Anti-helicobacter pylori adhesin protein a nanobody and preparation method and application thereof

Abstract

The application discloses anti-Helicobacter pylori adhesin protein A nanobody and a preparation method and application thereof, six strains of nanobody against Helicobacter pylori adhesin protein A (HpaA) are screened, the amino acid sequences of the six strains of nanobody are shown in SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13, the nanobody can specifically target the HpaA protein, has the characteristics of small molecular weight, high water solubility, high resistance, high stability, high antigen binding, low immunogenicity and the like, and is identified and characterized, and the nanobody can be applied to an HpaA protein detection reagent and can be used as a candidate nanobody for detecting Helicobacter pylori.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine detection, and particularly relates to an anti-Helicobacter pylori adhesin protein A nanobody and a preparation method thereof, and further relates to application of the nanobody in preparation of a product for detecting Helicobacter pylori. BACKGROUND

[0002] Helicobacter pylori (H.pylori, Hp) is a microaerophilic gram-negative bacterium with a spiral structure that colonizes in the stomach. It was first discovered in 1982. Hp infection is closely related to a variety of diseases, including chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma, gastric cancer and other digestive system diseases, as well as diseases outside the digestive system such as blood system, nervous system, cardiovascular system, skin, ophthalmology, etc. At present, new therapeutic drugs that can overcome the drug resistance of Helicobacter pylori are urgently needed, and accurate diagnosis of Hp infection is necessary for further treatment. At present, the detection of Helicobacter pylori mainly includes carbon 13 or carbon 14 element detection, or rapid urease test and pathological biopsy of gastric mucosa taken by gastroscope. Therefore, the development of new reliable and convenient diagnostic reagents for Hp is expected to become an effective means to prevent and control Hp infection.

[0003] One of the key steps of Hp infection is the binding of bacterial adhesin protein A (HpaA) to receptors on gastric epithelial cells. It plays an important role in the Hp infection process, as it not only helps the bacteria to colonize the gastric mucosa, but also enhances the pathogenicity of the bacteria. Nanobodies (Nbs) are a unique single-domain antibody found in camelids and sharks, i.e., the variable domain of the heavy chain of heavy-chain antibody (VHH), which has strong antigen targeting and binding capacity, as well as outstanding dense tissue penetration, high specificity, high affinity, and high stability. Based on the numerous advantages of nanobodies, they are very suitable for the development of diagnostic kits, targeted drug delivery, and neutralizing antibody therapy research.

[0004] The present application aims to solve the problems of traditional antibodies. The first purpose is to screen 6 nanobodies focusing on the HpaA target, which can be used to develop a high-sensitivity and high-specificity detection method. The second purpose is to provide a preparation method of nanobodies for detecting Helicobacter pylori. The third purpose is to provide the use of Helicobacter pylori anti-HpaA nanobodies. SUMMARY

[0005] 1. In view of the above, one of the purposes of the present application is to provide an anti-Helicobacter pylori adhesin protein A nanobody; another purpose of the present application is to provide a fusion protein; a third purpose of the present application is to provide a nucleic acid sequence encoding the anti-Helicobacter pylori adhesin protein A nanobody; a fourth purpose of the present application is to provide a method for preparing the anti-Helicobacter pylori adhesin protein A nanobody; a fifth purpose of the present application is to provide a product for detecting Helicobacter pylori; and a sixth purpose of the present application is to provide use of the anti-Helicobacter pylori adhesin protein A nanobody or the fusion protein in the preparation of a product for detecting Helicobacter pylori.

[0006] To achieve the above purposes, the present application provides the following technical solutions:

[0007] 1. An anti-Helicobacter pylori adhesin protein A nanobody, characterized in that the amino acid sequence thereof is as shown in SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 or SEQ ID No. 13.

[0008] 2. A fusion protein, characterized in that the fusion protein comprises the amino acid sequence of the anti-Helicobacter pylori adhesin protein A nanobody and horseradish peroxidase.

[0009] 3. A nucleic acid sequence encoding the anti-Helicobacter pylori adhesin protein A nanobody, characterized in that the nucleotide sequence thereof is as shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12.

[0010] 4. A method for preparing the anti-Helicobacter pylori adhesin protein A nanobody, characterized in that it mainly comprises the following steps:

[0011] (1) optimizing and synthesizing a DNA sequence encoding HpaA, constructing a prokaryotic expression vector, extracting a recombinant HpaA antigen protein after expression in Escherichia coli, immunizing a camel with the recombinant HpaA antigen protein, collecting peripheral blood of the camel for isolation of lymphocytes, and then detecting the antigen-specific response level of the camel after immunization by ELISA;

[0012] (2) taking peripheral blood lymphocytes of camels, extracting total RNA and reverse transcribing to obtain cDNA, taking the cDNA as a template to perform PCR reaction to amplify the VHH region of the antibody, connecting the amplification product to a phage display vector, and performing electrotransformation into E. coli to establish a phage display library, screening the required positive clone through antigen incubation and ELISA identification, and then sequencing the positive clone to obtain the sequence of the HpaA nanobody;

[0013] (3) constructing the sequence of the HpaA nanobody into a prokaryotic or eukaryotic expression vector, performing large-scale expression and purification in E. coli, yeast or a mammalian cell line to obtain the nanobody against H. pylori adhesin protein A.

[0014] In some embodiments of the present application, in step (1), the DNA sequence encoding HpaA is shown as SEQ ID No. 1.

