CN117603356A - anti-pIgR nano antibody and application thereof - Google Patents

anti-pIgR nano antibody and application thereof Download PDF

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CN117603356A
CN117603356A CN202311620103.3A CN202311620103A CN117603356A CN 117603356 A CN117603356 A CN 117603356A CN 202311620103 A CN202311620103 A CN 202311620103A CN 117603356 A CN117603356 A CN 117603356A
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pigr
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antibody
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伍江波
丁朝阳
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Haibo Huajian Huzhou Pharmaceutical Technology Co ltd
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Haibo Huajian Huzhou Pharmaceutical Technology Co ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • 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®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Abstract

The invention discloses an anti-pIgR nano antibody and application thereof, wherein the nano antibody is PI-19, PI-21, PI-28, PI-5 and PI-74, and the amino acid sequences of the nano antibody are respectively shown in SEQ ID NO: 10. SEQ ID NO: 3. SEQ ID NO: 2. SEQ ID NO:13 and SEQ ID NO: shown at 27. The invention screens by utilizing phage display library to obtain the nano antibody capable of combining with pIgR with high affinity. The nano-antibody against pIgR obtained by screening in the invention binds pIgR with high affinity, can be used for preparing medicines for treating diseases related to pIgR, and provides a new means for treating the diseases.

Description

anti-pIgR nano antibody and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to an anti-pIgR nano antibody and application thereof.
Background
The poly-immunoglobulin receptor (pIgR) belongs to type I transmembrane glycoprotein, can be specifically combined with poly-immunoglobulin A (IgA) and poly-immunoglobulin M (IgM), and can be transported from the basal side membrane of epithelial cells to the top membrane through transcytosis and finally secreted into exocrine fluid. During this process, the extracellular segment of the multimeric immunoglobulin receptor is hydrolyzed, releasing the extracellular segment (also known as the secretory component) that binds to multimeric immunoglobulin a or multimeric immunoglobulin M. Secretory components are important components of sIgA molecules, directly participate in mucosal defense functions of sIgA, and play an important role in passive mucosal immunity. The multimeric immunoglobulin receptor (pIgR) can prevent pathogen adhesion on the luminal surface of mucous membrane by mediating the transport of multimeric immunoglobulin in cells, neutralize virus in epithelial cells, and secrete antigens out of the lamina propria. Thus, efficient secretion of the multimeric immunoglobulin receptor (pIgR) is a prerequisite for the multimeric immunoglobulin to exert a mucosal defense function. The multimeric immunoglobulin receptor (pIgR) is highly N-glycosylated, and its unique sugar chain structure in the molecule may be related to transcytosis of the receptor, correct localization of sIgA at the mucosa, and adhesion of antigen to epithelial cells. The multiple molecular mechanisms involved in the multimeric immunoglobulin receptor (pIgR) and secretory components make them play a central role in mucosal immunity.
The primary function of pIgR is to transport dimeric IgA or multimeric IgM from the lamina propria of the mucosa across epithelial cells to the mucosal surface, a process called transcytosis: (1) After synthesis in secretory epithelial cells, the pIgR is transported to the basal side and binds to the pIg formed in the lamina propria (mainly pIgA, if IgM is present, it can also bind); (2) The pIgR-pIg complex enters cells through endocytosis and then reaches the top side of mucosal epithelial cells through transcytosis; (3) The binding domain of pIgR and pIg is cleaved by protease, and the extracellular region of pIgR and pIgA are released into exocrine fluid in the form of complex to form secretory IgA (SIgA); empty pIgR can also be transported to release free SC into secretion; the released SC and SIgA diffuse into the mucus layer. Thus, pIgR plays a key role in SIgs formation and functions as part of SIgs or as a free SC.
