VHH antibody capable of being combined with helicobacter pylori

Abstract

The invention discloses a VHH antibody capable of being combined with helicobacter pylori, and relates to the technical field of VHH.A target gene is inserted into a pPICZalpha A carrier through gene synthesis, a linearized recombinant plasmid is obtained through enzyme digestion and purification technologies, the plasmid is converted into an X33 yeast competent state, and the VHH antibody capable of being combined with helicobacter pylori is obtained. Then obtaining a stable helicobacter pylori VHH antibody through positive clone screening, inducible expression and protein identification; according to the invention, the stability of the helicobacter pylori antibody can be improved, the production process of the helicobacter pylori antibody is optimized, and the more stable helicobacter pylori VHH antibody can be obtained; the method is simple, effective, easy and practical.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibodies, and in particular relates to a VHH antibody capable of binding to Helicobacter pylori. Background Art

[0002] The preparation of Helicobacter pylori antibodies requires bacterial culture, antigen extraction and purification to obtain high-purity antigens, and then select animals for immunization, followed by antibody collection and purification. The overall process is cumbersome and complex, with a long cycle and high cost;

[0003] In order to obtain a VHH antibody capable of binding to Helicobacter pylori adhesin BabA through secretory expression of Pichia pastoris protein, so that the antibody preparation has the characteristics of simple operation, short cycle and low cost, a technical solution is now provided. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art; to this end, the present invention provides a VHH antibody capable of binding to Helicobacter pylori.

[0005] A VHH antibody capable of binding to Helicobacter pylori, comprising: a sequence of SEQ ID NO: 1.

[0006] Compared with the prior art, the present invention has the following beneficial effects:

[0007] The present invention inserts the target gene into the pPICZalphaA vector by gene synthesis, obtains a linearized recombinant plasmid by enzyme digestion and purification technology, transforms the plasmid into X33 yeast competent cells, and then obtains a stable Helicobacter pylori VHH antibody by positive clone screening, induced expression and protein identification;

[0008] The present invention can improve the stability of Helicobacter pylori antibodies, optimize the Helicobacter pylori antibody production process, and obtain more stable Helicobacter pylori VHH antibodies; the present invention is simple, effective, and easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a schematic diagram of the pPI CZaA-rFC-188 recombinant plasmid of the present invention;

[0010] Figure 2 This is a schematic diagram of the linearization of the recombinant plasmid pPI CZaA-rFC-188 of the present invention;

[0011] Figure 3 This is a schematic diagram of the purification and recovery of the pPI CZaA-rFC-188 plasmid of the present invention;

[0012] Figure 4 This is a schematic diagram of colony PCR of the present invention;

[0013] Figure 5 This is a schematic diagram of WB identification of the present invention. DETAILED DESCRIPTION

[0014] The technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] See also Figure 1 The present application provides a VHH antibody capable of binding to Helicobacter pylori, comprising:

[0016] SEQ I ND:1 sequence;

[0017] Specifically prepared by the following method:

[0018] Gene synthesis recombinant plasmid;

[0019] The recombinant plasmid vector is linearized by:

[0020] Linearize the pPI CZaA-rFC-188 plasmid with Sac I endonuclease

[0021] 1) Prepare the enzyme digestion system according to the following table:

[0022]

[0023]

[0024] 2) Enzyme digestion at 37°C overnight;

[0025] 3) Agarose gel electrophoresis was used to detect the undigested pPI CZaA-rFC-188 plasmid as a control. The detection results were as follows: Figure 2 As shown;

[0026] Linearized vector purification and recovery;

[0027] 1) Configure the purification system according to the following table:

[0028]

[0029] 2) Leave at -20℃ for more than 2 hours;

[0030] 3) Centrifuge at 12000 rpm for 15 min at 4°C and discard the supernatant. A white precipitate can be observed on the wall.