[0015] In some embodiments of the present application, in step (2), the phage display vector is pMECS.

[0016] In some embodiments of the present application, in step (3), the prokaryotic expression vector is pET-25b(+) and the E. coli is BL21(DE3).

[0017] In some embodiments of the present application, in step (3), the prokaryotic expression vector is pCMV-N1-HRP and the mammalian cell line is 293T cells.

[0018] 5. A product for detecting H. pylori, characterized by comprising the nanobody against H. pylori adhesin protein A or the fusion protein.

[0019] 6. Use of the nanobody against H. pylori adhesin protein A or the fusion protein in the preparation of a product for detecting H. pylori.

[0020] The beneficial effects of the present application are that: the subject group selects the immunogenic HpaA target protein from the Hp target protein in combination with literature research, downloads the protein sequence from the NCBI website, removes the signal peptide, analyzes the B cell dominant antigen, and optimizes the sequence through the above steps, constructs a pET-28a-HpaA prokaryotic expression vector, transforms it into an E. coli BL21 (DE3) strain, and purifies the recombinant HpaA protein by IPTG induction expression. The prepared HpaA recombinant protein is used to immunize camels, and a total of 5 immunizations are performed with an immunization interval of 2 weeks. The first immunization: complete Freund's adjuvant, 2-5 times using incomplete Freund's adjuvant. One week after the completion of the 5 immunizations, the camel peripheral blood serum is taken, and the anti-HpaA protein antibody titer is detected by ELISA. The results show that the specific antibody titer of HpaA protein in the camel serum after the last immunization is 1:1024000, and the immunization effect is good, which can be used for the next step of extracting mRNA, reverse transcribing into cDNA, and constructing a phage library. After two rounds of PCR, the VHH gene is successfully amplified, and the pMECS and VHH gene are double-digested and connected to the TG1 competent cells, and the nanolibrary is constructed. After counting the library, the library size is 8 x 10 7 PFU / ml, and the library positive rate is 95.8% by PCR identification of the bacterial liquid.

[0021] After sequencing 24 randomly selected strains by Shengong Company, the results show that the sequences of the 24 strains are different, reflecting the diversity of the library. During the nanobody screening process, it is found that the recovery rate of specific recombinant phage increases significantly with the increase of the number of screening rounds, and the proportion of specific phage increases significantly. The above data show that after three rounds of screening, the specific recombinant phage is significantly enriched. Randomly select 120 monoclonal antibodies, amplify and culture the anti-HpaA monoclonal antibodies for affinity detection, and the results show that the crude extracts of the 120 clones all have strong reaction with HpaA protein, and the selected OD is greater than 1.8, and P / N is greater than 5. There are 24 nanobodies with strong binding to HpaA protein, indicating that the screened nanobodies have specificity.

[0022] The recombinant plasmid pET-25b-HpaA-Nbs is constructed, transformed into BL21 (DE3) competent cells, and after expression induced by IPTG, the bacterial bodies are collected, ultrasonicated, and purified by Ni-NTA. SDS-PAGE identification shows that the sizes of the purified prokaryotic HpaA-Nb8, HpaA-Nb9, HpaA-Nb10, HpaA-Nb11, HpaA-Nb12 and HpaA-Nb15 are consistent with the expected size (about 20 kDa), and the Image J software scanning gray value shows that the purity is higher than 90%, and the His tag WB identification of the anti-HpaA nanobody after purification further verifies the correct expression of the nanobody. The affinity of the purified nanobody and HpaA is detected by ELISA, and it is found that the binding activity of the six nanobodies and HpaA is strong, and the EC 50 values are 0.002694 μM, 0.03004 μM, 0.003379 μM, 0.01380 μM, 0.009783 μM and 0.01027 μM, respectively.

[0023] The eukaryotic expression vector pCMV-N1-HpaA-Nbs-HRP of the nanobody is constructed, the constructed plasmid is transfected into 293T cells, and after 72 h, the cell supernatant is collected, and the expression of the recombinant Nbs-HRP is detected by WB and ELISA, and the results show that the nanobody with an HRP tag is successfully expressed, and the binding of the recombinant Nbs-HRP and HpaA is detected by ELISA, and it is found that the nanobody has high affinity and can be used as an effective detection probe candidate of Helicobacter pylori.

[0024] Based on the phage display technology, six nanobodies against Helicobacter pylori adhesin protein A (HpaA) are screened, the nanobodies can specifically target the HpaA protein, have the characteristics of small molecular weight, high water solubility, high resistance, high stability, high antigen binding, low immunogenicity and the like, are identified and characterized, and the binding sites of the recombinant HpaA protein and the nanobody are analyzed, the nanobody is related to the nanobody and a preparation method and application thereof, the nanobody can be applied to an HpaA protein detection reagent, and the high sensitivity and high specificity make the nanobody outstanding in the aspect of diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to make the purposes, technical solutions and beneficial effects of the present application clearer, the present application provides the following drawings for illustration:

[0026] Figure 1Construction and transformation of pET-28a-HpaA prokaryotic expression vector; a: schematic diagram of construction of pET-28a-HpaA prokaryotic expression vector; b: nucleic acid electrophoresis map of construction of pET-28a-HpaA prokaryotic expression vector; c: transformation of recombinant plasmid into E. coli BL21 (DE3) strain, d: plasmid map of construction of pET-28a-HpaA prokaryotic expression vector; from the nucleic acid electrophoresis map, it can be seen that the pET-28a-HpaA prokaryotic expression vector is successfully constructed, and the band at 1000 bp is consistent with the expectation, and the colony on the plate shows that the transformation is successful.