The nano antibody is an antibody which is naturally deleted in light chain and is found in the peripheral blood of alpaca in 1989. Nanobodies comprise only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as heavy chain single domain antibodies, due to light chain deletions. The nano antibody has good tissue penetrability due to small volume and molecular weight, can penetrate through a barrier to reach focus tissues, is easy to discharge, has short half-life and greatly avoids toxic effects. Therefore, by combining the medicine with the nano-antibody targeting the pIgR, the medicine can enter the mucous membrane tissue and blood circulation across the epithelial cells of the mucous membrane layer by utilizing the transportation function of the pIgR, thereby reaching the target organ to achieve the aim of treatment. However, nanobodies with high affinity for pIgR are currently lacking.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide an anti-pIgR nano antibody and application thereof.
The technical scheme for solving the technical problems is as follows: providing an anti-pIgR nano-antibody, wherein the nano-antibody is PI-19, PI-21, PI-28, PI-5 and PI-74;
the amino acid sequence of the VHH region of PI-19 is shown below:
EVQLVESGGGSVQAGGSLRLSCAASGITISNNCMGWFRQAPGKEREGVALLYSTSSTSSQKSHADSVKGRFTISRDNAKNTVYLQMNSLKPEDSAMYYCAAKSGVCRWNWEYGMDYWDWGKGTLVTISS(SEQ ID NO：10)；
the amino acid sequence of the VHH region of PI-21 is shown below:
QVKLVQSGGGLVQPGGSLRLSCAASGFTFENYAMGWLRQAPGEGLEWVSSINYNGDVTAYTTSVKGRFTVSRDNAKNTLYLQLNDLKIDDTAMYYCAKGRYGWALSREDYNTRGQGTQVTVSS(SEQ ID NO：3)；
the amino acid sequence of the VHH region of PI-28 is shown below:
EVKLVESGGGSVQAGGSLRLSCAASGYTYSSACMGWFRQAPGKERDWVALIDGIGSTTYTDSVKGRFTISQDNAKNTLYLQMNSLKPEDTATYYCAADLLSGGYCHPVGGGARDWGGPVVADFTYWGQGTQVTVSS(SEQ ID NO：2)；
the amino acid sequence of the VHH region of PI-5 is shown below:
QVQLVESGGGSVQAGGSLRLSCQVSGYTGSQHSMGWFRQARGKEREGVACININGMTSYKWYDDSVKGRFAISQDNTKNTLYLQMNSLKPEDTAMYYCAAG--RYQLGHCLALPAYTDWGRGTQVTVSS(SEQ ID NO：13)；
the amino acid sequence of the VHH region of PI-74 is shown below:
EVKLVESGGGSVQAGGSLRLSCTASGPTYGRTCMGWFRQAPGREREGVATIETKGLTRYTYYADSVKGRFTISQDDAKNTLYLQMNSLKPEDTAMYYCAADGPGCSGGAFIARGRWYPHWGQGTQVTVSS(SEQ ID NO：27)。
the nucleotide sequence of the gene for encoding the anti-pIgR nano antibody is specifically as follows:
the nucleotide sequence of the gene encoding PI-19 is as follows:
GAAGTACAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAATCACGATCAGTAACAACTGTATGGGCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGCGAGGGGGTCGCGCTGCTTTATAGTACTAGTAGTACTAGTAGTCAGAAATCCCATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGATAACGCCAAGAACACAGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACAGTGCCATGTACTACTGTGCGGCCAAAAGTGGTGTCTGCCGCTGGAATTGGGAGTACGGCATGGACTACTGGGATTGGGGCAAAGGAACCCTGGTCACCATCTCCTCA(SEQ ID NO：37)；
a gene encoding PI-21, the nucleotide sequence of which is:
CAGGTGAAGCTCGTGCAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCGAGAACTACGCCATGGGGTGGCTCCGCCAGGCTCCAGGAGAGGGACTCGAGTGGGTCTCAAGTATTAATTACAATGGTGATGTCACTGCCTACACAACCTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAATTGAACGACCTGAAAATTGATGACACGGCCATGTATTACTGTGCAAAAGGGCGGTATGGATGGGCACTATCACGCGAGGATTATAACACCCGGGGCCAGGGGACCCAGGTCACTGTCTCCTCA(SEQ ID NO：30)；
the nucleotide sequence of the gene encoding PI-28 is as follows:
GAGGTGAAACTCGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACACCTACAGTAGCGCCTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGACTGGGTCGCACTTATTGATGGTATTGGTAGCACAACATACACAGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAATACTCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCACGTACTACTGTGCGGCAGACCTTCTCAGTGGTGGTTACTGCCACCCCGTGGGTGGTGGGGCTCGCGATTGGGGTGGACCCGTGGTCGCTGACTTTACTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO：29)；
the nucleotide sequence of the gene encoding PI-5 is as follows:
CAAGTACAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGCCAAGTCTCTGGGTATACCGGCAGTCAGCACTCCATGGGCTGGTTCCGCCAGGCTCGTGGGAAGGAGCGCGAGGGGGTCGCATGTATTAACATTAATGGTATGACATCATATAAATGGTATGATGACTCCGTGAAGGGCCGATTCGCCATCTCCCAAGACAACACCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAAACCCGAGGACACTGCCATGTACTACTGTGCGGCAGGAAGGTATCAGTTGGGTCACTGTTTGGCTCTACCCGCCTATACAGACTGGGGCCGGGGGACCCAGGTCACCGTGTCCTCA(SEQ ID NO：40)；
the nucleotide sequence of the gene encoding PI-74 is as follows:
GAAGTGAAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACCGCCTCTGGACCTACCTACGGTAGAACCTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAGAGAGCGCGAGGGGGTCGCAACTATTGAGACTAAAGGGCTCACACGCTACACATATTACGCGGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACGACGCCAAGAACACTCTGTATCTCCAGATGAACAGCCTGAAACCAGAGGACACTGCCATGTACTACTGTGCAGCGGACGGGCCAGGTTGTAGTGGTGGTGCCTTCATTGCCCGTGGTCGATGGTATCCACACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO：54)。
an expression vector comprising the above-described gene encoding an anti-pIgR nanobody.
A recombinant cell comprising the expression vector described above.
The application of the anti-pIgR nano antibody in preparing medicaments for preventing and/or treating diseases related to pIgR.
Further, the pIgR related disease is an autoimmune disease of the body.
The invention has the following beneficial effects:
the invention provides a nanometer antibody for resisting pIgR, which is PI-19, PI-21, PI-28, PI-5 and PI-74, and the amino acid sequences of the nanometer antibody are respectively shown in SEQ ID NO: 10. SEQ ID NO: 3. SEQ ID NO: 2. SEQ ID NO:13 and SEQ ID NO: shown at 27. The invention screens by utilizing phage display library to obtain the nano antibody capable of combining with pIgR with high affinity. The nano-antibody against pIgR obtained by screening in the invention binds pIgR with high affinity, can be used for preparing medicines for treating diseases related to pIgR, and provides a new means for treating the diseases.
Drawings
FIG. 1 is a diagram showing SDS-PAGE results of recombinant proteins of PI antibodies.
FIG. 2 is a graph showing the affinity results of monoclonal antibodies with PI proteins.
Detailed Description
The examples given below are only intended to illustrate the invention and are not intended to limit the scope thereof. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1:
the preparation method of the anti-pIgR nano antibody comprises the following steps:
1. enrichment of phage
In a Maxi-sorp 96-well plate, using a Human-pIgR-ECD-his antigen recombinant protein as a target, solid phase screening a camel immune library LibP/T-camel, and solid phase screening for 2-3 rounds to obtain enriched phage, wherein the specific process is as follows:
coating: antigen was placed in Maxi-sorp plates at 500 ng/Kong Baobei, 4℃overnight;
closing: the Maxi-sorp plates were blocked with 1% PVA and 2h at room temperature;
combining: adding an antibody library, mixing, and reacting for 2 hours at room temperature;
cleaning: washing Maxi-sorp plates with PT;
eluting: 100. Mu.L of 100mM HCl was added to the Maxi-sorp plate and bound phage eluted;
and (3) neutralization: neutralization was performed using 1M Tris-HCl;
amplification: 1mL of OD600=0.8 was infected with 100. Mu.L of the neutralization solution5-alpha F' Iq component E.coli cells, and then shaking at 37℃for 1h; then adding M13KO7 helper phage, shaking at 37 ℃ for 1h; finally, transferring the strain into 40mL of 2YT/Carb/Kan culture medium, and performing overnight expansion culture at 37 ℃;
enrichment identification: drop method plating (performed in synchronization with the amplification step described above) uses PVA wells as negative controls. If the number of phage clones of antigen Kong Fuji was more than 5 times the number of phage clones of PVA Kong Fuji, this was defined as successful enrichment.
