CN108059679B

CN108059679B - Humanized single-chain antibody and application thereof

Info

Publication number: CN108059679B
Application number: CN201711449190.5A
Authority: CN
Inventors: 李桂英; 赵良中; 郜瑞娟; 赵佳亮; 刘子钰; 琚常青
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2017-12-27
Filing date: 2017-12-27
Publication date: 2020-03-24
Anticipated expiration: 2037-12-27
Also published as: CN108059679A

Abstract

The invention belongs to the field of antibody drugs, discloses a humanized single-chain antibody INP resisting PHD2 and a humanized intracellular single-chain antibody ER-INP resisting PHD2 which are prepared by screening by using a phage antibody library technology and using a genetic engineering method, and discloses a polynucleotide for coding the humanized single-chain antibody, a vector containing the polynucleotide and application. The humanized single-chain antibodies INP and ER-INP are low-toxicity, high-efficiency and specific active molecules for inhibiting PHD2 hydroxylation activity, and avoid toxic and side effects of heterologous antibodies on human bodies. Meanwhile, the humanized single-chain antibody is a small-molecule single-chain antibody and has the advantages of small molecular weight, low immunogenicity and the like. In addition, the introduction of endoplasmic reticulum localization signal enables human intracellular single-chain antibody ER-INP to efficiently and stably inhibit the hydroxylation activity of PHD2 in cells, can effectively increase the protein level of HIF, promote angiogenesis and repair of tissue injury such as liver, and has a protective effect on tissue injury.

Description

Humanized single-chain antibody and application thereof

Technical Field

The invention belongs to the field of antibody medicines, and particularly relates to a human single-chain antibody and application thereof.

Background

The dynamic balance of oxygen plays an important role in the physiological and developmental processes of the body, and the body also responds to hypoxia as the oxygen concentration changes in the environment under hypoxic conditions, hypoxia-inducible factors (hypoxia-induibles factors, HIFs) were found as the most important factor of hypoxia stress, promoting hypoxia adaptive response under hypoxic conditions HIF-1 was first recruited by Semenza G L et al in 1992 in gel migration analysis of nuclear extracts of hypoxia-treated Hep3B and HepG2 cell lines, and experiments showed that HIF-1 mediated transcription of inducible Erythropoietin (EPO) gene, HIF-1 was the most sensitive factor of acute hypoxia stress, as the major transcription factor of hypoxia response of the body, current studies showed that 1000 more of its downstream genes have a central role in acute or chronic hypoxia adaptive response, such as regulation of erythropoiesis and neovascularization, regulation of metabolism of cells, regulation of cell metabolism, and regulation of proliferation of cell proliferation and proliferation of prolyl-hydroxylase, and proliferation of HIF-hydroxylation protein through the two key pathways of Hep-hydroxylase genes (HIF-6326, HIF) and histone hydroxylase-94-1-cholesterol-binding to Hep-1, and prolyl-hydroxylase-cholesterol-degrading factor, and histone-cholesterol-1, which are suggested by the two key pathways under conditions of Hep-cholesterol-degrading enzyme, i-cholesterol-binding, kinase-binding to the protein-cholesterol-binding protein, and promoting factor-binding protein, and promoting angiogenesis-binding protein, and promoting protein-binding protein, and promoting angiogenesis-binding protein, and promoting angiogenesis-binding protein, and promoting cell-binding protein, and promoting angiogenesis-binding protein, and promoting angiogenesis-binding protein, and promoting cell-binding protein, and promoting angiogenesis-binding protein, and promoting cell line-binding protein-.

Prolyl hydroxylase (Prolyl hydroxylase domain, PHD), also known as HIF proline hydroxylase (HIF-Prolyl hydroxylases, HPHs), regulates HIF- α protein levels by hydroxylating 2 proline residues in The oxygen-dependent degradation region of HIF- α, mediating its degradation through The ubiquitination pathway, is The key rate-limiting enzyme in this catalytic process, TreC and coworkers (Genetics,1983,104(4): 619) in 1983), suggesting that they isolated from oviposition-deficient C.elegans Egl-9 Gene, Epstein A C R (Cell,2001,107(1):43-54) suggested that The protein family encoded by The Egl-9 Gene could hydroxylate- α. Martin S.taylor M S (Gene,2001,275, 1) suggested that The homology of The HIF 125 and PHGO-9 protein encoded by The murine PHYb-9 Gene could be increased by hydroxylating of The proline hydroxylase module, PHGO 19, PHG-11-33, PHG-11, PHG-33-11, thus The major proline hydroxylase activity was shown to be increased by The hydroxylating protein activity of The mouse phg-3978, PHG-19, PHG-33, PHG-11, thus increasing The proline-7-11, PHG-7, PHG-11, PHG-7, and PHG-7, PHG-11, which were found to be The most important genes, and E, which were found to be increased in The effects of The hydroxylating activity of The hydroxylating protein in The processes of The hydroxylating protein, which was shown by The hydroxylating protein which was found that The major effects of The proline hydroxylase, which was increased in The hydroxylating enzyme in The activity of The protein in The same or.

The role of PHD in hypoxia, ischemia, inflammation and injury has been reported, as Campbell EL (Immunity,2014,40(1):66-77.) in 2014, it is proposed that inhibition of PHDs activity and improvement of stability of HIFs in tissue hypoxia can serve as an intrinsic adaptive response to the relative balance of hypoxia-induced inflammation and restoration of normal cell function, Bernhardt W M (Journal of the American Society of neuroprology, 2010,21(12):2151-2156) in 2010, and as a clinical test, it is proposed that oral biological PHD inhibitors can improve stability of HIF in normoxic conditions, promote expression of EPO, and have been safely used in renal anemia patients, kEcle T (PLoS Biol,2013,11(9 e 1665) in 2013, it is proposed that inhibitors can significantly improve the stability of HIF in mice with acute lung injury and reduce lung inflammation, 2006, as well as a target for improving angiogenesis activities of endothelial cell repair, such as VEGF regeneration, VEGF-11 (Na) in 2006), and VEGF-35, Na-K, Na-K-Na-K-Na-K-Na-K-Na-K-Na-K-Na-K-Na-K-Na.

However, the above-mentioned PHD activity-inhibiting agents have the following problems and disadvantages in the research of the treatment of tissue damage repair caused by ischemia, hypoxia, inflammation, and the like: firstly, the PHD small molecule inhibitor lacks the problems of subtype specificity and tissue targeting, so that obvious toxic and side effects can be generated; secondly, the iron chelator or the iron competitor is used as a PHD inhibitor, can cause the iron metabolism disorder of the organism, has obvious toxic and side effects and is limited in clinical application; in addition, the target and efficient inactivation of PHD2 cannot be realized due to the problems of instability, narrow action range and the like.