[0027] Figure 2 Identification and characterization of HpaA recombinant protein; a: SDS-PAGE electrophoresis map of HpaA recombinant protein purification; b: WB identification of HpaA recombinant protein His tag. After ultrasonic disruption of the induced expression bacteria, affinity chromatography purification was carried out by using Ni column, the SDS-PAGE result showed that the size of the obtained target protein was about 30 kDa, which was consistent with the expected target protein molecular size, and the Image J software gray value scanning showed that the purity was more than 90%, the His tag was identified by WB, which further showed that the HpaA recombinant protein induction expression purification was completed, and could be used for immunizing camels.

[0028] Figure 3 Antibody titer determination of camel serum before and after immunization of bactrian camel; from Figure 3 It can be seen from that the specific antibody titer of HpaA protein in the camel serum taken one week after the last immunization was 1:1024000, and the immunization effect was good, which could be used for extracting RNA for subsequent experiments.

[0029] Figure 4 Construction of nanobody library; a: 700 bp fragments were obtained by first round PCR amplification, and 400 bp fragments were obtained by second round PCR amplification; c: 4000 bp fragments were obtained by double enzyme digestion of pMECS vector; d: PCR of transformed bacterial liquid. From Figure 4 It can be seen that through PCR technology, two rounds of amplification of VHH were completed, and pMECS was successfully connected after enzyme digestion, and the nucleic acid electrophoresis band of the bacterial liquid PCR after transformation was 700 bp, which was consistent with the expected target molecular band size, and the nanobody library was successfully constructed.

[0030] Figure 5 Phage library diversity identification; a: plate count of antibody library capacity; b: bacterial liquid PCR identification of library positive rate. Figure 5 It is shown that after library counting, the library size is 8×10 7PFU / mL, library positive rate PCR identification of bacterial solution The primers MP57 and GIII were used, and the positive was 700 bp. 24 bacteria were randomly selected, of which 23 were positive and 1 was negative, and the library positive rate was 95.8%.

[0031] Figure 6 The results of positive clone sequence analysis; 24 strains were selected for sequencing, and the results are shown in Figure 6 The sequences of the 24 strains are all different, reflecting the diversity of the library.

[0032] Figure 7 ELISA identification of anti-HpaA protein specific nanobodies; a: Elisa test of the binding of crude extracts 1-60 of anti-HpaA nanobodies; b: Elisa test of the binding of crude extracts 61-120 of anti-HpaA nanobodies. Figure 7 The results show that the crude extracts of 120 clones all have strong reaction with HpaA protein, and the OD of the selected strains is greater than 1.8, and the P / N is greater than 5. There are 24 nanobodies with strong binding to HpaA protein, indicating that the screened nanobodies have specificity.

[0033] Figure 8 HpaA protein specific nanobody amino acid sequence information; after screening, the amino acid sequence information of the final 6 nanobodies is obtained.

[0034] Figure 9 Nanobody identification and characterization; a: anti-HpaA nanobody purification SDS-PAGE electrophoresis map; b: anti-HpaA nanobody purification His tag WB identification map. Figure 9 The results show that the recombinant plasmid pET-25b-HpaA-Nbs is transformed into BL21(DE3) competent cells, and after IPTG induction expression, the bacterial cells are collected and ultrasonicated, and purified by Ni-NTA. SDS-PAGE identification shows that the purified prokaryotic HpaA-Nb8, HpaA-Nb9, HpaA-Nb10, HpaA-Nb11, HpaA-Nb12 and HpaA-Nb15 have the expected size (about 20 kDa) and high purity, and the His tag WB identification of the purified anti-HpaA nanobodies further verifies the correct expression of the nanobodies.

[0035] Figure 10For the determination of the binding activity of HpaA nanobody and HpaA antigen protein; a: HpaA-Nb8 and HpaA antigen protein binding activity; b: HpaA-Nb9 and HpaA antigen protein binding activity; c: HpaA-Nb10 and HpaA antigen protein binding activity; d: HpaA-Nb11 and HpaA antigen protein binding activity; e: HpaA-Nb12 and HpaA antigen protein binding activity; f: HpaA-Nb15 and HpaA antigen protein binding activity. Figure 10 The results show that: 6 strains of nanobodies bind strongly to HpaA recombinant antigen protein, and the EC 50 The results show that: 6 strains of nanobodies bind strongly to HpaA recombinant antigen protein, and the EC

[0036] Figure 11 For the construction of eukaryotic expression vector pCMV-N1-HpaA-Nbs-HRP; a: double enzyme digestion of pCMV-N1 electrophoresis map; b: bacterial liquid PCR identification of recombinant plasmid pCMV-N1-HpaA-Nbs-HRP. Figure 11 The results show that: the eukaryotic expression nanobody plasmid is successfully constructed, and can be used for transfection 293T cell experiment.