Harvesting: separating and purifying the phage cultured in expansion mode by using a PEG8000/NaCl precipitation method, and then entering a next round of elutriation and screening until enrichment is successful.
Colony cloning conditions of the above-described panning were as follows in table 1:
TABLE 1 colony clone statistics for panning
2. Phage ELISA identification
After successful phage enrichment, 96 monoclonal antibodies were randomly picked for phage ELISA identification, and the specific process was as follows:
coating: antigen was placed in ELISA plates at 100 ng/Kong Baobei, 4℃overnight;
shaking: selecting positive phage clones in a deep hole plate, and shaking at 37 ℃ and 220rpm for overnight;
closing: blocking ELISA plates with 1% PVA, blocking for 2h at room temperature;
combining: centrifuging the deep hole plate, obtaining a supernatant which is bacteriophage, transferring 50-100 mu L of the supernatant into an ELISA plate, and reacting for 2 hours at room temperature;
cleaning: washing the ELISA plate with PT, adding 100 mu L of Anti-M13 HRP antibody, and reacting for 1h at room temperature;
color development: washing ELISA plate with PT, washing with PBS, adding 100 μl TMB, standing at 37deg.C, and developing for 5-10min;
and (3) terminating: the reaction was stopped by adding 50. Mu.L of 1M phosphoric acid;
measuring: the absorbance at 450nm was measured with a microplate reader.
Clones with OD value of binding to Human-pIgR-ECD-his antigen recombinant protein/OD value of 2 or more binding to BSA were defined as positive clones, and 92 positive monoclonal antibodies were obtained in total in the process.
The statistical table of the cloning positive rate of the recombinant protein of the Human-pIgR-ECD-his antigen is shown in Table 2:
TABLE 2 statistical results of cloning Positive Rate of recombinant protein of Human-pIgR-ECD-his antigen
3. Positive clone sequencing and affinity sequencing
Phage ELISA positive monoclonal is selected for gene sequencing, sequencing results are analyzed, and sequences which are complete in sequencing, have no terminator mutation and no double peaks are selected for comparison, so that 27 unique sequences (sequences with more than 1 amino acid difference in CDR regions) are obtained in total. The amino acid sequences of CDRH3 were then separated into 16 groups and affinity ordered according to OD values of phage ELISA, see in particular table 3.
TABLE 3 ELISA affinity ranking of positive cloned phages
4. Eukaryotic transient expression
According to the affinity ranking table, 5 positive monoclonal are preferred for each target, the target fragment VHH sequence is amplified by PCR, and then inserted into pCIG-hIgG 1 eukaryotic expression plasmid (purchased from Chengdu Cheng Shijun Biotechnology Co., ltd.) to construct recombinant expression plasmid. The HEK293F expression system was used for 5 days, and the supernatant was collected and purified. The specific process is as follows:
1. constructing a recombinant expression plasmid: preferably positive monoclonal, amplifying target fragment by PCR, inserting into pCIG-hIgG 1 eukaryotic expression plasmid, constructing antibody recombinant expression plasmid, and sequencing to identify correctly inserted clone.
2. Plasmid extraction: competent cells containing plasmids were grown in expansion and recombinant plasmids were extracted using the endotoxin-removing plasmid extraction kit.
