Anti-helicobacter pylori adhesin protein A nano antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-helicobacter pylori adhesin protein A nano antibody as well as a preparation method and application thereof. Six anti-helicobacter pylori adhesin protein A (HpaA) nano antibodies are screened, the amino acid sequences of the six nano antibodies are as 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 nano antibodies can specifically target HpaA protein, and the HpaA protein can be specifically targeted to the HpaA protein. The nano antibody has the characteristics of small molecular weight, high water solubility, high tolerance, high stability, high antigen binding property, low immunogenicity and the like, is identified and characterized, can be applied to an HpaA protein detection reagent, and can be used as a candidate nano antibody for detecting helicobacter pylori.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technology, and in particular to an anti-Helicobacter pylori adhesin protein A nano antibody and a preparation method thereof, and also to the application of the nano antibody in the preparation of a product for detecting Helicobacter pylori. Background Art

[0002] Helicobacter pylori (H.pylori, Hp) is a microaerophilic Gram-negative bacterium with a spiral structure that colonizes 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 blood system, nervous system, cardiovascular system, skin, ophthalmology and other extra-digestive system diseases. At present, new therapeutic drugs that can overcome the drug resistance of Helicobacter pylori are urgently needed, and accurate diagnosis of Hp infection is very necessary for further treatment. At present, the detection of Helicobacter pylori is mainly through carbon 13 or carbon 14 element detection, or rapid urease test and pathological biopsy of gastric mucosa taken through gastroscopy. Therefore, the development of new reliable and convenient diagnostic reagents for Hp is expected to become an effective means of preventing and controlling Hp infection.

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

[0004] The present invention aims to solve the problems existing in traditional antibodies. The first purpose is to focus on the HpaA target and screen out 6 strains of nano antibodies, which can be used to develop high-sensitivity and high-specificity detection methods. The second purpose is to provide a method for preparing nano antibodies for Helicobacter pylori detection. The third purpose is to provide the use of Helicobacter pylori anti-HpaA nano antibodies. Summary of the invention

[0005] 1. In view of this, one of the objects of the present invention is to provide an anti-Helicobacter pylori adhesin protein A nanobody; the second object of the present invention is to provide a fusion protein; the third object of the present invention is to provide a nucleic acid sequence encoding the anti-Helicobacter pylori adhesin protein A nanobody; the fourth object of the present invention is to provide a method for preparing the anti-Helicobacter pylori adhesin protein A nanobody; the fifth object of the present invention is to provide a product for detecting Helicobacter pylori; the sixth object of the present invention is to provide the 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] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. An anti-Helicobacter pylori adhesin protein A nanobody, characterized in that: its amino acid sequence 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. The nucleic acid sequence encoding the anti-Helicobacter pylori adhesin protein A nanobody is characterized in that: its nucleotide sequence is shown as 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 the DNA sequence encoding HpaA, constructing a prokaryotic expression vector, and extracting the recombinant HpaA antigen protein after expressing it in Escherichia coli; after immunizing camels with the recombinant HpaA antigen protein, collecting peripheral blood from the camels for separating lymphocytes, and then detecting the antigen-specific response level of the camels after immunization with ELISA;

[0012] (2) extracting total RNA from peripheral blood lymphocytes of camels and reversely transcribing the RNA to obtain cDNA, using the cDNA as a template to perform PCR reaction to amplify the VHH region of the antibody, connecting the amplified product to a phage display vector, and electrotransforming it into Escherichia coli to establish a phage display library. The desired positive clones were screened by antigen incubation and ELISA identification, and then sequenced to obtain the sequence of the HpaA nanobody;

[0013] (3) The sequence of the HpaA nanobody is constructed into a prokaryotic or eukaryotic expression vector, and large-scale expression and purification are performed in Escherichia coli, yeast or mammalian cell lines to obtain an anti-Helicobacter pylori adhesin protein A nanobody.

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

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

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

[0017] In some embodiments of the present invention, 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 Helicobacter pylori, characterized in that it comprises the anti-Helicobacter pylori adhesin protein A nanobody or the fusion protein.