[0037] Figure 12 For the determination of the binding activity of HpaA nanobody and HpaA antigen protein; a: HpaA-Nb8 and HpaA antigen protein binding activity; b: HpaA-Nb9 and HpaA antigen protein binding activity; c: HpaA-Nb10 and HpaA antigen protein binding activity; d: HpaA-Nb11 and HpaA antigen protein binding activity; e: HpaA-Nb12 and HpaA antigen protein binding activity; f: HpaA-Nb15 and HpaA antigen protein binding activity. Figure 12 It can be seen that: the His tag of WB identified Nbs-HRP shows that the experimental group appears a band with a molecular size of about 75kDa and is consistent with the target band position of the control nanobody group, and the empty pCMV-N1 is not expressed, indicating that the Nbs-HRP is successfully expressed after transfection of the cells for 72h. ELISA detection of binding activity shows that: compared with the BSA control group, the purified Nbs-HRP has strong binding activity with HpaA recombinant protein. DETAILED DESCRIPTION

[0038] The present application will be further described below in conjunction with the drawings and specific examples, so that those skilled in the art can better understand the present application and implement it, but the examples are not limiting the present application.

[0039] The strains used in the present application and various reagents are as follows: plasmid pET-28a(+), pET-25b(+), E. coli strain BL21(DE3), E. coli strain DH5α, TG1 competent cells are preserved by the unit of the inventor, restriction endonuclease BamH I and Hind III are products of Dalian Takara Company; protein Marker is a product of Bio-Rad Company; plasmid extraction kit and gel recovery kit are products of Shanghai Sangon Company; column recovery kit is a product of Omega Company, and nickel ion affinity gravity chromatography column (Ni-NTA 5ml) is a product of Shanghai Sangon Company.

[0040] Material preparation:

[0041] 1. Ampicillin (Amp) stock solution (100 mg / mL): 1 g of ampicillin (Amp) was weighed and dissolved in 10 mL of sterile water to prepare a stock solution with a concentration of 100 mg / mL. The solution was filtered through a 0.22 μm bacterial filter to remove bacteria and stored in a -20°C refrigerator.

[0042] 2. Kanamycin (100 mg / mL): 1.0 g of kanamycin was weighed and dissolved in 10 mL of sterile water to prepare a stock solution with a concentration of 100 mg / mL. The solution was filtered through a 0.22 μm bacterial filter to remove bacteria and stored in a -20°C refrigerator.

[0043] 3. IPTG solution: 2.4 g of IPTG was weighed and dissolved in 10 mL of sterile water. The solution was filtered through a 0.22 μm filter to remove bacteria and stored in small portions at -20°C.

[0044] 4. Culture medium: (1) 2YT liquid medium: 16 g of tryptone, 10 g of yeast extract and 5 g of NaCl were weighed and added to 1 L of distilled water, and the pH was adjusted to 7.4. High-pressure steam sterilization. (2) 2YT-ATG medium: 1.5 g of agar powder per 100 mL of LB culture solution. After high-pressure sterilization, pour the plate. (3) 2YT-ATK medium: 1 mL of Amp and 1 mL of Kan antibiotics were added to 1 L of 2YT medium and 15 g of agar powder were added and shaken well. After high-pressure sterilization, pour the plate. (4) 2YT-T medium: 1.5 g of agar powder per 100 mL of 2YT culture solution was added to the 2YT medium, and after high-pressure sterilization, pour the plate.

[0045] 5. DNA electrophoresis buffer (50xTAE): 242 g of Tris, 37.2 g of Na2EDTA·2H2O and 57.1 mL of glacial acetic acid were weighed and added to 1 L of water. When used, dilute 50 times.

[0046] 6. SDS-PAGE electrophoresis buffer (5X): weigh Tris powder 15.1 g, glycine 94 g, SDS 5.0 g; add about 800 mL of deionized water, stir to dissolve; add deionized water to 1 L, store at room temperature; note: when adding water, the water should be allowed to flow slowly along the wall to avoid the generation of a lot of foam due to SDS.

[0047] 7. Coomassie brilliant blue protein staining reagent: (1) Coomassie brilliant blue G-250 staining solution (for protein quantification): Coomassie brilliant blue G-250 100 mg is dissolved in 50 mL of 95% ethanol, then 100 mL of 86% phosphoric acid is added, and diluted to 1000 mL with distilled water. (2) Decolorizing solution: 250 mL of ethanol, 80 mL of glacial acetic acid is diluted to 1000 mL with distilled water.

[0048] 8. ELISA reagent: (1) coating solution: 1.6 g of Na2CO3, 2.9 g of NaHCO3, 0.2 g of NaN3, add distilled water to 1 L, adjust the pH to 9.6. (2) washing solution: weigh 0.2 g of KH2PO4, 2.9 g of Na2HPO4·12H2O, 8.0 g of NaCl, 0.2 g of KCl, 0.5 mL of Tween-20, add ddH2O to 1 L (PBST). (3) blocking solution: weigh 0.2 g of skimmed milk powder and dissolve in 10 mL of washing buffer, filter sterilize and store at 4°C. (4) substrate solution: soluble single-component TMB substrate solution. (5) stop solution: measure 891.3 mL of distilled water, and add 108.7 mL of concentrated sulfuric acid (2M H2SO4) dropwise.

[0049] 9. Purification reagent: (1) 500 mM imidazole solution: weigh imidazole 34.04 g, add distilled water to 1 L. The rest of the concentration gradient imidazole solution can be obtained by dilution. (2) 20% ethanol: measure 200 mL of anhydrous ethanol, add deionized water to 1 L. (3) phosphate buffer (PBS): weigh 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, add distilled water to 1 L, adjust the pH to 7.4.