3. Transfection: the recombinant plasmid was transferred into HEK293F cells using transfection reagents.
4. Expression: HEK293F eukaryotic transient expression system is expressed for 5 days, and cell supernatant is collected to purify target protein.
5. SDS-PAGE: and (5) taking purified protein, running an SDS-PAGE gel, and identifying the purity and the size of the protein.
The expression of the above-mentioned recombinant protein of the pIgR antibody (also called recombinant protein of PI antibody) is shown in table 4:
TABLE 4 results of recombinant protein expression of pIgR antibodies
The hFC in the above table is a Human FC fragment, with the aim of facilitating antibody purification.
5. Antibody affinity ELISA detection
The affinity of a preferred monoclonal antibody (PI-5-hFc, PI-19-hFc, PI-21-hFc, PI-28-hFc, PI-74-hFc) to an antigen (also referred to as PI protein) was detected using ELISA as follows:
coating: antigen was placed in ELISA plates at 200 ng/Kong Baobei, 4℃overnight;
closing: blocking ELISA plates with 1% PVA, blocking for 2h at room temperature;
combining: starting at 10. Mu.g/mL, 7 concentration gradients were made at a 1:3 ratio, and then 10. Mu.L/well was added to ELISA plates and reacted for 2h at room temperature;
cleaning: washing the ELISA plate with PT, adding 100 mu L of Anti-Human IgG Fc HRP antibody, and reacting for 1h at room temperature;
color development: washing ELISA plate with PT, washing with PBS, adding 100 μl TMB, standing at 37deg.C, and developing for 5-10min;
and (3) terminating: the reaction was stopped by adding 50. Mu.L of 1M phosphoric acid;
measuring: the absorbance at 450nm was measured with a microplate reader.
Recording the obtainedOD value, combining antibody concentration to draw fitting curve, finding out half point of strongest signal, corresponding antibody concentration is EC 50 . Wherein EC is 50 Defined as the concentration that produces 50% efficacy or binding.
The affinity results of the monoclonal antibodies (PI-5-hFc, PI-19-hFc, PI-21-hFc, PI-28-hFc, PI-74-hFc) with PI proteins are shown in Table 5 and FIG. 2.
Table 5 affinity results of monoclonal antibodies with PI proteins
As can be seen from Table 5 and FIG. 2, monoclonal PI-19-hFc, PI-21-hFc, PI-28-hFc and PI protein affinity EC 50 1-10 nM; monoclonal PI-5-hFc, PI-74-hFc and PI protein affinity EC 50 10-30 nM.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (6)

1. An anti-pIgR nanobody, which is characterized in that the nanobody is PI-19, PI-21, PI-28, PI-5 or PI-74;
the amino acid sequence of PI-19 is as follows:
EVQLVESGGGSVQAGGSLRLSCAASGITISNNCMGWFRQAPGKEREGVALLYSTSSTSSQKSHA DSVKGRFTISRDNAKNTVYLQMNSLKPEDSAMYYCAAKSGVCRWNWEYGMDYWDWGKGTLVTI SS；
the amino acid sequence of PI-21 is as follows:
QVKLVQSGGGLVQPGGSLRLSCAASGFTFENYAMGWLRQAPGEGLEWVSSINYNGDVTAYTT SVKGRFTVSRDNAKNTLYLQLNDLKIDDTAMYYCAKGRYGWALSREDYNTRGQGTQVTVSS；
the amino acid sequence of PI-28 is as follows:
EVKLVESGGGSVQAGGSLRLSCAASGYTYSSACMGWFRQAPGKERDWVALIDGIGSTTYTDS VKGRFTISQDNAKNTLYLQMNSLKPEDTATYYCAADLLSGGYCHPVGGGARDWGGPVVADFTYW GQGTQVTVSS；
the amino acid sequence of PI-5 is as follows:
QVQLVESGGGSVQAGGSLRLSCQVSGYTGSQHSMGWFRQARGKEREGVACININGMTSYKW YDDSVKGRFAISQDNTKNTLYLQMNSLKPEDTAMYYCAAG--RYQLGHCLALPAYTDWGRGTQVT VSS；