The single-chain antibody is a small molecule antibody prepared by genetic engineering method, and is prepared by making the heavy chain variable region (V) of the antibody with elastic connecting peptide (generally 12-15 amino acids)_H) And the variable region of the light chain (V)_L) The molecular weight of the connected recombinant antibody is only one sixth of that of the original natural antibody, but the single-chain antibody contains all antigen binding sites, so that the single-chain antibody retains the antigen binding activity of the antibody to the maximum extent and is a small fragment with the antigen binding activity of the parent antibody. Because of its incomparable advantages, single-chain antibodies have been modified and modified by the prior art to improve their functions and develop new applications, among which intracellular antibody (intracellular antibody) technology has been developed in recent years,with the intensive research on antibody engineering and intracellular signal transduction, a novel antibody technology capable of blocking important target proteins in cells is derived. The intracellular antibody technology can properly modify antibody molecules by adding a cell nucleus positioning signal or an endoplasmic reticulum retention signal and other methods, so that the antibody molecules are directionally distributed in cell nucleus, cell cytoplasm or certain organelles, and the activity, the processing and the secretion process of certain biological macromolecules distributed in the part are specifically interfered or blocked, a series of biological processes of the cell are changed, so that the effect of combining and inactivating any cytoplasmic structure is achieved, and the phenotype knockout of important target molecules is realized. It is a new gene therapy approach following the technologies of antisense RNA, specific ribozyme, dominant negative mutation, suicide gene and the like. Compared with the newly developed RNAi technology, the intracellular antibody can act on the protein in the cell with high specificity and high stability, and has huge potential and application prospect in anti-tumor gene therapy.

Disclosure of Invention

In view of the above, the present invention provides a low-toxicity, high-efficiency, specific, and stable anti-PHD 2 humanized single-chain antibody, which can be used as a prolyl hydroxylase inhibitor to effectively knock out the phenotypic function of PHD2 in cells, thereby inhibiting hydroxylation of HIF-1 α by PHD2, up-regulating the HIF-1 level, promoting angiogenesis, and simultaneously achieving the purposes of protecting tissue damage and promoting repair of tissue damage such as liver, etc., aiming at the problems of toxic and side effects caused by lack of specificity and tissue targeting of the small molecule inhibitor for inhibiting PHD2 activity, and the problems of relative instability and narrow action range of antisense technology and RNA interference technology.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the human PHD2 gene is obtained by cloning from MCF-7 cells, and is cloned into a pET28a (+) prokaryotic expression vector to express and purify recombinant human PHD2 protein. The purified recombinant human PHD2 protein is used as an antigen to carry out the screening process of 'adsorption-washing-elution-collection-amplification' on a human phage antibody library, and the anti-PHD 2 human single-chain antibody is obtained by enrichment.

The amino acid sequence of the humanized single-chain antibody of the specific anti-PHD 2 is shown as SEQ ID NO. 1 and named as INP.

The amino acid sequence of the humanized single-chain antibody of the specific anti-PHD 2 is an amino acid sequence which is at least 80 percent homologous with the amino acid sequence shown in SEQ ID NO. 1 and has the same or similar functions by substituting, deleting or adding one or more amino acids.

In some embodiments of the invention, the plurality is 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.

In some embodiments of the invention, the human single chain antibody against PHD2 has an amino acid sequence that is 80%, 85%, 90%, 95%, or 97% homologous to the amino acid sequence set forth in SEQ ID NO. 1. Since PHD2 is mainly distributed in cytoplasm and exerts its hydroxylation function in cytoplasm, to achieve the function of INP-specific inhibition of PHD2, HIF levels are increased, and its angiogenesis promoting and tissue injury repairing functions are exerted. The invention further uses INP as a template, clones endoplasmic reticulum positioning signals, and obtains an anti-PHD 2 humanized intracellular single-chain antibody which comprises an endoplasmic reticulum positioning signal peptide ER and an E-tag label peptide through an E-tag detection signal, and is named as ER-INP.

Preferably, the amino acid sequence of the humanized single-chain antibody is shown as SEQ ID NO. 3.

The invention also provides a nucleotide for coding the human single-chain antibody.

In some embodiments, the nucleotides encoding the human single chain antibody INP of anti-PHD 2 are set forth in SEQ ID NO: 2.

In some embodiments, the nucleotides encoding the anti-PHD 2 human intracellular single chain antibody ER-INP are shown in SEQ ID NO: 4.

The invention provides an expression vector, which comprises nucleotide for coding the human single-chain antibody.

In some embodiments, the vector comprising the human intracellular single chain antibody ER-INP encoding anti-PHD 2 is pcDNA3.1-ER-INP.

The invention also provides a pharmaceutical composition which comprises the human single-chain antibody INP resisting PHD2 or the human intracellular single-chain antibody ER-INP resisting PHD 2.

In some embodiments, the invention analyzes the binding activity of the purified anti-PHD 2 single-chain antibody INP with the recombinant human PHD2 by ELISA and determines the affinity of the anti-PHD 2 single-chain antibody INP, and shows that the human single-chain antibody INP with anti-PHD 2 can specifically bind to the human recombinant PHD2 protein, and can competitively inhibit the binding of the anti-PHD 2 single-chain antibody with the recombinant human PHD2 protein, and can be used as prolyl hydroxylase inhibitor. Therefore, the invention provides the application of the human single-chain antibody INP resisting PHD2 and the human intracellular single-chain antibody ER-INP resisting PHD2 as prolyl hydroxylase inhibitors.

In some embodiments, the invention researches the expression condition, the location condition and the in vitro biological activity of the recombinant endoplasmic reticulum-located INP gene after transfecting a mouse macrophage Raw264.7 cell strain and a human embryonic kidney cell HER293 cell strain with the constructed eukaryotic expression vector pcDNA3.1-ER-INP, and the result shows that the endoplasmic reticulum-located anti-human PHD2 intracellular antibody INP obtained after adding an endoplasmic reticulum locating signal to the INP can be stably and effectively expressed in the mouse macrophage Raw264.7 cell and the human embryonic kidney cell HER293 cell, realizes the endoplasmic reticulum locating of the INP, obviously improves the protein level of the intracellular HIF-1 α, and inhibits the hydroxylation of the HIF-1 α.

In some embodiments, the mouse macrophage Raw264.7 cell and the human embryonic kidney cell HER293 cell transfected with the pcDNA3.1-ER-INP eukaryotic expression vector are respectively cultured with the human umbilical vein endothelial cell HUVEC, and the results show that the generation of blood vessels can be obviously promoted. Further, the culture medium supernatant of mouse macrophage Raw264.7 cell and human embryonic kidney cell HER293 cell transfected with pcDNA3.1-ER-INP eukaryotic expression vector is added into the yolk sac cavity of the chick embryo of 8-12 days old, and after the culture is carried out for 48 hours at 37 ℃, the result shows that the angiogenesis can be obviously promoted. Therefore, the invention provides the application of the human single-chain antibody INP resisting PHD2 and the human intracellular single-chain antibody ER-INP resisting PHD2 in preparing medicaments with the effect of promoting angiogenesis.

