Helicobacter pylori virulence protein Cag A antigen and antibody and application thereof

Abstract

The invention provides a helicobacter pylori virulence protein Cag A antigen, a helicobacter pylori virulence protein Cag A antibody and application of the helicobacter pylori virulence protein Cag A antigen and the helicobacter pylori virulence protein Cag A antibody. According to the invention, sequence analysis and combined screening are carried out on the antigen epitopes related to Cag A to obtain the Cag A antigen of a recombinant expression specific sequence, and the stability and specificity of the Cag A antigen are ensured. The invention also constructs a group of monoclonal antibodies specifically combined with the antigen based on the high-sensitivity Cag A antigen. The helicobacter pylori virulence protein Cag A antigen has relatively strong binding capacity with a Cag A antibody in a clinical sample, has relatively high detection sensitivity, can be combined with a specific antibody to serve as a raw material combination of a detection reagent, and has clinical detection superiority.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Helicobacter pylori virulence protein Cag A antigen and antibody and applications thereof. Background Art

[0002] When carrying out Helicobacter pylori eradication work, it is necessary to first perform Helicobacter pylori typing to determine whether the infection is a virulent strain, in order to guide the clinic to issue the correct Helicobacter pylori eradication plan and reduce the generation of drug-resistant strains. The detection antigens and antibodies designed for the Helicobacter pylori virulence protein Cag A as raw materials will promote the formation of new Helicobacter pylori typing detection kit products. Existing Helicobacter pylori detection patented technologies mostly use the full-length Cag A antigen as the coating antigen, which has a relatively complex recognition site and poor specific binding ability with the test sample, which can easily lead to missed detection. In addition, the accuracy of the relevant detection antibodies is limited, and the detection rate of the samples cannot be guaranteed.

[0003] Helicobacter pylori lives in the human stomach and reproduces. Helicobacter pylori may cause gastric ulcers and even gastric cancer, which brings great health burden to patients. During the process of adhesion and colonization, Helicobacter pylori can induce multiple genetic changes, express more than ten virulence factors, and trigger multiple adaptation mechanisms. Its virulence and pathogenicity are mainly affected by virulence factors.

[0004] The cytotoxin-associated gene product (Cag A) is an important virulence factor of Helicobacter pylori and one of the most studied virulence genes of Helicobacter pylori. The full length of the Cag A gene ranges from 3444 to 5925 bp, with a molecular weight between 128 and 140 kD, and contains two regions: the 5' highly conserved region and the 3' highly variable region.

[0005] Cag A protein is the encoded product of Cag A gene on cag pathogenicity island (cagPAI) of H.pylori. After infection, H.pylori injects Cag A into host cells through type IV secretion system encoded by cagPAI and phosphorylates it, causing a series of reactions such as intracellular signal transduction, leading to severe tissue inflammatory damage in the host, and is closely related to the formation of gastric adenocarcinoma. In addition to the interaction with host cells, the functional interaction between Cag A and other toxic proteins of Helicobacter pylori, such as vacuolating cytotoxin A (VacA), may further regulate the tumorigenic effect of Cag A.

[0006] 60%-70% of H.pylori strains have Cag A gene. According to the presence or absence of Cag A gene, H.pylori can be divided into two categories, namely type I and type II: Type I has the gene encoding Cag A, expresses Cag A protein, and has virulence. Type II does not have the gene encoding Cag A, does not express Cag A, and has no Vac A activity. At present, the breath test method is commonly used in clinical practice to detect H.pylori infection, and typing detection is often not performed. Once the infection is confirmed, drug treatment is performed immediately, which not only wastes medical resources and increases the economic burden, but also may cause bacterial resistance, aggravate the bacterial resistance crisis, and cause potential harm to human health. Therefore, typing detection of H.pylori infection is particularly necessary. Type I strains are highly toxic and pose a greater threat to patients. They are closely related to peptic ulcers, gastric cancer, etc. Infection with this type of strain requires eradication treatment.