[0050] Example 1: Synthesis and subcloning of HpaA gene

[0051] 1. Synthesis of DNA sequence encoding HpaA and vector construction

[0052] The subject group combined with literature research from Hp target protein to select immunogenic HpaA target protein, from NCBI website download the protein sequence XG12708F, through the removal of signal peptide analysis B cell dominant antigen analysis and other steps, optimize and synthesis of DNA sequence (SEQ ID NO. 1) encoding HpaA, the sequence is connected to pET-28a(+) empty plasmid BamHI and HindIII multiple cloning site between the construction of recombinant plasmid pET-28a-HpaA (such as Figure 1 , a, d).

[0053] 2. Transformation of recombinant plasmid: from -80℃ refrigerator to 1 tube of E. coli BL21 competent cells (the subject group made), add 3 μL of synthesized pET-28a-HpaA plasmid. Ice bath 30 min, 42℃ metal bath heat shock 90 s, ice bath 5 min. Add 800 μL of LB blank medium, mix, placed in 37℃ shaking table 220 rpm shaking 1 h. Take 20 μL of bacterial liquid coated on kanamycin resistant LB plate. Plate inverted in 37℃ incubator for 14 h. Pick up the well separated edge clear colonies on the transformation plate, inoculated in kanamycin resistant LB medium, 37℃ shaking culture overnight.

[0054] 3. Nucleic acid electrophoresis map identification of pET-28a-HpaA prokaryotic expression vector

[0055] Gel 1.0% agarose gel, add 1 μL 6xLoading buffer in proportion in the ligation product, through gel 100 V electrophoresis for 30 min, gel imaging instrument to collect nucleic acid electrophoresis results. The results show that: empty plasmid pET-28a, HpaA target gene fragment molecular size about 400 bp, positive clone plasmid pET-28a-HpaA, molecular size about 1000 bp, consistent with the target molecular size, successfully constructed recombinant HpaA protein expression vector (such as Figure 1 , b). The recombinant plasmid is transformed into BL21(DE3) strain, and the uniform colonies can be seen on the kanamycin resistant plate (such as Figure 1 , c), indicating that the transformation is successful.

[0056] Example 2: Identification of HpaA antigen protein expression and purification

[0057] 1. HpaA recombinant protein induction expression and purification

[0058] Take 100 μL of pET-28a-HpaA / BL21(DE3) bacteria solution cultured overnight and add to 10 mL of kanamycin-resistant LB medium, and culture at 220 rpm and 37°C to activate the bacteria solution once. Take 3 mL of the bacteria solution after the first activation and add to 300 mL of LB medium containing kanamycin for secondary activation, and culture at 37°C for 4-5 h until the OD600 is 1.0. Then, add 300 μL of 0.5 mM IPTG, and place in a 37°C shaker for induction. After 14 h, centrifuge at 10,000 rpm for 15 min to collect the bacteria, resuspend the bacteria in 5 mL of PBS, and then perform ultrasonic lysis of the bacteria solution for 5 min (120 V). Collect the supernatant for subsequent purification, filter the supernatant with a 0.22 μm bacterial filter, and purify the recombinant HpaA antigen protein with a nickel ion affinity gravity chromatography column (Ni-NTA 5 mL). First, wash away the impurities with 10 mM, 30 mM, and 50 mM imidazole, and then elute the target protein with 250 mM imidazole. After elution, detect the concentration of the recombinant protein with a BCA kit, and then store in 1.5 mL EP tubes at -80°C.

[0059] 2. SDS-PAGE electrophoresis and WB identification of HpaA recombinant protein

[0060] Insert a 10-hole comb into the prepared gel plate, and place at room temperature for 30 min to allow it to solidify. Treat the samples, and then load 10 μL of each sample for SDS-PAGE electrophoresis. First, perform electrophoresis at 80 V for 30 min, and then adjust to 120 V. After electrophoresis to the green scale line, remove the gel, and then shake in instant blue staining solution for staining. Then, shake in first-grade water for decolorization, and then observe the results under a gel imaging system. The pET-28a-HpaA / BL21(DE3) is expressed in a soluble form under the condition of induction at 37°C, and the molecular band of the target protein is about 30 kD, which is consistent with the expected results (as shown in Figure 2 , a), and the purity is more than 90% after scanning the gray value with Image J software. WB experiment further verifies the His tag. The previous step is the same as SDS-PAGE electrophoresis. After transferring the gel, perform constant current electrophoresis at 200 mA for 1 h. Remove the PVDF membrane, and then block with 2% skim milk for 1 h. After the time is up, wash the membrane with TBST for 3 times, 5 min each time. Incubate the HRP-labeled His tag antibody, and dilute the antibody with 2% skim milk at a ratio of 1:6000. Incubate at 4°C overnight. The next day, recover the primary antibody, wash the membrane with TBST for 3 times, 5 min each time, and then incubate with ECL developing solution. After exposure under a gel imaging system, the results further indicate that the HpaA recombinant protein is induced, expressed, and purified, and can be used for immunizing camels (as shown in Figure 2 , b).

[0061] Example 3: Camel immunization and antibody titer detection

[0062] Take 2 mg HpaA recombinant protein in a 10 mL syringe, another 10 mL syringe add 2 mL Freund's complete adjuvant, add connection conversion conversion head connection two syringes, left and right to push the emulsification device to emulsify, subcutaneous point immunity of adult bactrian camel, and then every two weeks 2 mg protein and Freund's incomplete adjuvant mixed emulsion immunization camel 4 times, after the last immunization, the 7th day, the peripheral blood of camel was collected for separation of lymphocytes. The level of antigen specific response of camel after immunization was detected by ELISA.