Furthermore, the invention also provides application of the human single-chain antibody INP resisting PHD2 and the human intracellular single-chain antibody ER-INP resisting PHD2 in preparing medicines with tissue injury protection and liver injury repair promotion effects.

According to the technical scheme, the invention discloses a humanized single-chain antibody, namely a humanized single-chain antibody INP resisting PHD2 and a humanized intracellular single-chain antibody ER-INP resisting PHD2, which are prepared by screening by using a phage antibody library technology and using a genetic engineering method, and also discloses a polynucleotide encoding the humanized single-chain antibody, a vector containing the polynucleotide encoding the humanized single-chain antibody and application of the polynucleotide.

The human single-chain antibody INP resisting PHD2 and the human intracellular single-chain antibody ER-INP resisting PHD2 are low-toxicity, high-efficiency and specific active molecules for inhibiting PHD2 hydroxylation activity, and the human single-chain antibody is a human antibody, so that toxic and side effects of a heterologous antibody on a human body are avoided. Meanwhile, the humanized single-chain antibody is a small-molecule single-chain antibody, has the advantages of small molecular weight, low immunogenicity and the like, and overcomes the defects of large molecular weight and high immunogenicity of the conventional monoclonal antibody. In addition, an endoplasmic reticulum localization signal is introduced and further recombined into a eukaryotic expression vector, so that the human intracellular single-chain antibody ER-INP can efficiently and stably inhibit the hydroxylation activity of PHD2 in cells, the protein level of HIF can be effectively increased, angiogenesis and liver and other tissue injury repair can be promoted, and tissue injury can be protected. In addition, tissue targeting of the human single-chain antibody INP resisting PHD2 and the human intracellular single-chain antibody ER-INP resisting PHD2 can be realized by using a tissue-specific eukaryotic expression vector, controllability of gene therapy is realized, angiogenesis promoting effect and tissue repair promoting effect can be fully exerted, tissue specificity is realized, targeted therapy is realized, and safety and effectiveness of therapy are ensured.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the amplification of PHD2 and identification of pET28a-PHD2 prokaryotic expression vector in example 1; wherein Panel A is an amplification of PHD2, lane 1 is BS2000DNA Marker;

lanes

2 and 3 are the PCR product of PHD 2; FIG. B is an identification chart of prokaryotic expression vector pET28a-PHD2, lane 1 is pET28a plasmid; lane 2 is pET28a/Hind III; lane 3 is pET28a/Hind III + EcoRI;

lanes

4 and 7 are recombinant plasmid pET28a-PHD 2;

lanes

5 and 8 are pET28a-PHD2/Hind III;

lanes

6 and 9 are pET28a-PHD2/Hind III + EcoRI; lane 10 is PHD2PCR product; lane 11 is a DNA Marker;

FIG. 2 shows the purification of PHD2 protein as analyzed by SDS-PAGE in example 1, wherein lane 1 is Marker; lane 2 shows the cell lysate before induction with pET28a-PHD2/BL21(DE3) IPTG; lane 3 shows the IPTG induction of 4h cell lysate from pET28a-PHD2/BL21(DE 3); lane 4 shows the supernatant of 4h cell disruption induced by pET28a-PHD2/BL21(DE3) IPTG; lane 5 is 10mM imidazole effluent; lane 6 is 40mM imidazole wash effluent; lanes 7-10 are 500mM imidazole eluents;

FIG. 3 shows a graph of the example 1ELISA for identifying PHD2 protein;

FIG. 4 is a graph showing the binding activity of the soluble single-chain antibody to PHD2 protein measured by ELISA method in example 2;

FIG. 5 shows the soluble expression and purification of INP by SDS-PAGE in example 2, lane 1 is Marker; lane 2 is the cell lysate before INP/HB2151IPTG induction; lane 3 is cell lysate after INP/HB2151IPTG induction; lane 4 is ammonium sulfate precipitation; lane 5 is the column effluent; lane 6 is 10mM imidazole effluent; lane 7 is 40mM imidazole wash effluent; lanes 8-10 are 500mM imidazole eluents;

FIG. 6 shows the scheme for identifying INP by Western blot in example 2, lane 1 is cell lysate before induction with pET28a-PHD2/BL21(DE3) IPTG; lane 2 shows the IPTG induction of 4h cell lysate from pET28a-PHD2/BL21(DE 3); lane 3 shows the supernatant of 4h cell disruption induced by pET28a-PHD2/BL21(DE3) IPTG; lane 4 is purified PHD2 protein;

FIG. 7 shows the binding activity of the example 3ELISA to the detection of INP and PHD 2;

FIG. 8 shows an affinity diagram of the non-competitive ELISA assay for INP of example 3;

FIG. 9 is a diagram showing the binding specificity of the anti-PHD 2 single-chain antibody to recombinant human PHD2 protein detected by competitive ELISA in example 3;

FIG. 10 is a graph showing the expression and localization of ER-INP in cells in example 4 by immunofluorescence;

FIG. 11 shows a Western-blot analysis of the expression profile of ER-INP in HEK293 cells of example 4;

FIG. 12 is a graph showing the intracellular binding of ER-INP and PHD2 detected by the co-immunoprecipitation assay of example 5;

FIG. 13 is a graph showing the effect of Western blot in example 5 on the hydroxylation of HIF-1 α, HIF-2 α and H IF-1 α by the intracellular antibody INP;

FIG. 14 is a graph of the effect of the intrabody ER-INP on angiogenesis examined in the HUVEV angiogenesis assay of example 6;

FIG. 15 is a graph showing the effect of intrabody ER-INP on angiogenesis in the chick embryo yolk sac assay of example 6;

FIG. 16 is a graph showing the effect of APAP of example 7 on ALT levels in mouse serum; wherein panel a is serum ALT levels in APAP-induced liver injury mice; panel B is the effect of tail vein injection of ER-INP intrabodies on APAP-induced liver damage mouse serum ALT;

FIG. 17 is a graph showing pathological changes of liver by HE staining of mouse liver in example 7; wherein, the graph A is APAP induced liver injury mouse liver tissue pathological change; FIG. B shows pathological changes of liver of APAP-induced liver injury mice injected with ER-INP intrabodies in tail vein, wherein a is a normal control group; b is APAP model group; c is the injection empty vector + APAP group; d is the group injected with intrabody ER-INP + APAP.

Detailed Description

The invention discloses a human single-chain antibody and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1: preparation of recombinant human PHD2 protein

Primer required for PCR

The PCR amplification PHD2 primer is synthesized by Shanghai biological engineering technical service company, and has the following sequence:

the forward primer HumanPHD2FP is 5'-CCGGAATTCATGCTGGCGCTCGAGTACATC-3'

The reverse primer HumanPHD2RP is 5'-CCCAAGCTTCTATACTTTAGCTCGTGCTCT-3'

Second, Experimental methods

Extracting total RNA of MCF-7 cells, and carrying out reverse transcription to obtain cDNA

Culturing MCF-7 cells in a 6-well plate until the confluency reaches 90-100%, collecting the cells, washing with precooled PBS for 2 times, adding 0.5ml Trizol extract, and extracting total RNA of the cells. Taking the extracted total RNA as an initiator, and adding various components required by reverse transcription reaction respectively according to the instruction of an RT-PCR kit to carry out RT reaction, wherein the conditions are as follows: 10min at 30 ℃, 30min at 45 ℃, 5min at 99 ℃ and 5min at 5 ℃; 94 ℃ for 2 min.