[0007] The detection of Cag A is an important indicator for typing detection. After being infected with type I strains, the human body will produce corresponding antibodies, and Cag A antigen can be detected in stool samples to further confirm the test results. However, the recombinant CagA protein antigen in the existing commercial kits is a full-length antigen. During the recognition process with the detection antibody, poor specific binding may occur, resulting in missed detection. Therefore, the development of a highly sensitive Cag A recombinant antigen protein and related monoclonal antibodies has important application value in the typing detection of Cag A. Summary of the invention

[0008] In view of the shortcomings of the prior art, the object of the present invention is to provide a Helicobacter pylori virulence protein CagA antigen and antibody and their application. The present invention performs sequence analysis and combination screening on the antigenic epitopes related to Cag A to obtain a recombinantly expressed Cag A antigen, ensures its stability and specificity during the recombinant expression process, and prepares a group of detection antibodies against the antigen based on the above-mentioned highly sensitive Cag A antigen, and the detection antibodies have good consistency compared with commercially available kits. It can be used as a new raw material for in vitro diagnostic reagents such as colloidal gold, immunochromatography, and chemiluminescence.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a Helicobacter pylori virulence protein Cag A antigen, wherein the Cag A antigen comprises an amino acid sequence as shown in SEQ ID NO.1.

[0011] The present invention performs sequence analysis on Cag A antigen, combines and screens out its clear antigen recognition sites and performs recombinant expression after sequence optimization, thereby improving the expression amount and purity of the antigen while also improving the detection signal sensitivity and reducing the possibility of missed detection.

[0012] In a second aspect, the present invention provides a nucleic acid molecule encoding the Helicobacter pylori virulence protein Cag A antigen described in the first aspect.

[0013] Preferably, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.2.

[0014] In a third aspect, the present invention provides a recombinant expression vector, wherein the recombinant expression vector contains the nucleic acid molecule described in the second aspect.

[0015] Preferably, after transfection / transduction / transformation of host cells, the recombinant expression vector enables the host cells to express the Helicobacter pylori virulence protein Cag A antigen described in the first aspect.

[0016] In a fourth aspect, the present invention provides a recombinant expression strain, wherein the recombinant expression strain contains at least one copy of the recombinant expression vector described in the third aspect, or at least one copy of the nucleic acid molecule described in the second aspect.

[0017] The present invention also provides the use of the Helicobacter pylori virulence protein Cag A antigen described in the first aspect in Helicobacter pylori typing detection.

[0018] In a fifth aspect, the present invention provides an anti-Helicobacter pylori virulence protein Cag A monoclonal antibody, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0019] The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.3, the amino acid sequence of CDR2 is shown in SEQ ID NO.4, and the amino acid sequence of CDR3 is shown in SEQ ID NO.5; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.6, the amino acid sequence of CDR2 is RAN, and the amino acid sequence of CDR3 is shown in SEQ ID NO.7;

[0020] Or, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown as SEQ ID NO.12, the amino acid sequence of CDR2 is shown as SEQ ID NO.13, and the amino acid sequence of CDR3 is shown as SEQ ID NO.14; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown as SEQ ID NO.15, the amino acid sequence of CDR2 is DTS, and the amino acid sequence of CDR3 is shown as SEQ ID NO.16.

[0021] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.8, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.9.

[0022] Alternatively, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.17, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.18.

[0023] Preferably, the heavy chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.10, and the light chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.11.

[0024] Alternatively, the heavy chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.19, and the light chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.20.

[0025] The present invention uses the above-mentioned Helicobacter pylori virulence protein Cag A antigen to prepare a group of specific monoclonal antibodies and completes preliminary sample screening tests. Compared with commercial antibodies, the detection sensitivity is improved, the number of missed samples is reduced, and it has high quality and clinical detection advantages.

[0026] In a sixth aspect, the present invention provides a nucleic acid molecule encoding the anti-Helicobacter pylori virulence protein Cag A monoclonal antibody described in the fifth aspect.

[0027] In a seventh aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the sixth aspect.

[0028] In an eighth aspect, the present invention provides a host cell, wherein the host cell contains at least one copy of the expression vector described in the seventh aspect, or the nucleic acid molecule described in the sixth aspect is integrated into the genome of the host cell.

[0029] The present invention also provides a kit for detecting Helicobacter pylori typing, wherein the kit contains the anti-Helicobacter pylori virulence protein Cag A monoclonal antibody described in the fifth aspect.

[0030] In a ninth aspect, the present invention provides a reference substance for detecting Helicobacter pylori typing, wherein the reference substance contains the anti-Helicobacter pylori virulence protein Cag A monoclonal antibody described in the fifth aspect.

