Camel source single-domain antibody GA4 for recognizing linear B cell epitope in clostridium difficile glutamate dehydrogenase and application of camel source single-domain antibody GA4

Abstract

The invention discloses a camel source single-domain antibody GA4 for recognizing linear B cell epitope in clostridium difficile glutamate dehydrogenase and application of the camel source single-domain antibody GA4. The nucleotide sequence of the camel source single-domain antibody GA4 is shown as SEQ ID NO: 1, and the amino acid sequence of the camel source single-domain antibody GA4 is shown as SEQ ID NO: 2. The camel source single-domain antibody GA4 has the advantages of high specificity, high sensitivity, good accuracy and the like, and it is proved that the camel source single-domain antibody GA4 can be used for detecting recombinant expressed CD-GDH and endogenous CD-GDH through enzyme-linked immunosorbent assay (ELISA), western blot hybridization (WB) and other methods.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a camel-derived single-domain antibody GA4 for recognizing a linear B cell antigen epitope in glutamate dehydrogenase (GDH) of Clostridium difficile (CD) and an application thereof. Background Art

[0002] Clostridium / Clostridioides difficile (CD) glutamate dehydrogenase (GDH) is a stable and highly expressed metabolic enzyme in Clostridium difficile cells. It unidirectionally catalyzes the conversion of L-glutamate into ketoglutarate. The enzyme protein is present in toxin-producing strains, attenuated strains, and non-toxin-producing strains, and is highly conserved in all types of CD. GDH is present in fecal samples of CD-infected patients, and has also been proven to exist in the cytoplasm of CD cultured in vitro in the form of homotrimers and is secreted in the supernatant of the culture fluid. Therefore, GDH has become the preferred target for rapid detection of enzyme immunoassays as a specific protein antigen of Clostridium difficile. Immunological rapid detection of GDH is currently used as a preliminary screening in clinical practice, and positive specimens are further tested for toxins or toxin genes of Clostridium difficile. However, the immunological rapid detection kits for Clostridium difficile GDH currently available on the market are all produced by foreign companies, and the antibodies targeting GDH used are also traditional antibodies such as classic mouse monoclonal antibodies.

[0003] There is a naturally missing light chain in camelids and other animals, which is composed only of heavy chain homodimers and contains only one heavy chain variable region domain VHH. Cloning and recombinant expression of this heavy chain variable region can prepare single domain antibody (SdAb), which is the smallest unit that can bind to the target antigen currently available. Because its relative molecular mass is about 1 / 10 of that of conventional antibodies, and its crystal size is 2.5nm wide and 4nm long, it is called nanobody. Compared with traditional IgG conventional antibodies, the above single domain / nanobody has many advantages such as high antigen binding ability, high tolerance, high stability, high expression and short production cycle, and can be widely used in diagnosis and treatment. The screening of single domain / nanobody includes antibody library construction and in vitro display, one of which is an efficient and rapid method for phage display of camel-derived natural nanoantibody library, from which target antigens such as recombinant CD-GDH can be used as bait to screen positive phage clones, and then the determined antibody gene sequence is recombinantly expressed and purified to prepare the corresponding nanoantibody. Summary of the invention

[0004] The purpose of the present invention is to address the shortcomings of the prior art and the lack of nanoantibodies targeting Clostridium difficile glutamate dehydrogenase (CD-GDH) in the current domestic and international markets, and to provide a camel-derived single-domain antibody GA4 that recognizes linear B cell antigen epitopes in CD-GDH, and its application in the detection of CD-GDH.

[0005] In order to achieve the above-mentioned object, the technical solution of the present invention is: to provide a camel-derived single domain antibody that recognizes the linear B cell antigen epitope in Clostridium difficile glutamate dehydrogenase, and the amino acid sequence of the camel-derived single domain antibody GA4 is shown in SEQ ID NO:2.

[0006] The present invention also provides a gene encoding the camel-derived single-domain antibody as described above.

[0007] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0008] The present invention also provides a vector comprising the above gene.

[0009] The present invention also provides a use of the camel-derived single-domain antibody in preparing a kit for detecting Clostridium difficile glutamate dehydrogenase (CD-GDH).

[0010] Furthermore, in the application, the detection methods include enzyme-linked immunosorbent assay (ELISA) and protein immunoblotting (WB).

[0011] The present invention also provides a kit for detecting Clostridium difficile glutamate dehydrogenase (CD-GDH), comprising the camel-derived single-domain antibody GA4.

[0012] Preferably, the kit is an ELISA detection kit or an immunochromatographic diagnostic kit using immunochromatographic test strips.

[0013] The beneficial effects of the present invention are as follows:

[0014] The camel-derived single-domain antibody GA4 of the present invention has strong specificity, high sensitivity and good accuracy because it recognizes the linear B cell antigen epitope in Clostridium difficile glutamate dehydrogenase (CD-GDH). It can be used in the kit of a fully automatic immunoassay analyzer and can also be applied to immunochromatographic test strips to detect Clostridium difficile glutamate dehydrogenase (CD-GDH), which is beneficial to large-scale Clostridium difficile detection and epidemiological surveys. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the SDS-PAGE electrophoresis diagram of the recombinantly expressed and purified CD-GDH in Example 1.