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

[0020] The beneficial effect of the present invention is that: the research group selected the HpaA target protein with strong immunogenicity from the Hp target protein in combination with literature research, downloaded the protein sequence from the NCBI official website, optimized the sequence by removing the signal peptide analysis and B cell dominant antigen analysis, constructed the pET-28a-HpaA prokaryotic expression vector, transformed it into the Escherichia coli BL21 (DE3) strain, and purified the recombinant HpaA protein by IPTG induction expression. The prepared HpaA recombinant protein was used to immunize camels for a total of 5 immunizations, with an immunization interval of once every 2 weeks. The first immunization: complete Freund's adjuvant, and incomplete Freund's adjuvant was used for the 2nd to 5th immunization. One week after the completion of the 5 immunizations, the camel peripheral serum was taken, and the anti-HpaA protein antibody titer was detected by ELISA. The results showed that the specific antibody titer of the HpaA protein in the camel serum after the last immunization was 1:1024000, and the immunization effect was good, which can be used for the next step of extracting mRNA, reverse transcription into cDNA, and constructing a phage library. After two rounds of PCR, the VHH gene was successfully amplified. After double restriction digestion of pMECS and VHH gene, they were connected and transformed into TG1 competent cells, and the nanolibrary was constructed. After library counting, the library size was 8×10 7 PFU / ml, positive rate of bacterial liquid PCR identification library: 24 bacteria were randomly selected, of which 23 were positive and 1 was negative, and the positive rate of the library was: 95.8%.

[0021] After 24 strains were randomly selected and sent to Biotech for sequencing, the results showed that the sequences of the 24 strains were all different, reflecting the diversity of the library. The enrichment of specific recombinant phages during the nano-antibody panning process was detected, and it was found that with the increase in the number of panning rounds, the phage recovery rate increased significantly; the specific phage ratio increased significantly; the above data showed that after three rounds of panning, the specific recombinant phages were significantly enriched. 120 monoclones were randomly selected, and the anti-HpaA monoclones were amplified and cultured for affinity detection. The results showed that the crude extracts of all 120 clones had a strong reaction with the HpaA protein and the OD was selected>1.8, and P / N>5. 24 nano-antibodies strongly bound to the HpaA protein, indicating that the screened nano-antibodies are specific.

[0022] The recombinant plasmid pET-25b-HpaA-Nbs was constructed and transformed into BL21 (DE3) competent cells. After IPTG induction, the cells were collected, sonicated, and purified using Ni-NTA. SDS-PAGE identification showed that the sizes of the purified prokaryotic HpaA-Nb8, HpaA-Nb9, HpaA-Nb10, HpaA-Nb11, HpaA-Nb12, and HpaA-Nb15 were consistent with expectations (about 20 kDa) and the grayscale values ​​scanned by Image J software showed that the purity was higher than 90%. After the anti-HpaA nanoantibody was purified, the His tag WB identification further verified the correct expression of the nanoantibody. The affinity of the purified nanoantibody and HpaA was detected by ELISA, and the binding activity of the 6 nanoantibodies to HpaA was strong, and the EC 50 They are 0.002694μM, 0.03004μM, 0.003379μM, 0.01380μM, 0.009783μM and 0.01027μM respectively.

[0023] The nanobody eukaryotic expression vector pCMV-N1-HpaA-Nbs-HRP was constructed, and the constructed plasmids were transfected into 293T cells respectively. The cell supernatant was collected after 72 hours, and the expression of recombinant Nbs-HRP was detected by WB and ELISA. The results showed that the HRP-tagged nanobody was successfully expressed, and the binding of recombinant Nbs-HRP and HpaA was detected by ELISA, showing high affinity, which can be used as an effective probe candidate for detecting Helicobacter pylori.

[0024] The present invention is based on phage display technology, and screens 6 strains of nano antibodies against Helicobacter pylori adhesin protein A (HpaA). The nano antibodies can specifically target HpaA protein, and have the characteristics of small molecular weight, high water solubility, high tolerance, high stability, high antigen binding, low immunogenicity, etc., and identify and characterize them, and analyze the binding sites of recombinant HpaA protein and nano antibodies. The present invention relates to nano antibodies and preparation methods and applications thereof. The nano antibodies can be applied to HpaA protein detection reagents, and the extremely high sensitivity and high specificity make the nano antibodies have outstanding advantages in diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

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

[0027] Figure 2 Identification and characterization of HpaA recombinant protein; a: SDS-PAGE electrophoresis of HpaA recombinant protein purification; b: WB identification of HpaA recombinant protein His tag. After the induced expression of the bacteria was ultrasonically broken, affinity chromatography was used for purification using a Ni column. The SDS-PAGE results showed that the size of the target protein was about 30kDa, which was consistent with the expected target protein molecular size. After grayscale scanning with Image J software, the purity was more than 90%. WB identified the His tag, further indicating that the induced expression and purification of the HpaA recombinant protein was completed and could be used to immunize camels.

[0028] Figure 3 To measure the serum antibody titer of Bactrian camels before and after immunization; Figure 3 It can be seen that the specific antibody titer of HpaA protein in the camel serum collected one week after the last immunization is 1:1024000, and the immunization effect is good, which can be used to extract RNA for subsequent experiments.