[0031] In the tenth aspect, the present invention provides the use of any one or a combination of at least two of the Helicobacter pylori virulence protein Cag A antigen described in the first aspect, the anti-Helicobacter pylori virulence protein Cag A monoclonal antibody described in the fifth aspect, the nucleic acid molecule described in the sixth aspect, the expression vector described in the seventh aspect, the host cell described in the eighth aspect, or the reference material for Helicobacter pylori typing detection described in the ninth aspect in the preparation of a product for Helicobacter pylori typing detection.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The preparation method of the recombinant Cag A protein provided by the present invention can prepare a large amount of soluble recombinant Cag A protein. The protein concentration is 2.2 mg / mL and the purity is 97.4%. The prepared antigen is used to immunize mice, and monoclonal antibodies are screened and prepared, which can specifically recognize the Cag A antigen without cross-reacting with other antigens, indicating that the Cag A antigen used for immunization has good immunogenicity and the monoclonal antibodies prepared by immunizing mice with the protein have good specificity, and can be used as raw materials for detection kits.

[0034] (2) The recombinant Cag A protein provided in the present invention has a high accuracy in the method of detecting Cag A antibody positive serotyping of Helicobacter pylori patients using enzyme-linked immunosorbent assay. The recombinant Cag A protein was used to detect positive serum and negative serum samples containing Cag A antibodies, and the results were compared with the German MIKROGEN reference kit. Among the 210 sera, 96 were negative in both detection methods. Among the remaining 114 cases, 13 were Cag A positive in the reference kit test results, while the indirect ELISA test results were negative. Ten were Cag A negative in the reference kit test results, while the indirect ELISA test results were positive. The remaining 91 were positive in both detection methods. The performance of the indirect ELISA test method was evaluated based on the results. Compared with the test results of the reference kit, the positive coincidence rate, negative coincidence rate and total coincidence rate were 87.5%, 90.5% and 89%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic diagram of the preparation process of the Cag A recombinant protein antigen and related monoclonal antibodies.

[0036] Figure 2 It is the electrophoresis diagram of the target Cag A expression protein nucleic acid (about 2.5 kbp) and the plasmid nucleic acid (about 5 kbp) after double enzyme digestion.

[0037] Figure 3 This is the SDS-PAGE picture of Cag A protein.

[0038] Figure 4 This is a Western blot identification image of Cag A protein.

[0039] Figure 5 This is the SDS-PAGE electrophoresis diagram of Cag A monoclonal antibody. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0041] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0042] The schematic diagram of the preparation process of the Cag A recombinant protein antigen and related monoclonal antibodies in the present invention is as follows Figure 1 shown.

[0043] Example 1

[0044] 1. PCR amplification and sequencing of Cag A gene

[0045] Purpose: To amplify the target sequence and complete sequencing comparison to prove the consistency between the actual sequence and the designed one.

[0046] The optimized and screened Cag A nucleic acid expression sequence (SEQ ID NO.2) was used as a template for PCR amplification. PCR was performed in a 50 μL reaction system, and the reaction system was as follows:

[0047] Join dd H 2 O 39.1μL, primers (10μmol / L) 2μL, expression sequence DNA 1μL, Platinum PFX DNA Polymerase 0.4μL, 10×PFX Amplification buffer 5μL, dNTP Mix (10mM) 1.5μL, MgSO 4 (50mM)1.0μL.

[0048] The PCR reaction conditions were as follows: 94°C for 5 min; 94°C for 15 s, 55°C for 30 s, 68°C for 1 min, 35 cycles; 72°C for 5 min; and storage at 4°C.

[0049] Detect the PCR results by 1% agarose gel electrophoresis, cut the gel and weigh it, recover the gel product according to the instructions of the DNA gel recovery kit, take 5-10 μL of the gel recovery product and detect the recovery efficiency by agarose gel electrophoresis, take 2 μL to determine the gene concentration, and store the rest at -20°C for plasmid construction operation.

[0050] 2. Construction and sequencing of recombinant plasmid

[0051] Purpose: To prepare a recombinant plasmid that can express a recombinant protein, which can then be expressed in host cells.

[0052] The Cag A gene fragment SEQ ID NO.2 amplified by PCR was connected to the linear pTrcHis2C vector after double restriction digestion to construct a recombinant plasmid. The recombinant plasmid was double-digested with Xho I and Kpn I, and the digestion products were identified by electrophoresis using 1% agarose gel. At the same time, sequencing primers were designed according to the gene length, and BigDye Terminator v3.1Cycle Sequencing Kit was used to sequence using ABI 3500XL genetic analyzer, and the sequencing results were spliced ​​and compared.