(II) construction of recombinant human PHD2 expression vector

2.1 acquisition of PHD2 Gene fragment

2.1.1. Polymerase Chain Reaction (PCR)

The PHD2 gene was amplified by PCR using cDNA obtained from MCF-7 cells as a template and HumanPHD2FP/HumanPHD2RP primers, PCR reaction program: pre-denaturation at ⑴ deg.C for 2min, at 8694 deg.C for 30s, at ⑶ deg.C for 30s, at ⑷ deg.C for 1min, and extension at ⑸ deg.C for 10min, wherein ⑵ - ⑷ was performed for 30 cycles.

Recovery of PCR amplification products

The PCR product was separated by 1% agarose gel electrophoresis, recovered according to the DNA recovery and purification kit and the purity and concentration of the recovered fragment were checked.

2.2 construction of PHD2 recombinant expression vector and prokaryotic expression purification

The prepared PHD2 fragment is positively cloned into pET28a, pET28a-PHD2 recombinant expression vector is constructed, and then after the sequence and reading frame are identified to be correct through EcoR I and Hind III enzyme digestion analysis and sequencing, BL21(DE3) competent cells are transformed, IPTG induced expression and corresponding condition exploration are carried out, PHD2 soluble expression is induced, recombinant human PHD2 protein is purified through affinity chromatography, and ELISA is used for identification and analysis.

Third, results and analysis

Construction and identification of pET28a-PHD2 recombinant expression vector

According to the known structural characteristics and the enzyme catalysis region of PHD2, a target fragment of human PHD2 with a catalysis structure domain is obtained by utilizing a designed primer through PCR amplification, and after a PCR product is subjected to 1% agarose gel electrophoresis, an obvious DNA band appears around 600bp, which is consistent with the experimental design (figure 1A). The PCR product and pET-28a (+) plasmid were subjected to EcoRI and HindIII double digestion, and the digestion product was successfully recovered. The digested pET-28a vector fragment is connected with a PHD2 target fragment by using T4 DNA ligase, escherichia coli JM109 is transformed, a single clone is selected the next day, recombinant plasmids are extracted after amplification culture, the obtained recombinant plasmids are digested and identified by EcoR I + Hind III, obvious DNA bands are found at 5000bp-6000bp and 500bp-1000bp respectively, and the sizes of the DNA bands are consistent with those of PCR fragments of the pET-28a vector fragment and the PHD2 (figure 1B). The constructed recombinant plasmid pET-28a-PHD2 is entrusted to Shanghai biological engineering Limited company for DNA sequencing analysis, and the result shows that the inserted sequence in the positive recombinant plasmid is consistent with the experimental design and the reading frame is correct. This shows that pET-28a-PHD2 prokaryotic expression vector has been successfully constructed.

2. Induced expression and purification of recombinant human PHD2 gene in colibacillus

Transforming BL21(DE3) competent cells with the recombinant plasmid pET-28a-PHD2 with correct sequencing, and finally determining the optimal induction condition of the soluble protein as OD₆₀₀0.6 IPTG was 0.5mM and induced at 37 ℃ for 4 h.

Corresponding analysis and further experiments were carried out to obtain recombinant human PHD2 protein, the pellet of pET28a-PHD2/BL21(DE3) was sonicated at 37 ℃ for 4h under induction with 0.5mM IPTG, the supernatant of the disrupted suspension was purified by Ni-column affinity chromatography, the protein was eluted with 500mM imidazole and analyzed by 15% SDS-PAGE to find a band of the desired protein at a molecular weight of about 25kDa, and the purity was found to be 98% or more by Bandscan scan (FIG. 2). Desalting by gel chromatography column, and concentrating with PEG20000 to obtain target protein.

3. ELISA identification of recombinant human PHD2 protein

To identify recombinant human PHD2 protein, purified recombinant human PHD2 protein and BSA were coated at a concentration of 10. mu.g/ml, respectively, incubated with anti-PHD 2 murine monoclonal antibody at 37 ℃ for 2h, then with HRP-anti murine IgG at 37 ℃ for 1h, after development of OPD, OD was detected with a microplate reader₄₉₂. The results are shown in FIG. 3, the binding activity of the recombinant human PHD2 protein and the anti-PHD 2 murine monoclonal antibody is significantly higher than that of BSA (P<0.01). The result shows that the purified recombinant human PHD2 protein has better binding activity with the antibody, and the binding is specific.

Example 2: preparation of specific anti-PHD 2 humanized single-chain antibody

Hereinafter, the "specific anti-human PHD2 humanized single-chain antibody" is abbreviated as "INP"

First, experimental material

Escherichia coli (Escherichia coli) JM109, HB2151 and XL1-Blue were purchased from Beijing Ding national Biotechnology, Inc.; the pUC119 plasmid was purchased from Dalibao bioengineering, Inc.; the HRP/Anti-M13 monoclonal antibody was purchased from Pharmacia.

Second, test method

Phage humanized single chain antibody libraries were obtained in Immunotechnology in 1998, specifically in Sbolstero D et al Nat Biotechnology, 2000,18:74-80 and Sbolstero D et aly,1998,3:271-278, and the final library volume was 1X 10 as indicated by the titer determination¹¹The phage human single-chain antibody library of (1).

Screening of antibody library an immobilized antigen immunoadsorption screening method was adopted, in which human PHD2 protein was diluted to 100. mu.g/ml with 0.05mol/L carbonate buffer solution, and 1ml was coated on an immune tube (NUNC), and the antibody library was subjected to 4 rounds of "adsorption-elution-amplification" screening as described in the journal of dermatological diseases, 2005,19: 388-. The secondary pool titer was determined for each round of screening and the phage input/output ratio (recovery) was calculated as an indicator of specific phage antibody enrichment.

Randomly picking 100 clones from colony culture plates of the 4 th round screening, inoculating the colonies into 2-YT for culture, superinfecting the colonies with the helper virus VCSM13 for induction culture overnight, and collecting the supernatant as phage antibody.

Third, results and analysis

1. Screening of anti-PHD 2 humanized phage antibody

After 4 rounds of screening, the recovery rate of the phage antibody is enriched by about 70 times, 100 clones picked from the last 1 round are used for preparing the phage antibody, an ELISA method is used for primary screening, and as a result, 15 clones capable of being combined with human PHD2 antigen are found, ferritin (Fer) and Ovalbumin (OA) are used as control antigens for carrying out phage ELISA identification, and a phage antibody monoclonal which can be specifically combined with human PHD2 and has no combination property with irrelevant antigens is obtained.