[0031] 4) Add 1 mL of pre-cooled 75% ethanol to resuspend the precipitate;

[0032] 5) Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, and dry the tube;

[0033] 6) Add 10 μl ddH2O to dissolve the precipitate;

[0034] 7) Agarose gel electrophoresis was used to detect the purification results, and the undigested plasmid was used as a control. The detection results were as follows: Figure 3 As shown;

[0035] pP I CZaA-rFC-188 plasmid was electroporated into yeast;

[0036] 1) Take 80u lX33 competent yeast, add all the purified pPICZaA-rFC-188 plasmid, mix well and transfer to a pre-cooled 0.2cm electroporation cuvette;

[0037] 2) Place on ice for 5 minutes;

[0038] 3) Electric shock according to the yeast electric shock parameter settings (1.5kV, 25uF, 200Ω);

[0039] 4) Immediately add 1 ml of pre-cooled 1 M sorbitol and transfer to a 2 ml sterile centrifuge tube;

[0040] 5) Incubate at 30°C for 1-2 hours;

[0041] 6) After diluting 10 times, spread 300 μl of the bacterial solution on a 15 cm YPDA plate containing Zeocin and culture at 30°C until a single colony grows.

[0042] Transformant screening;

[0043] 8 clones were randomly selected from the plate and subjected to colony PCR. The gel test results were as follows: 8 clones were randomly selected from the plate and stored on the corresponding plate, and colony PCR was performed. At the same time, colony PCR was also performed on the original plasmid. After the PCR was completed, 2 μl was taken from each of them for agarose electrophoresis detection. The results were as follows: Figure 4 , the bands of the five clones were all of the correct size.

[0044] Transformants were induced to express.

[0045] Monoclonal strains 4-6 were selected for expression experiments.

[0046] 1) Prepare three 50 ml conical flasks, add 5 ml YPG medium to each, inoculate monoclonal strains 1, 2, and 3 respectively, and culture at 30°C and 220 rpm for 1-2 days until the bacterial solution is saturated;

[0047] 2) Transfer to a 50 ml centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant;

[0048] 3) Resuspend the cells in 5 ml BMMY medium, transfer to a new sterile 50 ml conical flask, add methanol to a final concentration of 0.75%, and culture at 28°C and 220 rpm for 6 days;

[0049] 4) Methanol was added every 24 h (final concentration was 0.75%);

[0050] 5) On the morning of the 6th day, the bacterial culture was collected, centrifuged at 4000 rpm for 5 min, and the supernatant was collected;

[0051] 7. WB identification

[0052] 1) Take 20ul protein solution, add appropriate amount of 4ul 5× protein insertion buffer, and boil at 100℃ for 5-10min;

[0053] 2) Pipette 10 μl for protein electrophoresis;

[0054] 3) After transfer, wash the PVDF membrane with TBST once for 10 min and discard the TBST;

[0055] 4) Block with TBST containing 5% skim milk powder for 1 h at room temperature on a shaker at 50 rpm;

[0056] 5) Wash the membrane 3 times with TBST, 10 min each time;

[0057] 6) Dilute the secondary antibody with a blocking solution containing 5% skim milk powder and hybridize the membrane on a low-speed shaker at room temperature for 45 minutes;

[0058] 7) Wash the membrane three times with TBST, 10 min each time;

[0059] 8) After ECL exposure, the membrane was scanned. The results showed that all three clones were successfully expressed.

[0060] The above embodiments are only used to illustrate the technical method of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical method of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical method of the present invention.

Claims

1. A VHH antibody capable of binding to Helicobacter pylori, characterized in that: include: Sequence SEQ ID NO:

1.

2. A method for preparing a VHH antibody capable of binding to Helicobacter pylori, characterized in that: The method is used to produce the antibody as claimed in claim 1, and the specific method is: Gene synthesis recombinant plasmid, recombinant plasmid vector linearization; Linearized vector purification and recovery; pPICZaA-rFC-188 plasmid was electroporated into yeast; Transformant screening; Transformants induce expression; Western Blot identification.