[0053] The electrophoresis results are shown in Figure 2 , Figure 2 It is the electrophoresis diagram of the target Cag A expression protein nucleic acid (about 2.5kbp) and the plasmid nucleic acid (about 5kbp) after double enzyme digestion.

[0054] 3. Expression and purification of Cag A protein

[0055] Objective: To prepare the target recombinant protein and obtain high-purity protein through purification.

[0056] The constructed CagA-pTrcHis2C recombinant plasmid was transformed into competent cells Rosetta (DE3), and positive strains were screened using Amp resistance. The colonies containing the recombinant expression plasmid were plated in SOB medium (containing Amp 50 μg / mL, MgCl 2 10mmol / L) at 37°C and 220rpm. 600 When the pH value was 0.4-0.8, IPTG was added at a final concentration of 1 mmol / L. After induction at 37°C for 12 h, the bacteria were centrifuged and the supernatant was taken for SDS-PAGE analysis.

[0057] After Cag A protein induction, SDS-PAGE analysis showed that most of the proteins were soluble. We purified the soluble proteins using protein purification liquid chromatography. Cag A cells were collected and purified using 1× Ni column buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0), and then centrifuged to obtain the supernatant for later use. A Ni-separation column was used for subsequent purification.

[0058] The results of SDS-PAGE analysis are shown in Figure 3 , Figure 3 The SDS-PAGE image of Cag A protein is shown in Figure 1. Lane 1 is the target protein band, which is about 80 kDa and has a purity of 97%. The amino acid sequence of Cag A protein is shown in SEQ ID NO.1.

[0059] 4. Identification of Cag A protein

[0060] Purpose: To determine the consistency of the prepared recombinant protein with the target designed protein.

[0061] The purified Cag A protein was transferred to a PVDF membrane activated in methanol after SDS-PAGE gel electrophoresis and blocked with PBS containing 5% skim milk powder at room temperature for 2 h. The Cag A monoclonal antibody used for commercial research and development (Cag A antibody (A-10), sc-28368, SCBT-Santa Cruz Biotechnology USA), Cag A + Serum and Cag A - The serum was incubated at room temperature for 1 h, then rinsed with TBS-T (20 mM Tris buffered saline containing 0.1% Tween 20), and 10 mL of goat anti-mouse IgG-HRP conjugate and rabbit anti-human IgG-HRP conjugate secondary antibodies were added and incubated at room temperature for 1 h. After washing, HRP substrate was added, and protein bands were detected using a gel imager.

[0062] Western blot identification of Cag A protein Figure 4 As shown, Figure 4 This is a Western blot analysis of Cag A protein. Lane 1 is the commercial antigen result, and lane 2 is Cag A + (Positive) Serum results, lane 3 is Cag A - (negative) serum results. Western blot results showed that the Cag A protein of the present invention was negative for Cag A + Serum and commercial Cag A monoclonal antibodies can bind specifically, and a clear band at 80 kDa can be seen, which is similar to Cag A. - The serum did not react. The recombinant Cag A protein had good antigenicity.

[0063] 5. Use Cag A protein to detect serum antibodies

[0064] Purpose: To determine the biological activity of recombinant proteins, including the false negative and false positive rates in different sample populations.

[0065] 210 sera were collected, and the recomLine Helicobacter IgG, IgA 2.0 (MIKROGEN) serological test kit was used as a reference kit to perform a preliminary test on the Cag A antibody in the sera. The experimental operation was performed according to the instructions of the kit, and the results were confirmed according to the instructions after the experiment. The presence of Cag A antibodies in the 210 sera was statistically analyzed for later comparison of the results.

[0066] The Cag A protein prepared in the present invention is used to establish an indirect ELISA method to detect Cag A antibodies in patient serum.

[0067] First, the optimal coating concentration of Cag A and the optimal dilution multiple of serum in the indirect ELISA method were determined by the chessboard titration method. The purified Cag A protein was coated in a 96-well ELISA plate at a concentration of 1 μg / mL, 100 μL / well, incubated overnight at 4°C, washed three times with PBST, and then the ELISA blocking solution (0.05M carbonate buffer containing 2% BSA, pH 9.6) was added to each well at 250 μL / well, incubated at 37°C for 2 hours, washed three times with PBST, and the serum to be tested was added at a dilution of 1:100, 100 μL / well, incubated at 37°C for 1.5 hours, and washed three times with PBST. Add a 1:6000 diluted rabbit anti-human IgG-HRP conjugate antibody, incubate at 37°C for 40 minutes, and read using an ELISA reader after color development.