[0029] Figure 4 The construction of the nanobody library; a: The first round of PCR amplification obtained a 700 bp fragment, and the second round of PCR amplification obtained a 400 bp fragment; c: Double restriction enzyme digestion of the pMECS vector obtained a 4000 bp fragment; d: PCR of the transformed bacterial solution. Figure 4 It can be seen that two rounds of VHH amplification were completed through PCR technology, and it was successfully connected with the pMECS after enzyme digestion. The PCR nucleic acid electrophoresis band of the transformed bacterial solution was 700bp, which was consistent with the expected target molecule band size, and the nanolibrary was successfully constructed.

[0030] Figure 5 For the identification of phage library diversity; a: plate count antibody library capacity; b: bacterial liquid PCR identification library positive rate. Figure 5 After counting the library, the library size was 8×10 7PFU / mL, bacterial liquid PCR identification library positive rate identification using primers MP57 and GIII, positive 700bp. Randomly selected 24 bacteria, of which 23 bacteria were positive, 1 was negative, the library positive rate was 95.8%.

[0031] Figure 6 The results of positive clone sequence analysis are as follows: Figure 6 As shown, the sequences of the 24 strains were all different, reflecting the diversity of the library.

[0032] Figure 7 ELISA identification of anti-HpaA protein specific nanoantibodies; a: Elisa test of the binding of anti-HpaA nanoantibody crude extracts No. 1-60; b: Elisa test of the binding of anti-HpaA nanoantibody crude extracts No. 61-120. Figure 7 The results showed that crude extracts of all 120 clones reacted strongly with HpaA protein and were selected with OD>1.8 and P / N>5, and 24 nanobodies bound strongly to HpaA protein, indicating that the screened nanobodies were specific.

[0033] Figure 8 It is the amino acid sequence information of the HpaA protein-specific nanoantibody; after screening, the amino acid sequence information of the final 6 nanoantibodies was obtained.

[0034] Fig. 9 Identification and characterization of nanobodies; a: SDS-PAGE electrophoresis of purified anti-HpaA nanobodies; b: WB identification of purified His tag of anti-HpaA nanobodies. Fig. 9 The results showed that the recombinant plasmid pET-25b-HpaA-Nbs was transformed into BL21(DE3) competent cells, and the cells were collected and sonicated after IPTG induction, and purified by Ni-NTA. SDS-PAGE identification showed that the purified prokaryotic HpaA-Nb8, HpaA-Nb9, HpaA-Nb10, HpaA-Nb11, HpaA-Nb12 and HpaA-Nb15 were consistent with the expected size (about 20kDa) and had high purity. After the anti-HpaA nanoantibody was purified, the His tag WB identification further verified the correct expression of the nanoantibody.

[0035] Fig.10This is the determination of the binding activity of HpaA nanoantibody with HpaA antigen protein; a: HpaA-Nb8 binding activity with HpaA antigen protein; b: HpaA-Nb9 binding activity with HpaA antigen protein; c: HpaA-Nb10 binding activity with HpaA antigen protein; d: HpaA-Nb11 binding activity with HpaA antigen protein; e: HpaA-Nb12 binding activity with HpaA antigen protein; f: HpaA-Nb15 binding activity with HpaA antigen protein. Fig.10 The results showed that the six nanoantibodies strongly bound to the HpaA recombinant antigen protein. The EC values ​​of the six nanoantibodies were calculated by graphpad software. 50 They are: 0.002694μM, 0.03004μM, 0.003379μM, 0.01380μM, 0.009783μM, and 0.01027μM respectively.

[0036] Fig.11 The figure shows the construction of the eukaryotic expression vector pCMV-N1-HpaA-Nbs-HRP; a: electrophoresis of double-enzyme digestion of pCMV-N1; b: PCR identification of the recombinant plasmid pCMV-N1-HpaA-Nbs-HRP by bacterial liquid. Fig.11 The results showed that the eukaryotic expression plasmid of nanoantibodies was successfully constructed and could be used for transfection of 293T cells.

[0037] Fig.12 The expression of recombinant Nbs-HRP was detected by WB and ELISA; a: WB identification of fusion-expressed Nbs-HRP; b: ELISA detection of the binding activity of recombinant Nbs-HRP and HpaA. Fig.12 It can be seen that: WB identification of the His tag of Nbs-HRP showed that the experimental group had a molecular band of about 75kDa and the position was consistent with the target band of the control nanoantibody group, and the empty pCMV-N1 was not expressed, indicating that Nbs-HRP was successfully expressed after 72 hours of transfection. ELISA detection of binding activity showed that compared with the BSA control group, the purified Nbs-HRP had a strong binding activity with the HpaA recombinant protein. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0039] The strains and various reagents used in the present invention are as follows: plasmids pET-28a(+), pET-25b(+), Escherichia coli strain BL21(DE3), Escherichia coli strain DH5α, and TG1 competent cells are preserved by the inventor's unit; restriction endonucleases BamH I and Hind III are products of Dalian TakaRa Company; protein marker is a product of Polymeric Company; plasmid extraction kit and gel recovery kit are products of Shanghai Bioengineering 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 Bioengineering Company.

