Helicobacter pylori single-chain antibody and application thereof

Abstract

The invention relates to a helicobacter pylori single-chain antibody which is specifically combined with a helicobacter pylori vacuolar toxin protein (VacA) antigen and consists of a light chain variable region (VL), a linker peptide (Linker) and a heavy chain variable region (VH), the light chain variable region (VL) and the heavy chain variable region (VH) are entirely or partially derived from lgG1 or lgG2 or lgG3 or lgG4; the whole single-chain antibody is small, has good penetrability, can be expressed more easily in a common protein expression system, and is short in half-life period, so that rejection reaction does not exist, toxic and side effects are not easy to occur, and infection of helicobacter pylori on normal epithelial cells of the human stomach can be inhibited in an extremely short time; the adhesion of the helicobacter pylori in the stomach can be obviously reduced, and the infiltration of inflammatory cells and the damage of stomach tissues are reduced; the infection ability of helicobacter pylori can be quickly and remarkably reduced, and other harmful effects on stomach tissues and cells are avoided.

Description

Technical Field

[0001] The present invention relates to the field of antibodies against Helicobacter pylori, and particularly to a single-chain antibody of Helicobacter pylori and its uses. Background Art

[0002] Helicobacter pylori ( H p) is a Gram-negative spiral bacterium isolated from gastric mucosa in 1982, mainly transmitted among people through the oral-oral route. Helicobacter pylori is a highly contagious pathogenic bacterium of the digestive tract, which can colonize in human gastric epithelium, thus causing gastric mucosal inflammation. In some patients, it can also cause a series of diseases such as peptic ulcer and gastric cancer.

[0003] Antibiotic treatment is currently the main treatment method for Helicobacter pylori. However, due to the widespread abuse of antibiotics and the easy recurrence of Helicobacter pylori infection, drug resistance has become a major challenge for antibiotic treatment. The new methods for treating Helicobacter pylori mainly include: probiotic-assisted treatment, Helicobacter pylori vaccine, and Helicobacter pylori antibody. However, probiotics cannot completely cure Helicobacter pylori and can only be used as an auxiliary means. Vaccines require multiple immunizations and have a slow onset of action, being more suitable for preventing infection.

[0004] Therapeutic antibodies can specifically bind to target proteins and neutralize the activity of target proteins, thereby reducing the virulence of Helicobacter pylori in a short time or even killing Helicobacter pylori. However, the mechanism by which antibodies clear Helicobacter pylori is still unclear, and the effects are also uneven. There are few studies on existing therapeutic antibodies, and they all focus on chicken antibody IgY, with limited types and limited effects. There is a need for therapeutic antibodies with better effects, especially in the field of genetic engineering, where there is still a blank for Helicobacter pylori therapeutic antibodies. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a single-chain antibody of Helicobacter pylori against the VacA antigen of Helicobacter pylori in the field of genetic engineering.

[0006] A single-chain antibody of Helicobacter pylori, specifically binding to the vacuolating cytotoxin protein (VacA) antigen of Helicobacter pylori, consists of a light chain variable region (VL), a linker peptide (Linker), and a heavy chain variable region (VH); The whole or part of the light chain variable region (VL) is derived from the light chain variable region of IgG1 or IgG2 or IgG3 or IgG4; The whole or part of the heavy chain variable region (VH) is derived from the heavy chain variable region of IgG1 or IgG2 or IgG3 or IgG4.

[0007] Preferably, the single-chain antibody (ScFv) of Helicobacter pylori is, from the N-terminus to the C-terminus, the light-chain variable region (VL), the linker peptide (Linker), and the heavy-chain variable region (VH) in sequence; Alternatively, the single-chain antibody (ScFv) of Helicobacter pylori is, from the N-terminus to the C-terminus, the heavy-chain variable region (VH), the linker peptide (Linker), and the light-chain variable region (VL) in sequence.

[0008] Preferably, the light-chain variable region (VL) comprises the sequence shown in SEA ID NO.1; The heavy-chain variable region (VH) comprises the sequence shown in SEA ID NO.2.

[0009] Preferably, the sequence of the linker peptide (Linker) is shown in SEA ID NO.3; The nucleotide sequence of the single-chain antibody (ScFv) of Helicobacter pylori is shown in SEA ID NO.4.