[0068] Among the 210 sera, 96 were negative by both detection methods. Among the remaining 114, 13 were positive for Cag A by the reference kit and negative by the indirect ELISA test, 10 were negative for Cag A by the reference kit and positive by the indirect ELISA test, and the remaining 91 were positive by both detection methods. The performance of the indirect ELISA test was evaluated based on the results, and compared with the results of the reference kit test, the positive coincidence rate, negative coincidence rate and total coincidence rate were 87.5%, 90.5% and 89%, respectively. As shown in Table 1, Table 1 is the comparison of the results of the Cag A recombinant protein antigen in the detection of Cag A antibody in serum by indirect ELISA and the reference kit.

[0069] Table 1

[0070]

[0071] Example 2

[0072] 1. Preparation of monoclonal antibodies

[0073] Objective: To prepare monoclonal antibodies for detection using the prepared recombinant antigens, obtain mouse ascites containing antibodies using the hybridoma antibody preparation method, and purify and identify them.

[0074] After the Cag A antigen was mixed and emulsified with Freund's complete adjuvant, 5 6-week-old BALB / c female mice were subcutaneously injected with 30 μg / mouse, and then the emulsified antigen mixed with Freund's incomplete adjuvant and Cag A was injected for the 2nd, 3rd, and 4th time on the 15th, 30th, and 45th days, respectively. The antibody production of the mice was detected by ELISA. When the antibody titer reached the requirement, the spleen of the mouse was taken out to obtain B cells, and then fused with SP20 cells to prepare hybridoma cells, and the fusion method was PEG fusion method. Hybridoma cells were screened out using HAT culture medium, and the screened cells were cloned, and hybridoma cells capable of producing anti-Cag A specific antibodies were screened out by ELISA. The positive hybridoma cells were injected into the peritoneal cavity of the mouse, ascites was obtained, and then purified using a Protein-A affinity chromatography kit. These monoclonal antibodies can be detected by ELISA and Western blot using the Cag A recombinant protein mentioned in the present invention.

[0075] SDS-PAGE electrophoresis of Cag A monoclonal antibody Figure 5 As shown, lane 1 is monoclonal antibody 1, and lane 2 is monoclonal antibody 2.

[0076] 2. Sequencing results of monoclonal antibodies

[0077] Antibody 1:

[0078] Heavy chain CDR1: SEQ ID NO.3: GYTFTNYW

[0079] Heavy chain CDR2: SEQ ID NO.4: IYPGNSDV

[0080] Heavy chain CDR3: SEQ ID NO.5: LYGLYYNSSNYGSLDS

[0081] Light chain CDR1: SEQ ID NO.6: QDIYSY

[0082] Light chain CDR2: RAN

[0083] Light chain CDR3: SEQ ID NO.7: GCLKYDEFHT

[0084] Heavy chain variable region of antibody 1: SEQ ID NO.8:

[0085] EVQLQQSGTVLARPGASVKMSCKASGYTFTNYWMHWIKQRPGQGLEWIGSIYPGNSDVTYNQMFKGKAKLTTVTSTNTAFMELSTLTNEDSAVYYCLYGLYYNSSNYGSLDSWGQGTTLTVSS

[0086] Light chain variable region of Antibody 1: SEQ ID NO.9:

[0087] DIKMTQSPSSMYASLGERVTITCKASQDIYSYLSWFQQKPGKTPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCGCLKYDEFHTFGGGTKLEIK

[0088] Heavy chain of Antibody 1: SEQ ID NO.10:

[0089] EVQLQQSGTVLARPGASVKMSCKASGYTFTNYWMHWIKQRPGQGLEWIGSIYPGNSDVTYNQMFKGKAKLTTVTSTNTAFMELSTLTNEDSAVYYCLYGLYYNSSNYGSLDSWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVIPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0090] Light chain of Antibody 1: SEQ ID NO.11:

[0091] DIKMTQSPSSMYASLGERVTITCKASQDIYSYLSWFQQKPGKTPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCGCLKYDEFHTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECG

[0092] Antibody 2:

[0093] Heavy chain CDR1: SEQ ID NO.12: GFSLSTSGMG

[0094] Heavy chain CDR2: SEQ ID NO. 13: IWWDDDK

[0095] Heavy chain CDR3: SEQ ID NO.14: LYNRGNIYSMLTDAMDY

[0096] Light chain CDR1: SEQ ID NO.15: SSVNY

[0097] Light chain CDR2: DTS

[0098] Light chain CDR3: SEQ ID NO.16: QQWSSNPFT

[0099] Heavy chain variable region of antibody 2: SEQ ID NO.17:

[0100] QVTLKESGPGLLQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNTALKSGLTISKDTSKNQVFLKLANVNTADTATYYCLYNRGNIYSMLTDAMDYWGQGTSVTVSS

[0101] Light chain variable region of antibody 2: SEQ ID NO.18:

[0102] QIVMTQSPAIMSASPGEKVTMTCSSSSSVNYMHWYQQKSGISPKRWIYDTSKLTSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPFTFGSGTKLEIK

[0103] Heavy chain of antibody 2: SEQ ID NO.19:

[0104] QVTLKESGPGLLQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNTALKSGLTISKDTSKNQVFLKLANVNTADTATYYCLYNRGNIYSMLTDAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0105] Light chain of Antibody 2: SEQ ID NO.20:

[0106] QIVMTQSPAIMSASPGEKVTMTCSSSSSVNYMHWYQQKSGISPKRWIYDTSKLTSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0107] 3. Potency verification of monoclonal antibody

[0108] Dilute 10×ELISA coating buffer to 1×, dilute the CagA recombinant protein antigen of the present invention to 0.5μg / mL with 1×ELISA coating buffer, add the diluted sample coating solution to the ELISA plate, 100μL per well; seal the ELISA plate with a sealing film, and coat overnight at 4°C; after washing the plate, add ELISA blocking solution to the ELISA plate, 200μL per well, and block at 37°C for 1h. Dilute 1μg / mL commercial Cag A monoclonal antibody, monoclonal antibody 1 and monoclonal antibody 2 of the present invention at 1:2000 with 1×ELISA enzyme diluent, and then dilute to 1:256000 at a 2-fold ratio, for a total of 8 concentrations, 100μL per well, and incubate at 37°C for 1h; after washing the plate, add TMB substrate to the ELISA plate, 100μL per well, and incubate at room temperature away from light for 15min;

[0109] Add ELISA stop solution to the ELISA plate, 100 μL per well, to terminate the reaction. Detect the absorbance at a single wavelength of 450 nm; and analyze the absorbance data to obtain the antibody titer.

[0110] The ELISA results showed that the binding titers of the two monoclonal antibodies provided by the present invention to the antigen described in the present invention were as follows: the ELISA titer of monoclonal antibody 1 was 1:2.56 million, and the ELISA titer of monoclonal antibody 2 was 1:2.56 million; both were better than the ELISA titer of commercial antibodies of 1:1.28 million, and the titer of monoclonal antibody 1 described in the present invention was numerically better than antibody 2.

[0111] It can be seen from the results that the binding potency of the two monoclonal antibodies mentioned in the present invention to the antigen described in the present invention is better than that of commercial antibodies.

[0112] Table 2 shows the comparison results of the ELISA titer of the Cag A recombinant protein antigen described in the Cag A monoclonal antibodies 1 and 2 and the commercial monoclonal antibody detection.

[0113] Table 2

[0114]

[0115] 4. Functional verification of monoclonal antibodies

[0116] Microbial samples taken from the stomach of 20 patients were lysed and tested by qPCR. Using the cDNA of the lysate as a template, primers for amplification of the conserved gene UreB and the virulence factor gene Cag A of Helicobacter pylori were designed. 1 μL of cDNA was extracted from the reverse transcription product as a template for quantitative Real-Time PCR. 9 μL of Fast SYBR mixed reaction solution (ThermoFisher Scientific) was added to the reaction well to make the total reaction volume 20 μL. The reaction mixture contained 5 μL Fast SYBR, 0.5 μL forward primer sequence (10 μM), 0.5 μL reverse primer sequence (10 μM), 3 μL nuclease-free H 2 Quantitative Real-Time PCR was performed in a 96-well (0.1 mL) plate for 40 cycles (Applied Biosystems Step One Plus Real-Time PCR System) using 2 -ΔΔCt Methods Fold changes were calculated. Pre-incubation at 95°C for 20 s, amplification at 95°C for 15 s, amplification at 60°C for 20 s (40 cycles); annealing at 60°C for 60 s. qPCR detection was performed under the above conditions and the results were analyzed.