the amino acid sequence of PI-74 is as follows:
EVKLVESGGGSVQAGGSLRLSCTASGPTYGRTCMGWFRQAPGREREGVATIETKGLTRYTYYA DSVKGRFTISQDDAKNTLYLQMNSLKPEDTAMYYCAADGPGCSGGAFIARGRWYPHWGQGTQVT VSS。
2. a gene encoding the anti-pIgR nanobody of claim 1, characterized in that the nucleotide sequence of the gene is specifically:
the nucleotide sequence of the gene encoding PI-19 is as follows:
GAAGTACAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAATCACGATCAGTAACAACTGTATGGGCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGCGAGGGGGTCGCGCTGCTTTATAGTACTAGTAGTACTAGTAGTCAGAAATCCCATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGATAACGCCAAGAACACAGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACAGTGCCATGTACTACTGTGCGGCCAAAAGTGGTGTCTGCCGCTGGAATTGGGAGTACGGCATGGACTACTGGGATTGGGGCAAAGGAACCCTGGTCACCATCTCCTCA；
a gene encoding PI-21, the nucleotide sequence of which is:
CAGGTGAAGCTCGTGCAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCGAGAACTACGCCATGGGGTGGCTCCGCCAGGCTCCAGGAGAGGGACTCGAGTGGGTCTCAAGTATTAATTACAATGGTGATGTCACTGCCTACACAACCTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAATTGAACGACCTGAAAATTGATGACACGGCCATGTATTACTGTGCAAAAGGGCGGTATGGATGGGCACTATCACGCGAGGATTATAACACCCGGGGCCAGGGGACCCAGGTCACTGTCTCCTCA；
the nucleotide sequence of the gene encoding PI-28 is as follows:
GAGGTGAAACTCGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACACCTACAGTAGCGCCTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGACTGGGTCGCACTTATTGATGGTATTGGTAGCACAACATACACAGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAATACTCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCACGTACTACTGTGCGGCAGACCTTCTCAGTGGTGGTTACTGCCACCCCGTGGGTGGTGGGGCTCGCGATTGGGGTGGACCCGTGGTCGCTGACTTTACTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA；
the nucleotide sequence of the gene encoding PI-5 is as follows:
CAAGTACAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGCCAAGTCTCTGGGTATACCGGCAGTCAGCACTCCATGGGCTGGTTCCGCCAGGCTCGTGGGAAGGAGCGCGAGGGGGTCGCATGTATTAACATTAATGGTATGACATCATATAAATGGTATGATGACTCCGTGAAGGGCCGATTCGCCATCTCCCAAGACAACACCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAAACCCGAGGACACTGCCATGTACTACTGTGCGGCAGGAAGGTATCAGTTGGGTCACTGTTTGGCTCTACCCGCCTATACAGACTGGGGCCGGGGGACCCAGGTCACCGTGTCCTCA；
the nucleotide sequence of the gene encoding PI-74 is as follows:
GAAGTGAAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACCGCCTCTGGACCTACCTACGGTAGAACCTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAGAGAGCGCGAGGGGGTCGCAACTATTGAGACTAAAGGGCTCACACGCTACACATATTACGCGGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACGACGCCAAGAACACTCTGTATCTCCAGATGAACAGCCTGAAACCAGAGGACACTGCCATGTACTACTGTGCAGCGGACGGGCCAGGTTGTAGTGGTGGTGCCTTCATTGCCCGTGGTCGATGGTATCCACACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。
3. an expression vector comprising the gene encoding the anti-pIgR nanobody of claim 2.
4. A recombinant cell comprising the expression vector of claim 3.
5. Use of the anti-pIgR nanobody according to claim 1 for the preparation of a medicament for the prevention and/or treatment of pIgR related diseases.
6. The use according to claim 5, wherein the pIgR related disease is an autoimmune disease of the body.
CN202311620103.3A 2023-11-29 2023-11-29 anti-pIgR nano antibody and application thereof Pending CN117603356A (en)

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