[0040] Materials preparation:

[0041] 1. Ampicillin (Amp) stock solution (100 mg / mL): Weigh 1 g of ampicillin (Amp) and dissolve it in 10 mL of sterile water to prepare a stock solution with a concentration of 100 mg / mL. Filter and sterilize through a 0.22 μm bacterial filter and store the solution in aliquots at -20°C.

[0042] 2. Kanamycin (100 mg / mL): Weigh 1.0 g of kanamycin and dissolve it in 10 mL of sterile water to prepare a stock solution with a concentration of 100 mg / mL. Filter the solution through a 0.22 μm bacterial filter for sterilization and store the solution in aliquots at -20°C.

[0043] 3. IPTG solution: Weigh 2.4 g IPTG and dissolve it in 10 mL sterile water. Filter and sterilize with a 0.22 μm filter. Divide into small portions and store at -20°C.

[0044] 4. Culture medium: (1) 2YT liquid culture medium: weigh 16g of tryptone, 10g of yeast extract and 5g of NaCl, add distilled water to 1L, adjust the pH to 7.4, and sterilize by high pressure steam. (2) 2YT-ATG culture medium: 1.5g of agar powder / 100mL of LB culture medium, sterilize by high pressure, and pour into a plate. (3) 2YT-ATK culture medium: add 1mL of Amp and 1mL of Kan antibiotics and 15g of agar powder to 1L of 2YT culture medium and shake well. After high pressure sterilization, pour into a plate. (4) 2YT-T culture medium: add 1.5g of agar powder / 100mL of 2YT culture medium to 2YT culture medium, sterilize by high pressure, and pour into a plate.

[0045] 5. DNA electrophoresis buffer (50xTAE): weigh 242g Tris, 37.2g Na 2 EDTA·2H 2 O and 57.1 ml of glacial acetic acid, add water to 1 L, and dilute 50 times before use.

[0046] 6. SDS-PAGE electrophoresis buffer (5X): weigh 15.1 g of Tris powder, 94 g of glycine, and 5.0 g of SDS; add about 800 mL of deionized water and stir to dissolve; add deionized water to make up to 1 L and store at room temperature; Note: When adding water, let it flow slowly along the wall to avoid generating 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): Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 mL of 95% ethanol, then add 100 mL of 86% phosphoric acid and dilute to 1000 mL with distilled water. (2) Decolorizing solution: Dilute 250 mL of ethanol and 80 mL of glacial acetic acid with distilled water to 1000 mL.

[0048] 8. ELISA reagents: (1) Coating solution: 1.6 g Na 2 CO 3 , 2.9 g NaHCO 3 , 0.2 g NaN 3 , add distilled water to 1L and adjust the pH to 9.6. (2) Washing liquid: weigh 0.2g KH 2 PO 4 , 2.9 g Na 2 HPO 4 12H 2 O, 8.0 g NaCl, 0.2 g KCl, 0.5 mL Tween-20, add ddHO 2 O to 1L (PBST). (3) Blocking solution: Weigh 0.2g skim milk powder and dissolve it in 10mL washing buffer, filter and sterilize it, and store it at 4℃. (4) Substrate solution: Soluble single-component TMB substrate solution. (5) Stopping solution: Measure 891.3mL of distilled water and add concentrated sulfuric acid (2M H 2 SO 4 )108.7mL.

[0049] 9. Purification reagents: (1) 500mM imidazole solution: weigh 34.04g imidazole and add distilled water to 1L. The remaining concentration gradient imidazole solutions can be obtained by dilution. (2) 20% ethanol: weigh 200ml anhydrous ethanol and add deionized water to 1L. (3) Phosphate buffer (PBS): weigh 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 , 0.24 g KH 2 PO 4 , add distilled water to 1L and 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] Based on literature research, our research group selected the highly immunogenic HpaA target protein from the Hp target proteins. After downloading the protein sequence XG12708F from the NCBI official website, we optimized and synthesized the DNA sequence encoding HpaA (SEQ ID NO.1) by removing the signal peptide and analyzing the B cell dominant antigen. The sequence was ligated between the BamHI and HindⅢ multiple cloning sites of the pET-28a(+) empty plasmid to construct the recombinant plasmid pET-28a-HpaA (such as Figure 1 , a, d).