[0117] The test results showed that 20 samples were positive for the UreB gene, indicating that all 20 samples were Helicobacter pylori (Hp). Among the 20 Hp-positive samples, 11 samples were positive for the CagA gene, indicating that these 11 samples were pathogenic subtypes of Helicobacter pylori and infected patients needed eradication treatment; the remaining 9 samples were non-pathogenic Helicobacter pylori.

[0118] The above-mentioned Helicobacter pylori was lysed and protein was extracted, and the monoclonal antibody described in the present invention was combined by direct ELISA. The ELISA results showed that all 11 Helicobacter pylori protein lysates with positive Cag AqPCR results were also positive for binding with the monoclonal antibody described in the present invention. All Helicobacter pylori protein lysates with negative Cag AqPCR results were also negative for binding with the two monoclonal antibodies described in the present invention.

[0119] As shown in Table 3, Table 3 is a comparison of the results of monoclonal antibody detection of Helicobacter pylori lysis products and gene amplification. The experimental results show that the two monoclonal antibodies of the present invention can accurately detect highly pathogenic Helicobacter pylori samples.

[0120] Table 3

[0121]

[0122]

[0123] In summary, the present invention constructs a group of highly sensitive Cag A recombinant antigen proteins and related monoclonal antibodies, discloses gene fragments expressing the protein, recombinant expression vectors, recombinant expression strains and preparation methods; and specific preparation methods of detection monoclonal antibodies prepared based on this antigen. After comparison with commercial raw materials by enzyme-linked immunosorbent assay, the recombinant protein has a strong binding ability with Cag A antibodies in clinical samples and has a high detection sensitivity. The recombinant protein and monoclonal antibody can be used as a combination of raw materials for detection reagents, which has advantages in clinical detection.

[0124] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A Helicobacter pylori virulence protein Cag A antigen, characterized in that: The Cag A antigen comprises the amino acid sequence shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Helicobacter pylori virulence protein Cag A antigen according to claim 1; Preferably, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to claim 2; Preferably, after transfecting / transducing / transforming a host cell, the recombinant expression vector enables the host cell to express the Helicobacter pylori virulence protein Cag A antigen according to claim 1.

4. A recombinant expression strain, characterized in that: The recombinant expression strain contains at least one copy of the recombinant expression vector according to claim 3, or at least one copy of the nucleic acid molecule according to claim 2.

5. A monoclonal antibody against Helicobacter pylori virulence protein Cag A, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.3, the amino acid sequence of CDR2 is shown in SEQ ID NO.4, and the amino acid sequence of CDR3 is shown in SEQ ID NO.5; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.6, the amino acid sequence of CDR2 is RAN, and the amino acid sequence of CDR3 is shown in SEQ ID NO.7; Or, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown as SEQ ID NO.12, the amino acid sequence of CDR2 is shown as SEQ ID NO.13, and the amino acid sequence of CDR3 is shown as SEQ ID NO.14; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown as SEQ ID NO.15, the amino acid sequence of CDR2 is DTS, and the amino acid sequence of CDR3 is shown as SEQ ID NO.

16.

6. The anti-Helicobacter pylori virulence protein Cag A monoclonal antibody according to claim 5, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.8, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.9; Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.17, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.18; Preferably, the heavy chain amino acid sequence of the monoclonal antibody is as shown in SEQ ID NO.10, and the light chain amino acid sequence of the monoclonal antibody is as shown in SEQ ID NO.11; Alternatively, the heavy chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.19, and the light chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.

20.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-Helicobacter pylori virulence protein Cag A monoclonal antibody according to claim 5 or 6.

8. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 7.

9. A host cell, characterized in that The host cell contains at least one copy of the expression vector according to claim 8, or the nucleic acid molecule according to claim 7 is integrated into the genome of the host cell.

10. A reference substance for detecting Helicobacter pylori typing, characterized in that: The reference product contains the anti-Helicobacter pylori virulence protein Cag A monoclonal antibody according to claim 5 or 6.

11. Use of any one or a combination of at least two of the Helicobacter pylori virulence protein Cag A antigen according to claim 1, the anti-Helicobacter pylori virulence protein Cag A monoclonal antibody according to claim 5 or 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, or the reference material for Helicobacter pylori typing detection according to claim 10 in the preparation of a product for Helicobacter pylori typing detection.

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