[0010] Preferably, the nucleotide sequence of the light-chain variable region (VL) of the single-chain antibody (ScFv) of Helicobacter pylori is shown in SEAID NO.5; The nucleotide sequence of the heavy-chain variable region (VH) of the single-chain antibody (ScFv) of Helicobacter pylori is shown in SEA ID NO.6.

[0011] Preferably, the nucleotide sequence of the linker peptide (Linker) is shown in SEA ID NO.7; The nucleotide sequence of the single-chain antibody (ScFv) of Helicobacter pylori is shown in SEA ID NO.8.

[0012] Use of a single-chain antibody of Helicobacter pylori, any of the above single-chain antibodies (ScFv) of Helicobacter pylori, in the preparation of a medicament for treating Helicobacter pylori.

[0013] A pharmaceutical composition comprising any of the above single-chain antibodies of Helicobacter pylori.

[0014] A detection product comprising the single-chain antibody of Helicobacter pylori according to any one of the above, for detecting the presence or amount of Helicobacter pylori in a sample.

[0015] The amino acid sequence in any of the above sequences is substituted, deleted or added with one or several amino acids, has a high homology with the original sequence, preferably more than 80% homology, and retains the original properties and functions of the sequence. The derivative protein is considered that these mutant sequences can achieve the same function and the same effect as the original sequence and are the same protein.

[0016] The nucleotide sequence in any of the above sequences, after substitution, deletion or addition of one or several nucleotides, has a high homology with the original sequence, preferably more than 80% homology, and the derivative gene that retains the original properties and functions of the sequence is considered that these mutant sequences can achieve the same function and the same effect as the original sequence, and are the same gene.

[0017] The VacA protein can promote the function and expression of another virulence protein CagA that is highly related to the occurrence and development of cancer. Antibodies against VacA can effectively bind and neutralize the activity of VacA, reduce the virulence and carcinogenic risk of Helicobacter pylori, and even kill Helicobacter pylori. Therefore, our laboratory set out to design antibodies against Helicobacter pylori targeting VacA, especially single-chain antibodies against Helicobacter pylori targeting VacA. Because single-chain antibodies are relatively small in size, have good penetrability, can be more easily expressed in common protein expression systems, and have a short half-life, there is no rejection reaction and it is not easy to have toxic side effects, and they have a certain therapeutic effect on Helicobacter pylori. At the same time, humanized IgG was selected to increase the content of therapeutic antibodies against Helicobacter pylori in serum and improve the ability of the immune system to kill Helicobacter pylori.

[0018] The designed single-chain antibody of Helicobacter pylori (ScFv) has a stable structure, can be normally expressed, and has antigen-binding ability. Through the experiment of co-culturing the single-chain antibody of Helicobacter pylori and the normal human gastric epithelial cell GES-1 infected with Helicobacter pylori, it can be seen that the single-chain antibody of Helicobacter pylori can inhibit Helicobacter pylori ( H p) infection of normal human gastric epithelial cells GES-1 within a very short time.

[0019] According to the results of mouse experiments, it can be seen that this single-chain antibody of Helicobacter pylori (ScFv) is very successfully designed. This single-chain antibody of Helicobacter pylori (ScFv) can significantly reduce the adhesion of Helicobacter pylori in the stomach, reduce the infiltration of inflammatory cells and the damage of gastric tissue; the single-chain antibody of Helicobacter pylori (ScFv) can quickly and significantly reduce the infectivity of Helicobacter pylori, and it will not cause other harmful effects on gastric tissue and cells itself. Description of the Drawings

[0020] Figure 1 is the three-dimensional modeling result of the single-chain antibody of Helicobacter pylori (ScFv).

[0021] Figure 2 is the QMEANDisCo analysis result of the single-chain antibody of Helicobacter pylori (ScFv).

[0022] Figure 3It is the Ramachandran Plot analysis result of the single-chain antibody (ScFv) of Helicobacter pylori.

[0023] Figure 4 It is the result of protein-protein docking between the single-chain antibody (ScFv) of Helicobacter pylori and the Helicobacter pylori vacuolating toxin protein (VacA) antigen.

[0024] Figure 5 It is the structural diagram of the pCzn1 vector plasmid.

[0025] Figure 6 It is the structural diagram of the recombinant plasmid pCZN1-ScFv.

[0026] Figure 7 It is the result of double digestion of the recombinant plasmid pCZN1-ScFv with Nde I-Xba I and electrophoresis detection.

[0027] Figure 8 It is the sequencing result of the recombinant plasmid pCZN1-ScFv after double digestion with Nde I-Xba I.