[0053] 2. Transformation of recombinant plasmid: Take 1 tube of E. coli BL21 competent cells (self-made by the research team) from the -80℃ refrigerator and add 3μL of the synthesized pET-28a-HpaA plasmid. Ice bath for 30min, heat shock in a 42℃ metal bath for 90s, and quickly ice bath for 5min. Add 800μL LB blank culture medium, mix well, and shake at 220rpm in a 37℃ shaker for 1h. Take 20μL of the bacterial solution and spread it on a kanamycin-resistant LB plate. Invert the plate in a 37℃ incubator and culture for 14h. Pick the well-separated and clearly edged colonies on the transformation plate, inoculate them into kanamycin-resistant LB culture medium, and shake and culture at 37℃ overnight.

[0054] 3. Identification of nucleic acid electrophoresis of pET-28a-HpaA prokaryotic expression vector

[0055] A 1.0% agarose gel was prepared, and 1 μL 6× Loading buffer was added to the ligation product in proportion. After 30 minutes of gel electrophoresis at 100V, the results of nucleic acid electrophoresis were collected by gel imaging. The results showed that the molecular size of the HpaA target gene fragment of the empty plasmid pET-28a was about 400bp, and the molecular size of the positive cloned plasmid pET-28a-HpaA was about 1000bp, which was consistent with the target molecular size. The recombinant HpaA protein expression vector (such as Figure 1 , b). The recombinant plasmid was transformed into the BL21 (DE3) strain, and uniform colonies were observed on the kanamycin-resistant plate (e.g. Figure 1 , c), indicating that the conversion is successful.

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

[0057] 1. Induced expression and purification of HpaA recombinant protein

[0058] 100 μL of overnight cultured pET-28a-HpaA / BL21 (DE3) bacterial solution was added to 10 mL of LB medium containing kanamycin resistance, and cultured at 220 rpm and 37°C for primary activation of the bacterial solution. 3 mL of the primary activated bacterial solution was added to 300 mL of LB medium containing kanamycin resistance for secondary activation, and cultured at 37°C for 4-5 h until OD600 was 1.0, 300 μL of 0.5 mM IPTG was added, and the cells were induced in a shaker at 37°C for 14 h, and centrifuged at 10000 rpm for 15 min to collect the cells, and then 5 mL of PBS was added to resuspend the cells, and the bacterial solution was ultrasonically lysed for 5 min (120 V), and the supernatant was collected for subsequent purification, filtered with a 0.22 μm bacterial filter, and purified by nickel ion affinity gravity chromatography column (Ni-NTA 5mL), purify the recombinant HpaA antigen protein, first use 10mM, 30mM, 50mM imidazole to wash out the impurities, then use 250mM imidazole to elute the target protein, collect the eluate, use BCA kit to detect the concentration of the recombinant protein, divide it into 1.5mL EP tubes, and store it in a -80℃ refrigerator.

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

[0060] Pour 12.5% ​​separation gel and concentrated gel into the gel plate in turn, insert a 10-hole comb, and place it at room temperature for 30 minutes to solidify for use. Take 10μL of the processed samples and load them for SDS-PAGE electrophoresis. The voltage is first 80v for 30min, then adjusted to 120v. After electrophoresis to the green scale line, take out the gel, place it in the instant blue staining solution for shaking and staining, and then place it in the first-level water for shaking and decolorization. Observe the results under the imaging system. pET-28a-HpaA / BL21 (DE3) is expressed in a soluble form under 37℃ induction, and the molecular band appears as the target protein of about 30kD, which is in line with the expected results (such as Figure 2 , a), and after being scanned by the grayscale value of Image J software, the purity reached more than 90%. The WB experiment further verified the His tag. The previous steps were the same as SDS-PAGE electrophoresis. After taking the gel, transfer to the membrane. The constant current was 200mA and the membrane was transferred for 1h. The PVDF membrane was taken out and blocked with 2% skim milk for 1h. After the time was up, the membrane was washed 3 times with TBST, 5min / time. Incubate the His-tagged antibody with HRP. The antibody was diluted with 2% skim milk at a ratio of 1:6000 and incubated at 4°C overnight. The next day, the primary antibody was recovered, the membrane was washed 3 times with TBST, 5min / time, and after incubation with ECL colorimetric solution, the results were exposed under the gel imaging system, which further demonstrated that the induced expression and purification of the HpaA recombinant protein was completed and could be used to immunize camels (such as Figure 2 , b).

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

[0062] Take 2mg HpaA recombinant protein in a 10mL syringe, add 2mL Freund's complete adjuvant in another 10mL syringe, add a connecting conversion adapter to connect the two syringes, push the emulsification device back and forth to fully emulsify, and immunize adult Bactrian camels subcutaneously in the neck. Then, 2mg protein was mixed with Freund's incomplete adjuvant and emulsified every two weeks to immunize camels 4 times. On the 7th day after the last immunization, peripheral blood of the camels was collected for lymphocyte isolation. The antigen-specific response level of camels after immunization was detected by ELISA.