2. Expression and binding Activity of soluble Single chain antibodies

In order to detect the binding activity of the obtained phage antibody and human PHD2, the phage antibody supernatant of a positive clone is infected with the strain HB2151, IPTG induces the expression of a soluble single-chain antibody, an ELISA plate is coated with human PHD2, and the culture supernatant of scFv/HB2151 after IPTG induction, Anti-V5 monoclonal antibody and HRP-goat Anti-mouse IgG are used as antibodies for ELISA detection, and the result shows that the clone has specific binding activity with PHD2 protein (figure 4). Sequencing and subsequent analysis results show that the gene of the positive clone has a full length of 759bp (the sequence is shown in SEQ ID NO: 2), and the 253 amino acids are coded and can be specifically combined with PHD2, so that the positive clone is determined to be an anti-PHD 2 humanized single-chain antibody and named INP.

3. Soluble expression, purification and identification of anti-PHD 2 humanized single-chain antibody INP

IPTG-induced supernatant from INP/HB2151 was subjected to 40% ammonium sulfate precipitation, and the precipitate was subjected to PBS dissolution and dialysis for affinity purification. After purification with 10mM imidazole in binding buffer, 40mM imidazole in washing buffer, and 500mM imidazole in elution buffer, the solution eluted with 500mM imidazole showed a clear protein band at about 27kDa as shown in FIG. 5. Then removing imidazole by using a PD-10 desalting column to obtain purified anti-PHD 2 single-chain antibody protein. And (3) carrying out SDS-PAGE electrophoresis on the purified single-chain antibody of the PHD2, and carrying out Western blot analysis by using an anti-V5 Tag monoclonal antibody and an anti-V5 Tag monoclonal antibody as primary antibodies. As shown in FIG. 6, the IPTG-induced INP/HB2151 supernatant and purified INP sample lane showed a specific band of interest at 27kDa, whereas no band of interest was detected in the non-induced INP/HB 2151. These results indicate that soluble expression and purification of the anti-PHD 2 single chain antibody INP has been successfully achieved.

Example 3: activity characterization of anti-PHD 2 humanized single-chain antibody INP

First, experimental material

Reagents and materials refer to example 1 and example 2.

Second, Experimental methods

ELISA analysis of the binding activity of purified anti-PHD 2 single-chain antibody INP to recombinant human PHD 2.

2. The non-competitive ELISA method measures the INP affinity of the anti-PHD 2 single-chain antibody.

3. The competition ELISA experiment detects the binding specificity of the anti-PHD 2 single-chain antibody to PHD 2.

Third, results and analysis

ELISA detection of binding Activity of INP to purified PHD2 protein

In order to identify the binding activity of INP to recombinant human PHD2 protein, purified recombinant human PHD2 protein and BSA were coated at a concentration of 10. mu.g/ml, respectively, using anti-PHD 2 mouse monoclonal antibody as a positive control, INP and anti-V5 tag antibody as experimental groups, followed by incubation with HRP-anti-mouse IgG at 37 ℃ for 1h, and detecting OD492 with a microplate reader after OPD color development. The results are shown in fig. 7, the binding activity of INP to recombinant human PHD2 protein is significantly higher than that of BSA (P <0.01), indicating that purified INP has good binding activity to recombinant human PHD2 protein.

2. Determination of INP affinity of anti-PHD 2 single-chain antibody

Using a non-competitive ELISA method, a standard curve was prepared using the concentration of the antibody as the abscissa and the OD490 value of the antigen-antibody reaction as the ordinate (FIG. 8), and then the curve and the calculation formula (n [ Ab1 ]]-[Ab]) V (n-1) to calculate the affinity constant of the anti-PHD 2 scFv, n is the dilution factor of the anti-PHD 2 scFv, Ab and Ab1 respectively represent 1/2MaxOD when the antigen concentrations were Ag and Ag1₄₉₀The antibody concentration of (4). The value of affinity was found by calculation to be (2.43. + -. 0.16). times.10^-7mol/L。

3. anti-PHD 2 single-chain antibody INP specifically binds to PHD2 protein

To examine the specificity of binding of the anti-PHD 2 single-chain antibody to PHD2, ELISA plates were coated with purified PHD2 protein, anti-PHD 2 single-chain antibody was previously incubated with recombinant human PHD2 protein as experimental group, and anti-PHD 2 single-chain antibody was previously incubated with unrelated proteins (Cyclin D1, PHD2, CDK4, BSA) as control group. As a result, as shown in fig. 9, the binding ability of the anti-PHD 2 single-chain antibody previously incubated with PHD2 protein to the coated recombinant human PHD2 protein was significantly decreased (P <0.01), and this significant inhibition indicates that the binding of the anti-PHD 2 single-chain antibody to PHD2 was specific.

Example 4: preparation of PHD 2-resistant human intracellular single-chain antibody ER-INP

First, experimental material

1. Strains and plasmids

pcDNA3.1(+) plasmid was purchased from Invitrogen; coli JM109 was purchased from Biotechnology Ltd of Beijing ancient China.

2. Molecular cloning major related reagents

Restriction enzymes Hind iii, EcoRV, Taq DNA polymerase and their corresponding buffers, dNTP from gangbao bioengineering ltd, endotoxin-removing plasmid extraction kit from OMEGA, liposomes (Lipofectamine2000), G418, Trizol from Invitrogen, RIPA lysate from bi yun biotechnology ltd, anti-E-tag antibody from pharmacia, Annexin V apoptosis detection kit from nan kayak biotechnology ltd, anti- β -tubulin mouse monoclonal antibody from Sigma, the remainder of examples 1-3.

3. Primer design

The primers used in the experiment were synthesized by Shanghai Biotechnology engineering services, Inc. The method comprises the following specific steps:

ER-INP-FP1:	5’-GTATCAACAGCTACAGCTGTCCACTCCGCCGAAATTGTGATGACGCAGTCT-3’
		ER-INP-RP1:	5’-TTCCGGATCCGGATACGGCACCGGCGCACCTGAGGAGACAGTGACCAGGGT-3’
ER-INP-FP2:	5’-CCCAAGCTTGGATGGAGCTGTATCATCCTCTTCTTGGTATCAACAGCTACAGCTGTC-3’
		ER-INP-RP2:	5’-CCGGATATCTTACAGGTCGTCCTTACGCGGTTCCGGATCCGGATACGGCAC-3’

the forward primer and the reverse primer introduce an endoplasmic reticulum positioning sequence, a detection label and a corresponding enzyme cutting site to construct an intracellular antibody ER-INP.

Second, Experimental methods

(ii) acquisition of endoplasmic reticulum-localized ER-INP Gene fragments

1. Polymerase Chain Reaction (PCR)

In order to prepare an anti-human PHD2 intracellular INP single-chain antibody gene by a directional cloning method and introduce an endoplasmic reticulum localization signal, the gene is constructed into a eukaryotic expression vector pcDNA3.1, and two pairs of primers, namely ER-INP-FP1/ER-INP-RP1 and ER-INP-FP/ER-INP-RP, are designed. The primer design is based on PHD2 human single chain antibody gene sequence and different subcellular zone retention type intracellular antibody gene sequence, and according to PCR primer design principle, the start codon and stop codon and corresponding enzyme cutting site are introduced.