[0028] Figure 9 It is the SDS-PAGE protein expression identification result.

[0029] Figure 10 It is the SDS-PAGE identification result of the purified protein.

[0030] Figure 11 It is the Western Blot identification result of the purified protein.

[0031] Figure 12 It is the result diagram of Western Blot detecting the binding ability of ScFv to the Helicobacter pylori vacuolating toxin protein (VacA) antigen. G12 in the figure is the alias abbreviation of ScFv.

[0032] Figure 13 It is the result of detecting the binding ability of ScFv to the antigen VacA by indirect ELISA. (***, p<0.001; **, p<0.01; *, p<0.05) Figure 14 It is H The expression levels of p-16s-RNA and COX-2 in the H ScFv antibody-treated GES-1 cell model infected with p. (***, p<0.001; **, p<0.01; *, p<0.05) Figure 15 It is H The identification result of the mouse model infected with p.

[0033] Figure 16 The expression results of p-16sRNA and COX-2 in the stomachs of mice in each group were detected by RT-qPCR. H

[0034] Figure 17 The expression results of the inflammatory factor COX-2 in the stomachs of mice in each group were detected by ELISA.

[0035] Figure 18 The damage conditions of mouse gastric tissues were identified by HE staining.

[0036] Figure 19 The H infection conditions of mouse gastric tissues were identified by immunohistochemistry. Specific implementation manners

[0037] The technical solutions and technical effects of the embodiments of the present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the present invention.

[0038] The materials, reagents, etc. used in the following examples can be obtained from commercial channels without special instructions. For those not specifying specific technologies or conditions in the examples, they shall be carried out according to the technologies or conditions described in the literature in this field (for example, referring to "Molecular Cloning: A Laboratory Manual", Third Edition, J. Sambrook et al., translated by Huang Peitang et al., Science Press) or according to the product instructions.

[0039] VacA is one of the most important virulence factors in Helicobacter pylori-induced host cell lesions and is named because it can induce vacuole formation in eukaryotic cells. The vacuole toxin exists in all known Helicobacter pylori strains. The vacuole toxin has multiple functions. First, the vacuole toxin can cause vacuolization of target cells; second, the vacuole toxin can inhibit cell proliferation, thereby affecting the repair of gastric mucosal cells; third, the vacuole toxin can cause mitochondrial depolarization and thus induce apoptosis; fourth, the vacuole toxin can also inhibit the activation of T lymphocytes in the lamina propria to disrupt the immune response; fifth, VacA inhibits the expression of IL-23 in dendritic cells and induces the expression of IL-10 and TGF-β in macrophages. Therefore, Helicobacter pylori can create a tolerant environment through the immune regulatory activity of VacA. This may cause the response of T cells to Tregs to deviate. This mechanism helps Helicobacter pylori to persist in an environment that affects host cell immunity. VacA can inhibit autophagy of gastric mucosal epithelial cells, resulting in a weakened ability of cells to clear Helicobacter pylori and its virulence factors. Therefore, antibodies against VacA can effectively bind and neutralize the activity of VacA, reduce the virulence and carcinogenic risk of Helicobacter pylori, and even kill Helicobacter pylori.

[0040] ​Single-chain antibody is a new type of genetically engineered antibody, which consists of the variable region of the light chain, the variable region of the heavy chain and the linker peptide connecting the two variable regions, and can retain the ability of conventional antibodies to specifically bind antigens. Moreover, the single-chain antibody is relatively small in size, has good penetrability, is easier to express in common protein expression systems, has a short half-life, has no rejection reaction, and is not prone to toxic and side effects.

[0041] Retrieve the variable region of the light chain (VL) derived from IgG1 or IgG2 or IgG3 or IgG4 and the variable region of the heavy chain (VH) derived from IgG1 or IgG2 or IgG3 or IgG4 from the existing gene library in the laboratory, screen suitable variable regions of the light chain (VL) and variable regions of the heavy chain (VH), and design a linker peptide (Linker) to connect the variable region of the light chain (VL) and the variable region of the heavy chain (VH) into a single-chain antibody of Helicobacter pylori (ScFv). The single-chain antibody of Helicobacter pylori (ScFv) is abbreviated as G12 in the experiment.