[0063] 1. Preparation of Liquid

[0064] 1) Preparation of coating solution: weigh Na 2 CO 3 1.6 g, NaHCO 3 2.9 g, dissolved in 1 L ddH 2 O, adjust the pH to 9.6 using a pH meter;

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

[0066] 3) Preparation of antibody diluent: Dissolve phosphate in 1L ddHO 2 O, then add 500 μL Tween 20, and adjust the pH to 7.4 using a pH meter;

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

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

[0069] 6) Stop solution (2M H 2 SO 4 ) was prepared by pouring 22.2 mL of concentrated sulfuric acid into 177.8 mL of ddH 2 In O.

[0070] 2. ELISA to detect the antibody titer produced by camels immunized with HpaA recombinant protein

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

[0072] 2) Coating: Add the recombinant protein dilution to the ELISA plate, 100 μL / well, incubate at 4°C overnight, wash three times with washing solution, air dry, wrap with plastic wrap, and place in a 4°C refrigerator for later use;

[0073] 3) Blocking: Add 200 μL / well of blocking solution to the ELISA plate, place in a 37°C incubator for 1 hour, and wash 5 times with PBST;

[0074] 4) Dilute the serum in multiples of 1:1000, 1:2000, 1:4000, 1:8000, etc., until the dilution reaches 1:2048000;

[0075] 5) Take the blocked ELISA plate, add diluted serum in sequence, 100 μL / well, place in a 37°C incubator for 1 hour, wash 5 times with PBST, and air dry;

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

[0077] 7) Wash with PBST for 5 times, dry in air, incubate with HRP-labeled goat anti-rabbit antibody, add 100 μL of diluted secondary antibody (1:5000) to each well, and incubate at 37°C for 1 h;

[0078] 8) Wash 5 times with PBST, dry in air, add 100 μL / well of substrate color development solution (TMB), and incubate in a dark box at 37°C for 15 min;

[0079] 9) Add stop solution (2M H 2 SO 4 ), and immediately placed on a microplate reader to measure the OD value at a wavelength of 450 nm;

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

[0081] Results: The antibody titer reached 1:1024000 on the 7th day after the last immunization, indicating that the HpaA recombinant protein constructed in the present invention has good immunogenicity (such as Figure 3 ).

[0082] Example 4: Construction of a Nanolibrary Targeting HpaA

[0083] One week after the end of the 5 immunizations, 50 mL of camel peripheral lymphocytes were extracted for total RNA extraction and reverse transcription. The peripheral blood lymphocytes preserved by Trizo1 reagent were extracted with chloroform and isopropanol, and the precipitate was washed with 75% ethanol and dissolved in ultrapure water to obtain total RNA. The concentration was measured and the quality of RNA was evaluated by electrophoresis. After the quality was qualified, Takara reverse transcription was used. The cDNA obtained by reverse transcription was used as a template for PCR reaction, and two rounds of amplification were performed. After enzyme digestion and ligation, the VHH region of the antibody was cloned by PCR technology, and the second round of cloning products were inserted into the multiple cloning sites PstⅠ-HF and NotⅠ-HF of the phage display vector pMECS by homologous recombination. 100 ng of the ligation product pMECS-VHHs was taken and added to a pre-cooled electrotransfer cup containing 50 μL of Escherichia coli TG1 competent cells and placed in an electrotransfer instrument (voltage 1800 V, resistance 200 Ω, time 5 ms) for electrotransformation. The electrotransfer cup was removed, and the transformant pMECS-VHHs-TG1 was revived and cultured. 24 clones were randomly selected and identified by colony PCR using primers MP57 and GIII. The PCR product with a single band of about 700 bp was considered a positive clone to obtain a phage display library (such as Figure 4 ).

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

[0085] Take out 20 μL of the bacterial solution after electroporation for library identification, dilute it 10 times using LB / Amp-GLU medium, and then take 10 -4 , 10 -5 , 10 -6 100 μL of the diluted solution was evenly spread on the LB / Amp-GLU plate and incubated at 37°C for 6-8 hours. After the colonies grew on the plate, the number of colonies was calculated and the library capacity was estimated to be: 8×10 7 PFU / mL. Randomly pick 24 colonies with regular shapes and place them in LB / Amp-GLU medium, and culture them at 37°C and 220rpm for 3-4h. PCR identification of library positive rate: Randomly select 24 bacteria and perform PCR on the bacterial liquid. The results show that 23 of them are positive and 1 is negative. The library positive rate is: 95.8% (such as Figure 5 ).

[0086] Example 6: Screening of recombinant nanobodies against HpaA protein

[0087] 1. Affinity panning

[0088] 1) Dilute the recombinant HpaA protein with carbonate buffer at pH 9.6 to a final concentration of 5 μg / mL, and add 100 μL / well to the ELISA plate. The antigen coating amount for the first to third rounds of panning is 20 μg / well, 10 μg / well, and 5 μg / well, respectively. Select 2 non-adjacent wells and add Binding buffer as a no-antigen control.