[0063] 1. Preparation of liquid

[0064] 1) Preparation of coating solution: weigh Na2CO3 1.6 g, NaHCO3 2.9 g, dissolve in 1 L ddH2O, adjust the pH to 9.6 with pH meter;

[0065] 2) Preparation of blocking solution: 1 g bovine serum albumin, dissolved in 100 mL antibody diluent (1:100);

[0066] 3) Preparation of antibody diluent: dissolve phosphate in 1 L ddH2O, then add 500 μL Tween 20, and adjust the pH to 7.4 with pH meter;

[0067] 4) Preparation of washing solution: same as antibody diluent

[0068] 5) Color developing solution (TMB), product of Solerbio Company;

[0069] 6) Preparation of termination solution (2M H2SO4): pour 22.2 mL concentrated sulfuric acid into 177.8 mL ddH2O.

[0070] 2. ELISA detection of antibody titer produced by HpaA recombinant protein immunized camel

[0071] 1) Dilute the purified HpaA antigen protein to 5 μg / mL with coating solution;

[0072] 2) Coating: add the recombinant protein diluent to the enzyme-labeled plate, 100 μL / well, incubate overnight at 4°C, then wash 3 times with washing solution, dry and cover with plastic wrap, and store in 4°C refrigerator;

[0073] 3) Blocking: add 200 μL / well of blocking solution to the enzyme-labeled plate, and incubate in 37°C incubator for 1 hour, wash 5 times with PBST;

[0074] 4) Dilute the serum by 1:1000, 1:2000, 1:4000, 1:8000, etc. to 1:2048000 ratio;

[0075] 5) Take the closed enzyme-labeled plate, add diluted serum, 100 μL / well, place in 37℃ incubator for 1 h, wash with PBST for 5 times, and dry;

[0076] 6) Incubate rabbit anti-camel IgG serum (1:2000) 100 μL for 1 h at 37℃;

[0077] 7) Wash with PBST for 5 times, dry, incubate HRP-labeled goat anti-rabbit antibody, add diluted secondary antibody (1:5000) 100 μL per well, incubate for 1 h at 37℃;

[0078] 8) Wash with PBST for 5 times, dry, add substrate color developing liquid (TMB) 100 μL / well, incubate in 37℃ incubator for 15 min in dark;

[0079] 9) Add stop solution (2M H2SO4), immediately place on enzyme-labeled instrument to measure OD value at 450 nm wavelength;

[0080] 10) Result judgment: A sample / A negative value ≧2.1 is positive (negative control is camel pre-immune serum diluted at the same dilution multiple).

[0081] Result: The antibody titer on the 7th day after the last immunization reached 1:1024000, which indicated that the HpaA recombinant protein constructed in the application has good immunogenicity (as shown in Figure 3 ).

[0082] Example 4: Constructing a nano library against HpaA

[0083] One week after the end of the 5th immunization, 50 mL of peripheral lymphocytes of the camel were extracted for total RNA extraction and reverse transcription. The peripheral blood lymphocytes preserved by Trizo 1 reagent were added with chloroform and isopropyl alcohol for extraction, and the precipitate was washed with 75% ethanol and dissolved in ultrapure water to obtain total RNA. The concentration and electrophoresis were used to evaluate the quality of the RNA, and the Takara reverse transcription was used after the quality was qualified. The cDNA obtained by reverse transcription was used as a template for PCR reaction, and two rounds of amplification were performed. The VHH region of the antibody was cloned by PCR technology after enzyme digestion and ligation, and the second round of cloning products were inserted into the multiple cloning site Pst I-HF and Not I-HF of the phage display vector pMECS by homologous recombination. 100 ng of the ligation product pMECS-VHHs was added to a pre-cooled electroporation cup containing 50 μL of E. coli TG1 competent cells, and was placed in an electroporation instrument (voltage 1800 V, resistance 200 Ω, time 5 ms) for electroporation. The electroporation cup was taken out, and the transformant pMECS-VHHs-TG1 was recovered and cultured. 24 clones were randomly selected, and colony PCR identification was performed using primers MP57 and GIII. The PCR product with a single band of about 700 bp was considered as a positive clone to obtain the phage display library (as shown in Figure 4 ).

[0084] Example 5: Identification and diversity analysis of nanolibrary

[0085] 20 μL of the bacterial liquid after the culture by electroporation was taken out for library identification. The LB / Amp-GLU medium was used for 10-fold dilution, and 100 μL of the dilution liquid with dilution degrees of 10 -4 , 10 -5 , and 10 -6 were uniformly coated on the LB / Amp-GLU plate, and were cultured at 37°C for 6-8 h. After the colonies grew on the plate, the number of colonies was calculated to obtain the library capacity of 8×10 7 PFU / mL. 24 regular-shaped colonies were randomly selected into the LB / Amp-GLU medium, and were cultured at 37°C and 220 rpm for 3-4 h. The library positive rate was identified by PCR. 24 bacteria were randomly selected, and the bacterial liquid PCR was performed. The results showed that 23 bacteria were positive and 1 was negative, and the library positive rate was 95.8% (as shown in Figure 5 ).