[0042] Perform three-dimensional modeling on the single-chain antibody of Helicobacter pylori (ScFv) through trRosetta, and the results are as Figure 1 shown. After modeling, its structure is evaluated. The Siwss-model uses the GMQE (Global Model Quality Estimate) score to evaluate the protein model. The GMQE credibility range is 0-1, and the closer to 1, the better the quality. As Figure 2 can be seen, the score of the single-chain antibody of Helicobacter pylori (ScFv) is 0.82±0.05. Then, perform a Ramachandran plot analysis on the single-chain antibody of Helicobacter pylori (ScFv), and the results are as Figure 3 shown. More than 90% of the residues of both fall into the optimal region. In summary, the predicted three-dimensional structure of the single-chain antibody of Helicobacter pylori (ScFv) has sufficient confidence and can be used for subsequent protein docking.

[0043] Obtain the spatial structure information of the antigen domain (PDB ID: 2QV3) of Helicobacter pylori vacuolating cytotoxin protein (VacA) from the RCSB Protein Data Bank (https: / / www.rcsb.org / ), input it together with the predicted model of the single-chain antibody of Helicobacter pylori (ScFv) into Z-Dock (https: / / zdock.umassmed.edu / ) for protein docking, and use the bioinformatics software PyMOL to analyze the binding surface residues of the protein docking model to predict the binding ability and binding mode of the single-chain antibody of Helicobacter pylori (ScFv) to the antigen of Helicobacter pylori vacuolating cytotoxin protein (VacA).

[0044] The results of protein-protein docking between the single-chain antibody (ScFv) of Helicobacter pylori and the vacuolating cytotoxin protein (VacA) antigen of Helicobacter pylori are as follows Figure 4 As shown, the sky blue color represents the VacA-P55 domain, the ball-and-stick model represents the binding site of VacA-P55 and ScFv, the yellow dashed line represents the hydrogen bond, and the blue dashed line represents the π-bond interaction. It can be seen that the structure of the single-chain antibody of Helicobacter pylori does not affect its function of binding to the antigen.

[0045] Based on the above design and the process of structural simulation verification, the inventor finally determined a single-chain antibody (ScFv) of Helicobacter pylori that specifically binds to the vacuolating cytotoxin protein (VacA) antigen of Helicobacter pylori, which consists of a light-chain variable region (VL), a linker peptide (Linker), and a heavy-chain variable region (VH). The light-chain variable region (VL) as a whole or in part is derived from the light-chain variable region of IgG1 or IgG2 or IgG3 or IgG4; the heavy-chain variable region (VH) as a whole or in part is derived from the heavy-chain variable region of IgG1 or IgG2 or IgG3 or IgG4.

[0046] The single-chain antibody (ScFv) of Helicobacter pylori is, from the N-terminus to the C-terminus, the light-chain variable region (VL), the linker peptide (Linker), and the heavy-chain variable region (VH) in sequence; Alternatively, the single-chain antibody (ScFv) of Helicobacter pylori is, from the N-terminus to the C-terminus, the heavy-chain variable region (VH), the linker peptide (Linker), and the light-chain variable region (VL) in sequence.

[0047] The light-chain variable region (VL) contains the sequence shown in SEA ID NO.1; The heavy-chain variable region (VH) contains the sequence shown in SEA ID NO.2.

[0048] The linker peptide (Linker) has the sequence shown in SEA ID NO.3; In summary, the sequence of the single-chain antibody (ScFv) of Helicobacter pylori is shown in SEA ID NO.4.

[0049] Specifically, the nucleotide sequence of the light-chain variable region (VL) of the single-chain antibody (ScFv) of Helicobacter pylori is shown in SEA ID NO.5; the nucleotide sequence of the heavy-chain variable region (VH) of the single-chain antibody (ScFv) of Helicobacter pylori is shown in SEA ID NO.6; the nucleotide sequence of the linker peptide (Linker) is shown in SEA ID NO.7; the nucleotide sequence of the single-chain antibody (ScFv) of Helicobacter pylori is shown in SEA ID NO.8.

[0050] For the above-mentioned single-chain antibody (ScFv) of Helicobacter pylori obtained by design, specific construction, expression purification and immunological analysis will be carried out next. The specific experimental procedures are as follows: Among them, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed. The vector can transform, transduce or transfect host cells so that the genetic material elements carried by it can be expressed in the host cells. The vector can contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes, etc. In addition, the vector can also contain an origin of replication. The vector may also include components to assist its entry into cells, such as virus particles, liposomes or protein coats, but not only these substances. The vector can be selected from, but not limited to: plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosome YAC, bacterial artificial chromosome BAC or P1-derived artificial chromosome PAC), phages (such as λ phage or M13 phage), and animal viruses used as vectors, for example, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). Regarding the "host cell", that is, the host cell containing the above vector, it can be selected from, but not limited to: prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cell models such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells.