[0089] 2) Discard the coating solution, wash 5 times with PBST, 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 5 times with PBST, and dilute the recombinant phage solution to 5×10 11 pfu / mL, 100 μL was added to each well and incubated at room temperature for 2 h.

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

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

[0093] 6) Take 10 μL for gradient dilution, determine the titer, calculate the panning and recovery rates, and mix the remaining eluates for amplification and purification 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 TG1 in the logarithmic growth phase, mix by inversion, let stand at 37°C for 30 min, add 8 mL of 2×TY / Amp-GLU culture medium, and culture in a shaker at 37°C at 220 rpm until the logarithmic growth phase.

[0096] 2) Rescue of phagemid: Add 20 MOI of helper phage M13K07 to the culture medium, mix by inversion, stand at 37°C for 30 min, centrifuge at 3800×g for 10 min at room temperature, discard the supernatant, resuspend the precipitate in 300 mL of 2×TY / Amp-Kan medium, and culture at 37°C and 220 rpm for 14 h.

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

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

[0099] Table 1: Enrichment of specific recombinant phages during panning

[0100]

[0101] The enrichment of specific recombinant phages during the panning process is shown in Table 1. The results show that: with the increase in the number of panning rounds, the phage recovery rate increased significantly; the specific phage ratio increased significantly; the above data show that after three rounds of panning, the specific recombinant phages were significantly enriched.

[0102] 3. ELISA Identification of Anti-HpaA Protein Specific Nanobodies

[0103] The 24 strains of nanoantibodies were sent for sequencing, and the sequencing results were returned by the biotechnology company after completion. The differences between the sequences were analyzed, and the results of sequencing 24 strains were selected. The sequences of the 24 strains were different, reflecting the diversity of the library (such as Figure 6 ). 120 colonies randomly selected from the third round of elution titer determination plate were induced with IPTG, and the cells were collected and freeze-thawed to obtain crude nanobody extracts. Nanobodies that specifically reacted with HpaA protein were screened by indirect ELISA. The results showed that all crude extracts of 120 clones reacted with HpaA protein. Among the 120 clones, crude extracts had a strong reaction with HpaA protein and OD>1.8 and P / N>5. 24 nanobodies strongly bound to HpaA protein, indicating that the screened nanobodies have specificity (such as Figure 7 After screening, 6 strains of HpaA-specific nanoantibodies were finally obtained (such as Figure 8 ).

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

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

[0106] 1) The VHH fragments of different clones obtained by 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, spread on LB plates (50 μg / mL kanamycin).

[0108] 3) Select a single colony and inoculate it into LB liquid medium containing 10 mL of kanamycin, and culture it in a shaking incubator at 37°C overnight.

[0109] 4) Inoculate 300 μL of the overnight strain into 300 mL of LB medium and culture in a shaking incubator at 37°C until the logarithmic growth phase. Add IPTG for induction and culture in a shaking incubator for 16-20 h at different induction temperatures.

[0110] 5) Collect the bacteria by centrifugation and break the bacteria to obtain the crude antibody extract;

[0111] 6) Purify the antibody protein by nickel ion affinity gravity chromatography column (Ni-NTA 5mL), using imidazole gradient elution method, low concentration imidazole eluent is used for washing impurities, high concentration imidazole eluent 250mM can finally prepare protein with purity of more than 90%. Anti-HpaA nanobodies were obtained by the above method, a total of 6 strains. 2. Identification of expressed and purified nanobodies

[0112] The recombinant plasmid pET-25b-HpaA-Nbs was transformed into BL21 (DE3) competent cells, and the cells were collected and sonicated after IPTG induction, and purified by Ni-NTA. SDS-PAGE identification showed that the purified prokaryotic expression proteins HpaA-Nb8, HpaA-Nb9, HpaA-Nb10, HpaA-Nb11, HpaA-Nb12 and HpaA-Nb15 were consistent with the expected size (about 20 kDa) and had high purity (such as Fig. 9 , a), His tag WB identification after purification of anti-HpaA nanobody further verified the correct expression of the nanobody (e.g. Fig. 9 , b), the nucleotide sequences and amino acid sequences of the 6 nanobodies are shown in SEQ ID No. 2 to 13, respectively.

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

[0114] The 6 nanoantibodies purified by prokaryotic expression need to bind to HpaA recombinant protein and measure the EC of the binding 50 value to evaluate its binding activity.

[0115] 1) Dilute the recombinant HpaA protein with carbonate buffer at pH 9.6 to a final concentration of 5 μg / mL, add 100 μL / well to a 96-well ELISA plate, set up a PBS control well, and coat overnight at 4°C.