[0086] Example 6: Screening of anti-HpaA protein recombinant nanobody

[0087] 1. Affinity panning

[0088] 1) The recombinant HpaA protein was diluted with carbonate buffer at pH 9.6 to a final concentration of 5 μg / mL, and 100 μL / well was added to the enzyme-labeled well plate. The antigen coating amount of the first to third rounds of panning was 20 μg / well, 10 μg / well, and 5 μg / well, respectively. Two non-adjacent wells were selected and 100 μL of binding buffer was added as an antigen-free control.

[0089] 2) Discard the coating solution, wash with PBST for 5 times, add 200 μL of 2% skim milk to each well, and block at 37°C for 1 h.

[0090] 3) Discard the blocking solution, wash with PBST for 5 times, dilute the recombinant phage solution with 2% skim milk to 5 x 10 11 pfu / mL, add 100 μL to each well, and incubate at room temperature for 2 h.

[0091] 4) Discard the recombinant phage, wash with PBST for 15 times, and wash with PBS for 10 times.

[0092] 5) Add 100 μL of freshly prepared 0.1 M triethylamine, stand at room temperature for 10 min, elute the specifically bound phage; transfer the eluate to a 1.5 mL sterile centrifuge tube, and quickly neutralize with 100 μL of Tris-HCL buffer;

[0093] 6) Take 10 μL for gradient dilution, determine the titer, calculate the panning and recovery rate, mix the rest of the eluate, and then amplify and purify for the next round of affinity panning.

[0094] 2. Amplification of the library after panning

[0095] 1) Infection of TG1 cells: take 200 μL of the panning eluate, infect 2 mL of logarithmic growth phase TG1, invert and mix well, stand at 37°C for 30 min, add 8 mL of 2 x TY / Amp-GLU medium, and incubate at 37°C on a 220 rpm shaker until logarithmic growth phase.

[0096] 2) Rescue of phagemids: add 20 MOI of helper phage M13K07 to the culture, invert and mix well, stand at 37°C for 30 min, centrifuge at 3800 x g at room temperature for 10 min, discard the supernatant, resuspend the pellet with 300 mL of 2 x TY / Amp-Kan medium, and incubate at 37°C for 14 h at 220 rpm.

[0097] 3) Concentration of recombinant phage: 4°C, 4000xg centrifugation for 40 min, collect the supernatant, add 1 / 5 volume of PEG / NaCl, mix well, ice bath for 30 min; 4°C, 4000xg centrifugation for 40 min, discard the supernatant; add 0.9 mL PBS to resuspend the precipitate, incubate at 4°C overnight; 4°C, 15,000xg centrifugation for 30 min to precipitate bacterial cells, cell debris and phage aggregates, collect the supernatant and store at 4°C. This is the amplification product, determine the titer, and use for the next round of panning or analysis.

[0098] 4) Repeat the affinity panning step until the three rounds of panning process are completed.

[0099] Table 1: Enrichment of specific recombinant phage during the panning process

[0100]

[0101] The enrichment of specific recombinant phage during the panning process is shown in Table 1. The results show that with the increase of panning rounds, the phage recovery rate increases significantly, and the proportion of specific phage increases significantly. The above data show that after three rounds of panning, the specific recombinant phage is significantly enriched.

[0102] 3. ELISA identification of anti-HpaA protein specific nanobodies

[0103] The 24 strains of nanobodies were sent for sequencing, and the sequencing results were returned by Shenguo Company. The differences between the sequences were analyzed, and 24 strains were selected for sequencing. The results showed that the sequences of the 24 strains were different, reflecting the diversity of the library (e.g. Figure 6 ). IPTG was used to induce 120 randomly selected colonies from the third eluate titer determination plate, and the bacterial cells were collected and freeze-thawed to obtain nanobody crude extract. Indirect ELISA was used to screen nanobodies that specifically reacted with HpaA protein. The results showed that the crude extracts of 120 clones all reacted with HpaA protein, and among the 120 clones, 24 strains of nanobodies had strong binding to HpaA protein, indicating that the screened nanobodies had specificity (e.g. Figure 7 ). After screening, 6 strains of specific nanobodies against HpaA were finally obtained (e.g. Figure 8 ).

[0104] Example 7: Identification and characterization of anti-HpaA nanobodies

[0105] 1. Expression and purification of anti-HpaA nanobodies in prokaryotic expression system

[0106] 1) The VHH fragments of different clones obtained from the previous sequencing analysis were cloned into the prokaryotic expression vector pET-25b(+) to obtain the recombinant plasmid pET-25b-HpaA-Nbs, which was then transformed into BL21(DE3).

[0107] 2) After transformation, the LB plate (50 μg / mL kanamycin) was coated.

[0108] 3) A single colony was selected and inoculated in 10 mL of LB liquid medium containing kanamycin, and cultured at 37°C overnight.

[0109] 4) 300 μL of the overnight strain was inoculated into 300 mL of LB medium, and cultured at 37°C until the logarithmic growth phase, and then IPTG was added for induction. After shaking culture at different induction temperatures for 16-20 h.

[0110] 5) The bacteria were centrifuged and the bacterial bodies were broken to obtain the crude antibody extract;

[0111] 6) The antibody protein was purified by nickel ion affinity gravity chromatography column (Ni-NTA 5 mL), and imidazole gradient elution was used. The low concentration imidazole eluate was used for washing, and the high concentration imidazole eluate 250 mM could prepare a protein with a purity of more than 90%. Six anti-HpaA nanobodies were obtained by the above method.