[0051] The single-chain antibody gene of Helicobacter pylori initially obtained was predicted for signal peptides using Signal P 4.0 and 5.1, and the signal peptide sequence was removed; the target gene was codon-optimized; the full gene sequence of the determined single-chain antibody (ScFv) of Helicobacter pylori was sent to a gene company for synthesis.

[0052] Extract the pCzn1 vector plasmid. The pCzn1 vector plasmid is as Figure 5 shown, and double-digest the pCZN1 vector plasmid and the single-chain antibody (ScFv) gene of Helicobacter pylori with Nde I-Xba I; at 37 °C for 2 h; use a DNA recovery kit to recover the double-digested products of the ScFv gene and the pCzn1 vector. The recovered linearized pCZN1 vector and the ScFv gene fragment are ligated into a closed circular DNA molecule through complementary sticky ends under the action of T4 DNA ligase, that is, the recombinant plasmid pCZN1-ScFv. The structure of the recombinant plasmid pCZN1-ScFv is as Figure 6 shown.

[0053] The completed recombinant plasmid pCZN1-ScFv was digested with double enzymes and detected by electrophoresis. The results are as follows Figure 7 shown. Lane 1 in the figure is the recombinant plasmid pCZN1-ScFv, lane 2 is the result of double enzyme digestion, and lane M is the maker. A large fragment of 4340 bp containing the ScFv gene and a small fragment of 816 bp without the ScFv gene were formed. The double enzyme digestion result was consistent with the actual situation. The sample was sent for sequencing. The results are as follows Figure 8 shown, that is, the nucleotide sequence of the single-chain antibody (ScFv) of Helicobacter pylori is as shown in SEA ID NO.8. The sequencing result was consistent with the prediction, and there was no frameshift mutation.

[0054] Construction of the expression vector DE3-pCZN1-SV. 1 μL of the recombinant plasmid pCZN1-ScFv was added to 100 μL of competent bacteria and placed on ice for 20 min. Heat shock at 42 °C for 90 s, quickly place on ice for 5 min, and add 600 μL of LB culture medium. Shake at 37 °C and 220 r / min for 1 h, centrifuge and coat on an LB plate containing 50 μg / mL Amp, and incubate upside down at 37 °C overnight.

[0055] Then, cell culture and protein induction and secretion experiments were carried out. A single clone on the LB plate was picked and inoculated into a test tube containing 50 μg / mL Amp LB culture medium, and shaken at 37 °C and 220 r / min overnight. The next day, it was inoculated at a ratio of 1:100 into 30 mL of LB culture medium containing 50 μg / mL Amp, and shaken at 37 °C and 220 r / min until the OD600 of the bacteria was 0.6 - 0.8. 1 mL of the culture was taken out, centrifuged at 10000 r / min at room temperature for 2 min, the supernatant was discarded, and the bacterial cell pellet was resuspended with 100 μL of 1× loading buffer. IPTG (final concentration of 0.5 mM) was added to the remaining culture, and shaken at 37 °C and 220 r / min for 4 h. 1 mL of the culture was taken out, centrifuged at 10000 r / min at room temperature for 2 min, the supernatant was discarded, and the bacterial cell pellet was resuspended with 100 μL of 1× loading buffer. The remaining culture was centrifuged at 4000 r / min for 10 min, the supernatant was discarded, and the bacterial cell pellet was resuspended with PBS; after the resuspended solution was ultrasonically disrupted, the supernatant and the pellet were respectively taken and resuspended with loading buffer, and SDS-PAGE detection was carried out. The results are as follows Figure 9 shown. Lane M is the Maker; lane 1 is the group containing the empty plasmid; lane 2 is the uninduced group; lane 3 is the induced expression group; lane 4 is the supernatant after disruption of the induced group. The band size was consistent with the expectation, indicating that the recombinant Escherichia coli DE3-pCzn1-ScFv successfully expressed the target protein.