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

[0117] 3) Discard the blocking solution, wash with PBST 5 times, add 100 μL of nanoantibodies diluted twice into each well, dilute each antibody into 16 concentration gradients, and incubate at 37°C for 1 h.

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

[0119] 5) Wash with PBST for 5 times, incubate with 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 5 times with PBST, add 100 μL TMB color development solution to each well, and incubate at 37°C in the dark for 15 min.

[0121] 7) Termination: Add 100 μL 2M H 2 SO 4 Stop the color development reaction and read the OD450 value.

[0122] The results showed that the six nanoantibodies strongly bound to the HpaA recombinant antigen protein. The EC values ​​of the six nanoantibodies were calculated by graphpad software. 50 They are: 0.002694μM, 0.03004μM, 0.003379μM, 0.01380μM, 0.009783μM, 0.01027μM (such as Fig.10 ).

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

[0124] The eukaryotic expression vector pCMV-N1-HRP also has two restriction endonuclease cleavage sites, BamH I and HindIII. Therefore, the vector pCMV-N1-HRP can be double-digested, and the target gene VHH sequence fragment and the digested vector can be connected to obtain the pCMV-N1-HpaA-Nbs-HRP plasmid, which is then transformed into DH5α competent cells. After that, the positive clone monoclonal strain is picked, and the bacterial liquid PCR method is used to identify whether the target gene is successfully inserted into the vector (such as Fig.11 ). After amplifying the eukaryotic expression recombinant plasmid, the eukaryotic expression plasmid was extracted using the Vinozan endotoxin-free plasmid extraction kit for use in transfection.

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

[0126] Expression and 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 hours to characterize and identify HpaA-Nbs-HRP. WB identified the His tag, and ELISA detected the affinity of recombinant Nbs-HRP to HpaA (such as Fig.12 ). Fig.12 It can be seen that: WB identification of the His tag of Nbs-HRP showed that the experimental group had a band of about 75kDa and the position of the target band was consistent with that of the control nanoantibody group, and the empty pCMV-N1-HRP was not expressed, indicating that Nbs-HRP was successfully expressed after 72 hours of transfection. ELISA detection of binding activity showed that compared with the BSA control group, the purified Nbs-HRP had a strong binding activity with the HpaA recombinant protein.

[0127] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. An anti-Helicobacter pylori adhesin protein A nanobody, 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 anti-Helicobacter pylori adhesin protein A nanobody according to claim 1 and the amino acid sequence of horseradish peroxidase.

3. The nucleic acid sequence encoding the anti-Helicobacter pylori adhesin protein A nanobody according to claim 1, characterized in that: The nucleotide sequence thereof is shown as 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 method for preparing the anti-Helicobacter pylori adhesin protein A nanobody according to claim 1, characterized in that: The main steps include: (1) Optimizing and synthesizing the DNA sequence encoding HpaA, constructing a prokaryotic expression vector, and extracting the recombinant HpaA antigen protein after expressing it in Escherichia coli; after immunizing camels with the recombinant HpaA antigen protein, collecting peripheral blood from the camels for separating lymphocytes, and then detecting the antigen-specific response level of the camels after immunization with ELISA; (2) Extracting camel peripheral blood lymphocytes, extracting total RNA and reversely transcribing it to obtain cDNA, using the cDNA as a template to perform PCR reaction to amplify the VHH region of the antibody, connecting the amplified product to a phage display vector, and electrotransforming it into Escherichia coli to establish a phage display library, screening the desired positive clones through antigen incubation and ELISA identification, and then sequencing them to obtain the sequence of the HpaA nanobody; (3) The sequence of the HpaA nanobody is constructed into a prokaryotic or eukaryotic expression vector, and large-scale expression and purification are performed in Escherichia coli, yeast or mammalian cell lines to obtain an anti-Helicobacter pylori adhesin protein A nanobody.

5. The method according to claim 4, characterized in that: In step (1), the DNA sequence encoding HpaA is shown as SEQ ID No.

1.

6. The method according to claim 4, characterized in that: In step (2), the phage display vector is pMECS.

7. The method according to claim 4, characterized in that: In step (3), the prokaryotic expression vector is pET-25b(+), and the Escherichia coli is BL21(DE3).

8. The method according to claim 4, characterized in that: In step (3), the prokaryotic expression vector is pCMV-N1-HRP, and the mammalian cell line is 293T cells.

9. A product for detecting Helicobacter pylori, characterized in that: It comprises the anti-Helicobacter pylori adhesin protein A nanobody according to claim 1 or the fusion protein according to claim 2.

10. Use of the anti-Helicobacter pylori adhesin protein A nanobody according to claim 1 or the fusion protein according to claim 2 in the preparation of a product for detecting Helicobacter pylori.

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