[0112] The recombinant plasmid pET-25b-HpaA-Nbs was transformed into BL21(DE3) competent cells, and the bacterial bodies were collected and ultrasonicated after IPTG induction expression. The purified prokaryotic expression protein HpaA-Nb8, HpaA-Nb9, HpaA-Nb10, HpaA-Nb11, HpaA-Nb12 and HpaA-Nb15 were identified by SDS-PAGE to be consistent with the expected size (about 20 kDa) and high purity (e.g. Figure 9 , a), and the His tag WB identification of the purified anti-HpaA nanobody further verified the correct expression of the nanobody (e.g. Figure 9 , b), the nucleotide sequences and amino acid sequences of the six nanobodies are shown in SEQ ID Nos. 2-13.

[0113] Example 8: ELISA detection of the binding activity of anti-HpaA nanobodies

[0114] The six prokaryotic expression purified nanobodies need to be combined with HpaA recombinant protein to determine the EC 50 value of the combination and evaluate the binding activity.

[0115] 1) Recombinant HpaA protein was diluted with carbonate buffer at pH 9.6 to a final concentration of 5 μg / mL, and added to a 96-well enzyme plate at 100 μL / well, with PBS control wells, and coated overnight at 4°C.

[0116] 2) Discard the coating solution, wash with PBST 5 times, add 200 μL 2% skim milk per well, and block at 37°C for 1 h.

[0117] 3) Discard the blocking solution, wash with PBST 5 times, add 100 μL of two-fold dilution of nanobodies per well, with 16 concentration gradients for each antibody, and incubate at 37°C for 1 h.

[0118] 4) Wash with PBST 5 times, and incubate the antibody: add 100 μL of mouse anti-HSV (diluted 1:6000 with 2% skim milk) per well, and incubate at 37°C for 1 h.

[0119] 5) Wash with PBST 5 times, and incubate the antibody: add 100 μL of goat anti-mouse antibody with HRP label (diluted 1:7000 with 2% skim milk), and incubate at 37°C for 1 h.

[0120] 6) Color development: discard the antibody, wash with PBST 5 times, add 100 μL of TMB color development solution per well, and incubate at 37°C for 15 min in the dark.

[0121] 7) Termination: add 100 μL of 2M H2SO4 per well to terminate the color development reaction, and read the OD450 value.

[0122] The results show that 6 nanobodies bind strongly to the HpaA recombinant antigen protein, and the EC 50 values of the 6 nanobodies calculated by graphpad software are 0.002694 μM, 0.03004 μM, 0.003379 μM, 0.01380 μM, 0.009783 μM, and 0.01027 μM, respectively. Figure 10

[0123] Example 9: Construction of the eukaryotic expression vector pCMV-N1-HpaA-Nbs-HRP

[0124] The pCMV-N1-HRP eukaryotic expression vector also has two enzyme cleavage sites of restriction endonuclease BamH I and HindIII, so the vector pCMV-N1-HRP can be double-enzyme cleaved, and then the VHH sequence fragment of the target gene and the enzyme-cleaved vector can be ligated to obtain the pCMV-N1-HpaA-Nbs-HRP plasmid, which is transformed into DH5α competent cells. After that, a positive clone single clone strain is picked, and the method of bacterial liquid PCR is used to identify whether the target gene is successfully inserted into the vector. Figure 11 ​). After amplification of the eukaryotic expression recombinant plasmid, the eukaryotic expression plasmid was extracted by Venozen endotoxin-free plasmid extraction kit for transfection standby.

[0125] Example 10: WB and ELISA detection of expression of recombinant Nbs-HRP

[0126] Purification of anti-HpaA nanobody in eukaryotic 293T cells: the nanobody eukaryotic expression vector pCMV-N1-HpaA-Nbs-HRP plasmid constructed in Example 9 was transfected into 293T cells, and the cell supernatant was collected after 72 h, and HpaA-Nbs-HRP was characterized and identified. WB identified the His tag, and ELISA detected the affinity of recombinant Nbs-HRP to HpaA (as shown in Figure 12 ). Figure 12 It can be seen that: the WB identification of the His tag of Nbs-HRP showed that the experimental group appeared a band with a molecular size of about 75 kDa and was consistent with the target band position of the control nanobody group, and the empty pCMV-N1-HRP was not expressed, indicating that Nbs-HRP was successfully expressed after transfection of the cells for 72 h. ELISA detection of binding activity showed that compared with the BSA control group, the purified Nbs-HRP had strong binding activity to HpaA recombinant protein.

[0127] The above examples are only preferred embodiments for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation of the present application made by those skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A nanobody against Helicobacter pylori adhesin A, characterized in that: Its amino acid sequence is shown in SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 or SEQ ID No.

13.

2. A fusion protein, characterized in that: The fusion protein comprises the amino acid sequence of the anti-Helicobacter pylori adhesin A nanobody and horseradish peroxidase as described in claim 1.

3. The nucleic acid encoding the anti-Helicobacter pylori adhesin protein A nanobody as described in claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10 or SEQ ID No.

12.

4. A product for detecting Helicobacter pylori, characterized in that, It contains the anti-Helicobacter pylori adhesin A nanobody of claim 1 or the fusion protein of claim 2.

5. The use of the anti-Helicobacter pylori adhesin A nanobody of claim 1 or the fusion protein of claim 2 in the preparation of products for detecting Helicobacter pylori.

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