[0056] Subsequently, ScFv protein was isolated and purified. Using a low-pressure chromatography system, the supernatant solution was loaded onto a Ni-IDA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min. It was rinsed with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min until the OD280 value of the effluent reached the baseline. It was rinsed with Ni-IDA Washing-Buffer at a flow rate of 1 mL / min until the OD280 value of the effluent reached the baseline. The target protein was eluted with Ni-IDA Elution-Buffer at a flow rate of 1 mL / min, and the effluent was collected. The collected protein solution was added to a dialysis bag and dialyzed overnight using PBS. The protein concentrate was collected, diluted with an appropriate amount of loading buffer, and analyzed by SDS-PAGE. As Figure 10 shown, lane 1 in the figure is the positive control, lane 2 is the purified SV-1 protein, and lane M is the maker. The collected supernatant was subjected to protein electrophoresis. As Figure 11 shown, the size of the protein is 29KD. It can be seen that the purified protein ScFv was successfully obtained.

[0057] Subsequently, it was detected whether the single-chain antibody (ScFv) of Helicobacter pylori could bind to the target antigen in vitro, and its in vitro binding ability was analyzed by Western Blot and indirect ELISA methods.

[0058] Specifically for Western Blot, after subjecting the VacA antigen to SDS-PAGE electrophoresis, it was transferred to a nitrocellulose membrane (NC) at 100V for 1h, and incubated in a 5% skim milk blocking solution for 1.5 hours; it was reacted with 1:1000 purified SV-1 protein for 1h, and washed three times with PBST, 5min each time; then it was reacted with an HRP-labeled his-tag antibody at 1:80000 at room temperature for 1h. After washing three times with PBST, a color developer was used for color development. As Figure 12 shown, Western Blot showed that the ScFv protein could specifically bind to the VacA antigen.

[0059] Specifically for indirect ELISA, the VacA antigen was added to a 96-well plate and coated overnight at 4°C; washed five times with PBST, 5min each time; 5% skim milk blocking solution was added and incubated for 1.5 hours; after washing five times with PBST, SV-1 was added and reacted for 1h; then it was reacted with an HRP-labeled his-tag antibody at 1:80000 at room temperature for 1h. After washing three times with PBST, TMB color developer was used for color development for 5 - 10min, and an Elisa stop solution was added to terminate the reaction. The OD value at 450nm was read using an enzyme-linked immunosorbent assay reader. As Figure 13As shown, indirect ELISA showed that the ScFv protein (G12) could specifically bind to VacA.

[0060] After the single-chain antibody of Helicobacter pylori (ScFv) was determined to be correctly expressed and have antigen-binding ability in the above experiments, the therapeutic effect on Helicobacter pylori was then verified at the cell culture level and in a mouse model. The specific experimental procedures are as follows: Verification of the therapeutic effect on Helicobacter pylori at the cell culture level. First, the human gastric normal epithelial cell line GES-1 was cultured. The human gastric normal epithelial cell line GES-1 stored in liquid nitrogen was resuscitated. The GES-1 cells stored in the liquid nitrogen tank were taken out and thawed quickly in a 37 °C water bath. After centrifuging the cells in a laminar flow hood, the supernatant was discarded and an appropriate amount of pre-prepared complete medium (DMEM: fetal bovine serum (FBS): penicillin-streptomycin = 89:10:1) was added and mixed well. The mixed cells were transferred to a 25T culture flask and supplemented with an appropriate amount of DMEM complete medium (final volume about 5 - 6 mL). After pipetting and mixing well and labeling, they were placed in a cell culture incubator for culture.

[0061] Then, the medium replacement and subculture of human gastric normal epithelial cell line GES-1 were carried out.

[0062] At the same time, Helicobacter pylori ( H p) was cultured to the logarithmic growth phase and the bacterial liquid was collected.

[0063] To study the inhibitory effect of the single-chain antibody of Helicobacter pylori (ScFv) on H p-infected GES-1 cells, H p and GES-1 were co-cultured by the co-culture technique. Specifically, the GES-1 cells were cultured until their cell confluence reached 80%. The cells were counted using a hemocytometer and 3×106 cells / well were added to a six-well plate. After the cells adhered to the wall, fresh H p bacterial liquid was added at a ratio of MOI = 100:1, and then PBS / ScFv was added respectively. They were placed in an incubator at 37 °C and 5% CO2 to allow H p to co-culture with the cells for 12 - 24 h.