The ER-INP gene fragment positioned by endoplasmic reticulum is amplified by a PCR method by taking an anti-PHD 2 human single-chain antibody gene INP sequence screened from a human phage single-chain antibody library as a template.

A first round PCR reaction system for amplifying the ER-INP gene fragment:

second round PCR reaction System:

recovery of PCR amplification product

(II) construction and identification of ER-INP recombinant eukaryotic expression vector

After the ER-INP fragment is cut and recovered, it is forward cloned between plasmid pcDNA3.1hind III and EcoRV enzyme cutting site to obtain recombinant plasmid pcDNA3.1-ER-INP. The recombinant is transformed into JM109 competent cells, plasmid DNA is extracted by an alkaline lysis method, enzyme digestion identification and sequencing analysis by Shanghai Biotechnology engineering Limited company are correct, and then plasmid for transfection is prepared according to the specification of the endotoxin-removing plasmid extraction kit. After the constructed eukaryotic expression vector pcDNA3.1-ER-INP is transfected into a human liver cancer cell HepG2 cell, a mouse macrophage Raw264.7 cell and a human embryonic kidney cell HER293 cell, the expression and the positioning of the recombined INP gene positioned by the endoplasmic reticulum are researched through an indirect immunofluorescence experiment and a Western-blot analysis experiment.

(III) cell culture

HepG2 cells, HEK293 cells and Raw264.7 cells were cultured in DMEM medium containing 10% calf serum and incubated at 37 ℃ in a saturated humidity incubator with 5% CO 2. The cells grow adherently, and are trypsinized every 2-4 days for subculture.

(IV) cell transfection assay

HepG2 cells, HEK293 cells and Raw264.7 cells in good growth state were treated at 1X 10⁶The density of cells/well is respectively inoculated into a 6-well plate for culture, and when the cell growth reaches 90-95% confluence, the transfection can be carried out by using Lipofectame 2000. Cell pellets were harvested 48h after cell transfection for subsequent experiments.

Third, results and analysis

Successfully cloning endoplasmic reticulum localization type anti-PHD 2 human intracellular single-chain antibody ER-INP

In order to obtain the ER-INP gene of the human intracellular single-chain antibody positioned in the endoplasmic reticulum anti-PHD 2, the ER-INP-FP1/ER-INP-RP1 and primers containing an endoplasmic reticulum positioning signal, Hind III and EcoR V enzyme cutting site sequences and an E-tag label peptide sequence are respectively used as templates to amplify the ER-INP gene of the human intracellular single-chain antibody positioned in the endoplasmic reticulum anti-PHD 2 by a PCR method. Forward cloning the PCR product between HindIII and EcoRV of eukaryotic expression plasmid pcDNA3.1; the positive recombinant plasmid is subjected to double enzyme digestion by restriction enzymes Hind III and EcoRV, and agarose gel electrophoresis shows that an obvious DNA band appears at about 5400bp and about 867bp respectively. Further, the target fragment on the vector pcDNA3.1-ER-INP is detected in a positive and negative two-way mode through a T7 and BGH universal primer, a sequencing result shows that an insertion Sequence is consistent with an experimental design and a reading frame is completely correct, which shows that the pcDNA3.1-ER-INP eukaryotic expression vector introduced with an endoplasmic reticulum locating signal and an E-tag detection label is successfully constructed through PCR amplification, the full length of the ER-INP gene is 867bp (the Sequence is shown in Sequence No.4), and 288 amino acids are coded. This indicates that the eukaryotic expression vector pcDNA3.1-ER-INP of the recombinant intracellular antibody INP gene has been successfully obtained.

(II) stable expression of ER-INP in cells

In order to detect the expression of ER-INP, HepG2 cell, HER293 cell and Raw264.7 cell were transfected with liposome Lipofectam 2000 mediated recombinant plasmid pcDNA3.1-ER-INP and empty control plasmid pcDNA3.1, and the following experiments were performed after 48 hours of culture.

1. Indirect immunofluorescence assay

HepG2 cell and Raw264.7 cell cotransfected by pcDNA3.1-ER-INP and endoplasmic reticulum positioning plasmid pDsRed2-ER are taken as materials, and are subjected to a series of treatments such as cell slide, and the like, and the indirect immunofluorescence experiment analysis is carried out by taking an anti-E-tag mouse monoclonal antibody as a primary antibody and taking FITC-goat anti-rabbit IgG as a secondary antibody. In cells transfected with ER-INP, the cytoplasmic region emitted bright green light, whereas the empty vector control group did not fluoresce, and the endoplasmic reticulum localization plasmid pDsRed2-ER emitted red light, which co-localized with green light, as observed by immunofluorescence microscopy (FIG. 10). Indicating that ER-INP can be effectively expressed in cells and distributed in endoplasmic reticulum region.

Western-blot analysis

In order to further detect the expression of ER-INP in the cells, HEK293 cell lysates transfected with pcDNA3.1-ER-INP and pcDNA3.1, respectively, were collected and subjected to Western-blot detection using an anti-E-tag murine monoclonal antibody with the internal reference being the expression level of β -tubulin in each sample, the results are shown in FIG. 11, in which β -tubulin was expressed in each sample in a uniform amount, and only about 35kDa of the desired band was detected in the pcDNA3.1-ER-INP transfected HEK293 cells, and no relevant band was detected in the pcDNA3.1 transfected cells.

Example 5: activity of anti-PHD 2 intracellular single-chain antibody ER-INP for inhibiting PHD2

First, experimental material

anti-HIF- α antibody was obtained from Novus, Hydroxy-HIF-1 α (Pro564) antibody was obtained from CST, Hydroxy-HIF-1 α (Pro402) antibody was obtained from Millipore, and the remainder of examples 1-4.

Second, Experimental methods

After the constructed eukaryotic expression vector pcDNA3.1-ER-INP is transfected into mouse macrophage Raw264.7 cells and human embryonic kidney cell HER293 cells, the binding activity of the ER-INP and an intracellular target antigen PHD2 is analyzed through a co-immunoprecipitation experiment, and the influence of the ER-INP on the PHD2 activity is analyzed through Western-blot detection of the total protein level and the hydroxylation level of HIF-1 α.