3. The method for preparing a VHH antibody capable of binding to Helicobacter pylori according to claim 2, characterized in that: The specific method of linearizing the recombinant plasmid vector is: The pPICZaA-rFC-188 plasmid was linearized with SacI endonuclease; Prepare the enzyme digestion system according to the plasmid, 10× buffer, SacI, and ddH20 in the following order: 6ug, 5ul, 1ul, and make up to 50ul volume; Enzyme digestion at 37°C overnight; Agarose gel electrophoresis was used to detect the expression of pPICZaA-rFC-188 plasmid without enzyme digestion as a control.

4. The method for preparing a VHH antibody capable of binding to Helicobacter pylori according to claim 2, characterized in that: The specific method for purification and recovery of linearized vector is as follows: According to the enzyme digestion product, nucleic acid precipitation agent, 3M NaAc, pH=5.2, and anhydrous ethanol, the purification system was configured in volumes of 50ul, 10ul, 6ul, and 165ul in sequence; -20℃ for more than 2h; Centrifuge at 4℃12000rpm for 15min, discard the supernatant, and a white precipitate can be observed on the wall; Add 1 mL of pre-cooled 75% ethanol to resuspend the pellet; Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, and dry the mixture with the lid open; Add 10ul ddH2O to dissolve the precipitate; The purification results were checked by agarose gel electrophoresis.

5. The method for preparing a VHH antibody capable of binding to Helicobacter pylori according to claim 2, characterized in that: The pPICZaA-rFC-188 plasmid was electroporated into yeast in the following way: 1) Take 80ul of X33 competent yeast, add all the purified pPICZaA-rFC-188 plasmid, mix well and transfer to a pre-cooled 0.2cm electroporation cuvette; 2) Place on ice for 5 minutes; 3) Electric shock according to the yeast electric shock parameter settings (1.5kV, 25uF, 200Ω); 4) Immediately add 1 ml of pre-cooled 1 M sorbitol and transfer to a 2 ml sterile centrifuge tube; 5) Incubate at 30°C for 1-2 hours; 6) After diluting 10 times, spread 300ul of the bacterial solution on a 15cm YPDA plate containing Zeocin and culture at 30℃ until a single colony grows.

6. The method for preparing a VHH antibody capable of binding to Helicobacter pylori according to claim 2, characterized in that: The specific method for transformant screening is: 8 clones were randomly selected from the plate and subjected to colony PCR. The gel test results were as follows: 8 clones were randomly selected from the plate and saved on the corresponding plate, and colony PCR was performed. At the same time, colony PCR was also performed on the original plasmid. After the PCR was completed, 2ul was taken from each of them for agarose electrophoresis detection.

7. The method for preparing a VHH antibody capable of binding to Helicobacter pylori according to claim 2, characterized in that: The specific method of transformant induction is as follows: Monoclonal strains 4-6 were selected for small-scale expression tests; Prepare three 50 ml conical flasks, add 5 ml YPG medium to each, inoculate monoclonal strains 1, 2, and 3 respectively, and culture at 30°C and 220 rpm for 1-2 days until the bacterial solution is saturated; Transfer to a 50 ml centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant; Resuspend the cells in 5 ml BMMY medium, transfer to a new sterile 50 ml conical flask, add methanol to a final concentration of 0.75%, and culture at 28°C and 220 rpm for 6 days; Methanol was added every 24 h (final concentration 0.75%); On the morning of the 6th day, the bacterial culture was collected, centrifuged at 4000 rpm for 5 min, and the supernatant was collected.

8. The method for preparing a VHH antibody capable of binding to Helicobacter pylori according to claim 2, characterized in that: The specific method of WB identification is: Take 20ul protein solution, add appropriate amount of 4ul 5× protein loading buffer, and boil at 100℃ for 5-10min; Pipette 10ul for protein electrophoresis; After transfer, wash the PVDF membrane once with TBST for 10 min, and discard the TBST; Block with TBST containing 5% skim milk powder for 1 h at room temperature on a shaker at 50 rpm; Wash the membrane three times with TBST, 10 min each time; The secondary antibody was diluted with blocking solution containing 5% skim milk powder, and the membrane was hybridized on a low-speed shaker at room temperature for 45 min; Wash the membrane three times with TBST, 10 min each time; After ECL exposure, the film is scanned on the machine.

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