8. Wherein the sequence of the framework region 1 is shown as a sequence 4, the sequence of the framework region 2 is shown as a sequence 5, the sequence of the framework region 3 is shown as a sequence 6, and the sequence of the framework region 4 is shown as a sequence 7, the sequence of the complementarity determining region 1 is shown as a sequence 1, the sequence of the complementarity determining region 2 is shown as a sequence 2, and the sequence of the complementarity determining region 3 is shown as a sequence 3;
The nucleotide sequence of the encoding PCSK9 single-domain antibody protein sdAb-C12 is shown as sequence 9;
step three, induced expression and purification of anti-PCSK 9 single domain antibody sdAb-C12
(31) Construction of PCSK9 single-domain antibody expression bacterium
Firstly, a PCSK9 single-domain antibody monoclonal transfer culture medium is cultured at 37 ℃ overnight, and after the next day, plasmid is extracted, agarose gel electrophoresis is carried out, and the concentration is measured, the plasmid containing the PCSK9 single-domain antibody sequence is transformed into an expression bacterium HB2151, a flat plate is coated, and the culture is carried out at 37 ℃ overnight;
(32) Inducible expression of anti-PCSK 9 single-domain antibody sdAb-C12
Selecting 5 clones from a flat plate, performing clone PCR to verify whether plasmid is transferred into an expression strain, selecting positive clones, culturing at 37 ℃ until OD 600 is 0.6-0.8, adding IPTG for induced expression, centrifuging bacterial liquid, collecting bacterial precipitate, re-suspending the precipitate with a lysis buffer, ultrasonically crushing the bacterial, centrifuging and collecting the crushed bacterial supernatant;
(33) Purification of anti-PCSK 9 Single-domain antibody sdAb-C12
PCSK9 single domain antibody sdAb-C12 was obtained by Ni column affinity purification.
9. The method of claim 8, wherein in step (14), the names and sequences of the post-PCR primers are as follows:
in the first round, the primer name is CALL001, the sequence of which is shown as sequence 10, the primer name is CALL002, the sequence of which is shown as sequence 11;
In the second round, the primer was named sdAb-Back, whose sequence is shown as sequence 12, and the primer was named sdAb-For, whose sequence is shown as sequence 13.
10. The application of the single domain antibody capable of targeting and binding PCSK9 in preparing anti-PCSK 9 protein monoclonal antibodies according to any one of claims 1-3 or in immunological detection of PCSK9 for the purpose of non-disease diagnosis and treatment.
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