Third, results and analysis

The intracellular antibody ER-INP can bind to PHD2 protein in cells

To determine whether intracellular expressed intracellular antibody ER-INP could bind intracellular PHD2, co-immunoprecipitation experiments were performed. The method comprises the steps of taking HEK293 cells transfected with pcDNA3.1-ER-INP as an experimental group, taking HEK293 cells transfected with empty pcDNA3.1 vectors as a control, cracking the cells, adding an anti-E-tag mouse monoclonal antibody, incubating, adding Protein G-Sepharose for combination, and performing Western-blot experiment by taking an anti-PHD 2 rabbit polyclonal antibody as a primary antibody and an HRP-labeled goat anti-rabbit IgG as a secondary antibody. As shown in FIG. 12, a significant band was observed at the 46kDa position in the cell transfected with pcDNA3.1-ER-INP, but no band was detected in the cell transfected with pcDNA3.1, and in the Input control, a significant band was observed at the 46kDa position in both the vector group and the intrabody group, which is the intracellular PHD2 protein. This indicates that the ER-INP intracellular antibody against PHD2 can effectively bind to PHD2 protein in cells, i.e., the antibody can effectively recognize and bind to its target antigen PHD2 protein in cells.

(II) ER-INP inhibits hydroxylase activity of PHD2, and obviously up-regulates HIF-1 α level

In order to determine whether the intracellular antibody of ER-INP could inhibit PHD2 enzyme activity after binding to PHD2 protein, inhibit hydroxylation of proline in HIF-1 α, inhibit degradation of HIF-1 α, thereby up-regulating HIF-1 α level, the Westernblot method was used to determine the HIF-1 α 1 total protein level and the level of hydroxylated HIF-1 α (Pro402) and HIF-1 α (Pro564) in HEK293 and Raw264.7 cells transfected with pcDNA3.1-ER-INP, and β -Tubulin in each sample was used as an internal reference, as shown in FIG. 13, the HEK293 and Raw264.7 cells transfected with pcDNA3.1-ER-INP were found to have substantially increased HIF-1 α levels, and Hydroxy α (HIF 402-1) and Hydroxy 5961-Pro- α cells were found to have significantly decreased HIF-2 level, while inhibiting the intracellular antibody expression of HIF-2-Pro 638, thereby significantly reducing the level of HIF-2-Pro 2-LR expression in the cells.

Example 6: anti-PHD 2 intracellular single-chain antibody ER-INP angiogenesis promoting effect

First, experimental material

1. Cell culture, transfection and Activity detection reagents and materials refer to examples 1-5.

Second, Experimental methods

(I) Raw264.7 cells and HUVEC coculture in vitro analysis of ER-INP angiogenesis promoting effect

(II) in vivo analysis of ER-INP angiogenesis promoting effect of chicken embryo yolk sac angiogenesis model

Third, results and analysis

In order to examine the effect of ER-INP on angiogenesis, the HEK293 and Raw264.7 cells transfected with pcDNA3.1-ER-INP were co-cultured with HUVEC on cell Matrigel gel, and the angiogenesis promoting effect was observed, and as a result, the HEK293 and Raw264.7 cells transfected with pcDNA3.1-ER-INP were found to have tube formation starting at 6h, with 12h tube formation being most significant, and to have a significant difference compared with the empty vector control group (FIG. 14). The experimental result shows that the ER-INP has the function of promoting angiogenesis in vitro.

To further examine the effect of INP on angiogenesis in vivo, pcDNA3.1 and pcDNA3.1-ER-INP were transfected into HEK293 cells and Raw264.7 cells, and after culturing for 48h, culture supernatants were collected. The culture medium supernatant was dropped into the yolk sac of 8-12 day old chick embryos, cultured at 37 ℃ for 48 hours, and then observed for its effect on angiogenesis during development of the chick embryos, the results are shown in FIG. 15. Compared with the cells of the empty vector control group, the culture medium supernatants of the HEK293 and Raw264.7 cells transfected with pcDNA3.1-ER-INP can obviously promote the angiogenesis of chick embryos, and the ER-INP has the function of promoting the angiogenesis in vivo.

Example 7 anti-PHD 2 intracellular antibody ER-INP has the effects of protecting liver injury and promoting liver injury repair

First, experimental material

APAP (acetaminophen) was purchased from Sigma, USA; the Thinhua-SofastTM in vivo transfection reagent was purchased from Xiamen Sun horse bioengineering, Inc.; the ALT detection kit is purchased from Nanjing to build a bioengineering institute; the rest of the examples are the same as examples 1 to 6.

Experimental animals: balb/c mice, male, 8 weeks old, SPF grade, body weight 22-24g, purchased from Liaoning Biotechnology Ltd.

Second, Experimental methods

Establishment of APAP mouse liver injury animal model

Selecting SPF male Balb/c mice of 8 weeks old, injecting APAP with different doses of 250mg/kg and 300mg/kg into the abdominal cavity, and determining the appropriate liver injury model dose.

(II) measuring serum ALT levels

And after APAP injection, performing retroorbital venous blood collection for 8h, 24h, 48h and 72h respectively, and detecting the ALT level in serum by operating according to the ALT detection kit instruction.

(III) analysis of liver injury degree by HE Pathology of liver tissue

Mice were sacrificed 24h, 48h and 72h after APAP injection, liver tissues of the same sites were taken and fixed in 10% neutral formalin, wax blocks were prepared, sectioned, HE stained, and liver injury conditions were observed.

(IV) the Effect of ER-INP intrabodies in promoting liver injury repair in mice

Balb/c mice were randomly divided into four groups (normal control group, APAP model group, empty vector control + APAP group, intracellular antibody therapy + APAP group), the first two groups were both injected with 5% glucose injection into the tail vein, the third group was injected with empty vector plasmid wrapped with transfection reagent, the last group was injected with intracellular antibody plasmid wrapped with transfection reagent, and the amount of plasmid DNA injected per mouse was determined to be 50ug according to the instructions. APAP is injected into the abdominal cavity 48h after the plasmid injection, and the injection dose is 250 mg/kg.

Third, results and analysis

Establishment of APAP mouse liver injury animal model

To determine the appropriate dose of APAP liver injury, two injections of 250mg/kg and 300mg/kg were selected according to the pre-experiment. Detecting the ALT level of serum at 8h, 24h, 48h and 72h after injection; and observing the pathological changes of the liver tissues for 24h, 48h and 72 h.

As shown in FIG. 16A, ALT assay results showed that both 250mg/kg and 300mg/kg injections caused liver damage and both peaked at 24h, while serum ALT decreased significantly at 48h at 250mg/kg, but only slightly at 300mg/kg, indicating severe liver damage. As shown in FIG. 17A, the HE staining results show that both doses can cause liver damage, the damage is most serious at 24h, and the liver damage is repaired in mice injected with 250mg/kg dose at 48h, but the liver of the mice injected with 300mg/kg dose has large liver cell necrosis, so that the liver damage model is established by selecting 250mg/kg APAP dose.

(II) protective action and repair promoting action of ER-INP intracellular antibody on liver injury

Crowther M et al 2001(J Leukoc Biol, 2001; 70: 478-.