[0064] The H p and GES-1 were co-cultured with uninfected normal cells as a control. Samples were collected after 12 h and 24 h, and the mRNA expression levels of H p-16sRNA and the inflammatory factor COX-2 in different groups were identified. The results were as Figure 14 described: After treatment with the single-chain antibody of Helicobacter pylori (ScFv) for 12 h and 24 h HThe expressions of p-16sRNA and COX-2 were significantly decreased, indicating that the single-chain antibody of Helicobacter pylori (ScFv) could inhibit H the infection of p to GES-1 and protect GES-1 cells from H the infection of p. It shows that while ScFv retains the binding ability to the antigen, it does not lose the neutralizing ability to the antigen.

[0065] Then, the effect of treating Helicobacter pylori was verified in a mouse model.

[0066] Take out the freshly cultured H p, and use an ultraviolet spectrophotometer to adjust the bacterial liquid concentration to 1x108 CFU / mL. On the 1st and 8th days, intragastrically administer H 3 mL of p to the mice. On the 15th day, randomly select one mouse for sacrifice and take the stomach. Through quantitative culture ( Figure 15 -A) and H the p identification test paper to identify H the p infection situation ( Figure 15 -B). The results show that H the mouse model infected with p was successfully constructed.

[0067] Group the mice infected with H p according to their body weights: ScFv treatment group; PBS control group. According to the grouping, intragastrically administer 1 mL of ScFv and PBS (the antibody concentration was pre-diluted to 50 μg / mL) to the mice on the 15th, 22nd, 29th, and 36th days respectively. Sacrifice the mice on the 43rd day and take out the gastric tissues. Weigh the first portion of gastric tissue after removing the contents, transfer it to a sterile test tube, add an appropriate amount of physiological saline, and homogenize it with an electric homogenizer. Collect the homogenate for H the detection of p; Transfer the second portion of gastric tissue after removing the contents to a sterile test tube and fix it with 4% paraformaldehyde for histopathological detection of the mouse stomach.

[0068] To prove whether the single-chain antibody of Helicobacter pylori (ScFv) can treat H the C57 mice infected with p, use the Trizol method to extract the total RNA in the gastric homogenate of each group of mice, and detect the expressions of H p-16sRNA and the inflammatory factor COX-2 in the stomachs of each group of mice by RT-qPCR. The results are shown in Figure 16: The expression of H p-16sRNA in the antibody treatment group was significantly lower than that in the H p infection group, showing the same trend as the expression of the inflammatory factor COX-2, and there was no significant difference from the negative control group not infected with H p. It shows that the single-chain antibody of Helicobacter pylori (ScFv) has an inhibitory effect on the HIt has a therapeutic effect on C57 mice with p.

[0069] To further verify the inflammatory levels in the stomachs of mice in each group, total RNA and total protein were extracted from the mouse stomach homogenates, and the expression of the inflammatory factor COX-2 in the mouse stomach homogenates was detected using the ELISA method. The results were as Figure 17 shown: After treatment with the ScFv antibody, the levels of inflammatory factors in the mouse stomach were significantly reduced, indicating that the single-chain antibody against Helicobacter pylori (ScFv) reduced the expression of COX-2 protein in the mouse stomach by inhibiting H the infection of p, thus alleviating the gastritis condition in mice.

[0070] For histopathological examination of the mouse stomach tissues, the mouse stomach tissues were cut in half along the greater curvature, washed 2-3 times with PBS, fixed with 4% paraformaldehyde, and then subjected to HE staining. The results were as Figure 18 shown: In the treatment group with the single-chain antibody against Helicobacter pylori (ScFv), compared with the H p infection group, the infiltration of inflammatory cells was significantly reduced, the gastric mucosal epithelium was more complete, and the damage and exfoliation were less severe, indicating that the single-chain antibody against Helicobacter pylori (ScFv) could reduce H the infection of p, thereby reducing the inflammatory response of the mouse gastric mucosa.

[0071] To further observe the H infection situation of p in the mouse stomach, immunohistochemistry was continued on the paraffin blocks of the gastric tissues prepared above. The results were as Figure 19 shown: In the treatment group with the single-chain antibody against Helicobacter pylori (ScFv), the infection of H p was hardly visible, which further indicated that the single-chain antibody against Helicobacter pylori (ScFv) could treat H p infection in the mouse model, reducing H the adhesion of p in the mouse stomach.