In order to research whether the ER-INP intracellular antibody has the protection effect and the repair promotion effect on liver injury in vivo, 50 mu g/mouse of plasmid wrapped by a fushua transfection reagent is injected into tail vein, 250mg/kg of APAP is injected into abdominal cavity after transfection in vivo for 48h, and the ALT level change in serum and the pathological change of liver injury are detected at different time points after injection. As shown in FIG. 16B, the ALT assay results showed that the ALT levels in each group were increased to various degrees from 8h, peaked at 24h, and then gradually decreased, in addition to the normal control group. ALT level of APAP group is obviously higher than that of normal control group, which indicates the success of liver damage model. At 24h of APAP administration, ALT levels were higher in both the empty vehicle and APAP model groups, while ALT levels were significantly lower in the intrabody group than in the empty vehicle and APAP model groups. The HE results (FIG. 17B) show that in APAP administration for 24h, which is the most serious liver injury, the APAP group shows a great deal of hepatocyte necrosis, the damage degree of the empty vector group is similar to that of the APAP group, while the ER-INP intrabody group only has a small amount of hepatocyte necrosis, and the ER-INP plays a role in protecting the liver and promoting the liver injury repair.

The results show that the ER-INP intracellular antibody has obvious liver protection effect in mice and promotes liver injury repair.

Sequence listing

<110> Jilin university

<120> human single-chain antibody and application thereof

<130>MP1728112

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>253

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>1

Glu Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

2025 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Leu Gln Thr Pro Thr Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Ser Gly Gly Ser Thr Ile Thr Ser Ser Asp Val Tyr His Thr Lys Leu

115 120 125

Ser Ser Ser Gly Thr Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val

130 135 140

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

145 150 155 160

Thr Phe Ser Asp Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys

165 170 175

Gly Leu Glu Trp Val Ala Val Thr Ser Tyr Asp Gly Ser Asp Lys Tyr

180 185 190

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

195 200 205

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met

210 215 220

Ala Val Tyr Tyr Cys Ala Lys Ser Gly Gly Tyr Asn Gly Tyr Gly Val

225 230 235 240

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210>2

<211>759

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>2

gaaattgtga tgacgcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcttgca ggtctagtca gtccctcctg cattctaatg gatataacta tttggactgg 120

tacctgcaga agccagggca gtctccacag ctcctgattt atttgggtag caatcgggcc 180

tctggagtcc cagacaggtt tagtggcagt gggtcaggta ctgatttcac actgaaaatc 240

agcagggtgg aggctgagga tgttggagtt tattactgca tgcaaggtct acaaactccg 300

acgttcggcc aagggaccaa gctcaccgtc ctaggttccg gagggtcgac cataacttcg 360

tctgatgtat accatacgaa gttatcctcg agcggtaccc aggtgcagct ggtggagtct 420

gggggaggcg tggtccagcc tgggaggtcc ctgagactct cctgtgcagc gtctggcttc 480

accttcagtg actacggaat gcactgggtc cgccaggctc caggcaaggg gctggagtgg 540

gtggcagtta catcatatga tggatctgat aagtactacg gagactccgt gaagggccga 600

ttcaccatct ccagagacaa ttccaagaac acgctgtacc tgcaaatgaa cagcctgaga 660

gccgaggaca tggctgtgta ctattgtgcg aagtcaggcg gatataatgg ttacggagtt 720

gactactggg gccagggaac cctggtcact gtctcctca 759

<210>3

<211>288

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>3

Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ser Thr Ala Thr Ala Val

1 5 10 15

His Ser Ala Glu Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val

20 25 30

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

35 40 45

Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

100 105 110

Met Gln Gly Leu Gln Thr Pro Thr Phe Gly Gln Gly Thr Lys Leu Thr

115 120 125

Val Leu Gly Ser Gly Gly Ser Thr Ile Thr Ser Ser Asp Val Tyr His

130 135 140

Thr Lys Leu Ser Ser Ser Gly Thr Gln Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala

165 170 175

Ser Gly Phe Thr Phe Ser Asp Tyr Gly Met His Trp Val Arg Gln Ala

180 185 190

Pro Gly Lys Gly Leu Glu Trp Val Ala Val Thr Ser Tyr Asp Gly Ser

195 200 205

Asp Lys Tyr Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

210 215 220

Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

225 230 235 240

Glu Asp Met Ala Val Tyr Tyr Cys Ala Lys Ser Gly Gly Tyr Asn Gly

245 250 255

Tyr Gly Val Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

260 265 270

Gly Ala Pro Val Pro Tyr Pro Asp Pro Glu Pro Arg Lys Asp Asp Leu

275 280 285

<210>4

<211>867

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>4

ggatggagct gtatcatcct cttcttggta tcaacagcta cagctgtcca ctccgccgaa 60

attgtgatga cgcagtctcc actctccctg cccgtcaccc ctggagagcc ggcctccatc 120

tcttgcaggt ctagtcagtc cctcctgcat tctaatggat ataactattt ggactggtac 180

ctgcagaagc cagggcagtc tccacagctc ctgatttatt tgggtagcaa tcgggcctct 240

ggagtcccag acaggtttag tggcagtggg tcaggtactg atttcacact gaaaatcagc 300

agggtggagg ctgaggatgt tggagtttat tactgcatgc aaggtctaca aactccgacg 360

ttcggccaag ggaccaagct caccgtccta ggttccggag ggtcgaccat aacttcgtct 420

gatgtatacc atacgaagtt atcctcgagc ggtacccagg tgcagctggt ggagtctggg 480

ggaggcgtgg tccagcctgg gaggtccctg agactctcct gtgcagcgtc tggcttcacc 540

ttcagtgact acggaatgca ctgggtccgc caggctccag gcaaggggct ggagtgggtg 600

gcagttacat catatgatgg atctgataag tactacggag actccgtgaa gggccgattc 660

accatctcca gagacaattc caagaacacg ctgtacctgc aaatgaacag cctgagagcc720

gaggacatgg ctgtgtacta ttgtgcgaag tcaggcggat ataatggtta cggagttgac 780

tactggggcc agggaaccct ggtcactgtc tcctcaggtg cgccggtgcc gtatccggat 840

ccggaaccgc gtaaggacga cctgtaa 867

Claims

1. A humanized single-chain antibody has an amino acid sequence shown as SEQ ID NO. 1.

2. The human single chain antibody of claim 1, further comprising an endoplasmic reticulum localization signal peptide, an E-tag peptide.

3. The human single-chain antibody of claim 2, wherein the amino acid sequence is represented by SEQ ID NO 3.

4. A nucleotide sequence encoding the human single-chain antibody of any one of claims 1 to 3.

5. The nucleotide according to claim 4, wherein the nucleotide sequence is shown as SEQ ID NO. 2 or SEQ ID NO. 4.

6. An expression vector comprising nucleotides encoding the human single-chain antibody of any one of claims 1 to 3.

7. A pharmaceutical composition comprising the human single chain antibody of any one of claims 1-3.

8. Use of a human single chain antibody according to any one of claims 1 to 3 for the preparation of a prolyl hydroxylase inhibitor.

9. Use of a human single chain antibody according to any one of claims 1 to 3 for the preparation of a medicament having a pro-angiogenic effect.

10. Use of the human single-chain antibody of any one of claims 1 to 3 for the preparation of a medicament having effects of protecting liver injury and promoting liver injury repair.