[0072] In summary, the single-chain antibody against Helicobacter pylori (ScFv) can significantly reduce H the adhesion of p in the stomach, reduce the infiltration of inflammatory cells and the damage of gastric tissues. Combining with the results in the cell model, the single-chain antibody against Helicobacter pylori (ScFv) can rapidly and significantly reduce H the infectivity of p, and it will not cause other harmful effects on gastric tissues and cells itself. The research on the single-chain antibody against Helicobacter pylori (ScFv) shows that the single-chain antibody against Helicobacter pylori (ScFv) can have a therapeutic effect on H p infection.

[0073] The application of the above-mentioned single-chain antibody against Helicobacter pylori in the preparation of drugs for treating Helicobacter pylori.

[0074] A pharmaceutical composition comprising the above-mentioned single-chain antibody against Helicobacter pylori.

[0075] A detection product comprising the above-mentioned single-chain antibody against Helicobacter pylori, which is used to detect the presence or quantity of Helicobacter pylori in a sample. Specifically, it can be labeled by chemical methods or genetic engineering methods, and the labeled antibody or its antigen-binding fragment can be used to detect the presence or quantity of Helicobacter pylori in a sample. The detection product includes, but is not limited to, detection reagents, detection kits, detection chips or test strips. For the specific detection method, the following steps can be adopted: 1) Provide a sample; 2) Contact the sample with the above-mentioned single-chain antibody against Helicobacter pylori of the present invention and its uses or its antigen-binding fragment; 3) Detect the immune reaction between the sample and the antibody or its antigen-binding fragment. 4) Obtain the result.

[0076] The above-disclosed are only the preferred embodiments of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Those of ordinary skill in the art can understand all or part of the processes of implementing the above embodiments, and the equivalent changes made according to the claims of the present invention still fall within the scope covered by the invention.

Claims

1. A single-chain antibody against Helicobacter pylori, characterized in that: It specifically binds to the Helicobacter pylori vacuolating toxin protein (VacA) antigen and consists of a light chain variable region (VL), a linker peptide (Linker) and a heavy chain variable region (VH); The light chain variable region (VL) is derived in whole or in part from the light chain variable region of IgG1, IgG2, IgG3 or IgG4; The heavy chain variable region (VH) is derived in whole or in part from the heavy chain variable region of IgG1, IgG2, IgG3 or IgG4.

2. The Helicobacter pylori single-chain antibody according to claim 1, characterized in that: The Helicobacter pylori single-chain antibody (ScFv) comprises a light chain variable region (VL), a linker peptide (Linker) and a heavy chain variable region (VH) from the N-terminus to the C-terminus; Alternatively, the Helicobacter pylori single-chain antibody (ScFv) comprises a heavy chain variable region (VH), a linker peptide (Linker) and a light chain variable region (VL) from the N-terminus to the C-terminus.

3. The Helicobacter pylori single-chain antibody according to claim 1, characterized in that: The light chain variable region (VL) comprises the sequence shown in SEA ID NO.1; The heavy chain variable region (VH) comprises the sequence shown in SEA ID NO.

2.

4. The Helicobacter pylori single-chain antibody according to claim 3, characterized in that: The linker peptide sequence is shown in SEA ID NO.3; The sequence of the Helicobacter pylori single-chain antibody (ScFv) is shown in SEA ID NO.

4.

5. The Helicobacter pylori single-chain antibody according to claim 3, characterized in that: The nucleotide sequence of the light chain variable region (VL) of the Helicobacter pylori single-chain antibody (ScFv) is shown in SEA ID NO.5; The nucleotide sequence of the heavy chain variable region (VH) of the Helicobacter pylori single-chain antibody (ScFv) is shown in SEA ID NO.

6.

6. The Helicobacter pylori single-chain antibody according to claim 5, characterized in that: The nucleotide sequence of the linker peptide is shown in SEA ID NO.7; The nucleotide sequence of the Helicobacter pylori single-chain antibody (ScFv) is shown in SEA ID NO.

8.

7. A use of a Helicobacter pylori single-chain antibody, characterized in that: Use of any one of the Helicobacter pylori single-chain antibodies (ScFv) according to claims 1 to 6 in the preparation of a drug for treating Helicobacter pylori.

8. A pharmaceutical composition, characterized in that: The invention comprises the Helicobacter pylori single-chain antibody according to any one of claims 1 to 6.

9. A detection product, characterized in that: The single-chain antibody against Helicobacter pylori according to any one of claims 1 to 6 is used for detecting the presence or amount of Helicobacter pylori in a sample.

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