[0016] Figure 2This is the result of SDS-PAGE analysis of the recombinant expression and purification of camel-derived single-domain antibody GA4 in Example 3.

[0017] Figure 3 The camel-derived single-domain antibody GA4 in Example 4 was used for ELISA to detect recombinantly expressed CD-GDH.

[0018] Figure 4 This is a schematic diagram of the ELISA assay for binding of camel-derived single-domain antibody GA4 to recombinant CD-GDH protein in Example 4.

[0019] Figure 5 This is the result of using camel-derived single-domain antibody GA4 in Example 5 to detect recombinant and endogenous CD-GDH by WB. DETAILED DESCRIPTION

[0020] The present invention develops and obtains a camel-derived single-domain antibody GA4 that recognizes the linear B cell antigen epitope of Clostridium difficile glutamate dehydrogenase (CD-GDH).

[0021] The following are preferred embodiments of the present invention. The embodiments are used to explain the present invention, not to limit the present invention. Within the spirit of the present invention and the protection scope of the claims, any modifications and changes made by ordinary technicians in the technical field on the basis of the present invention fall within the protection scope of the present invention. The experimental methods without specific conditions in the examples of the present invention are usually based on conventional conditions; unless otherwise specified, the reagents and consumables used are commercially available products.

[0022] Example 1: Recombinant expression and purification of CD-GDH

[0023] According to the protein sequence of CD-GDH in GenBank (P27346), the DNA fragment was artificially synthesized after codon optimization, the N-terminal Nco I restriction site (including the start codon) was introduced, and the C-terminal His tag, stop codon and Xho I restriction site were introduced in sequence, and cloned into the Nco I-Xho I site of the Escherichia coli expression plasmid pET-28a (+) after double restriction digestion. The above recombinant plasmids were transformed into Escherichia coli HB101 competent cells, positive clones were screened and recombinant plasmids were extracted, and restriction endonuclease digestion identification and nucleotide sequence determination of the inserted fragment were performed to confirm that they were correct.

[0024] The correctly identified recombinant plasmid was transformed into competent cells of Escherichia coli BL21 (DE3), inoculated into LB plates containing kanamycin (5 mg / L), and cultured at 37 degrees Celsius overnight. The monoclonal strain was picked and added to LB liquid medium containing kanamycin (5 mg / L), cultured at 37°C and 200rpm until OD600 reached about 0.6, and IPTG with a final concentration of 1mM was added to induce the expression of the recombinant protein. After culturing at 37°C and 200rpm for 3h, the Escherichia coli cells were collected by low-temperature centrifugation, and 10 times the volume of PBS containing lysozyme and protease inhibitors was added, and the cells were resuspended and ultrasonically broken. After low-temperature centrifugation at 11000rpm for 15mins, the supernatant and precipitate were collected, and the expression form of the recombinant CD-GDH was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the recombinant CD-GDH ( Figure 1 ), the molecular weight was consistent with the expected value (46.8 kD) and the protein purity was >95%.

[0025] Example 2: Screening of camel-derived single-domain antibody phage-positive clones recognizing CD-GDH

[0026] Selection: 1 mL of purified recombinant CD-GDH at a concentration of 40 μg / mL was used to coat the immunotubes. After overnight at 4°C, the immunotubes were washed 3 times with 5 mL of PBST, blocked with 5 mL of 5% milk / PBST at 30°C for 1 h, and washed once with 5 mL of PBS. 12 pfu, add 50μg / mL NC-His protein and 1% skim milk / PBST, add PBS to a total volume of 1mL, incubate at room temperature for 0.5h for negative screening, then add to an immunotube coated with CD-GDH, wash 4 times with 5mL PBST, add 1mL of Gly-HCl (pH=2.2) to the tube to elute the phage, incubate at room temperature with shaking for about 6-8min, add Tris-HCl (pH=9.6) to neutralize the solution to pH=7.0-8.0, dilute the eluted phage, infect Escherichia coli TG1 in the logarithmic phase for amplification, and spread the plate to determine the titer. After harvesting the amplified phage, add it to an immunotube coated with CD-GDH, wash, elute, neutralize, dilute and infect amplify as above, and repeat 4 rounds. At the same time, the phage harvested in each round was verified by polyclonal ELISA.

[0027] Polyclonal ELISA test verification: The purified recombinant CD-GDH was coated on the immunoplate at a concentration of 4 μg / mL, 100 μL per well, overnight at 4°C. At the same time, 4 μg / mL NC-His protein and PBS were used as negative controls to coat the immunoplate. After washing 3 times with 300 μL PBST, 300 μL of 5% skim milk was added and blocked at 37°C for 2 hours. The phages after each round of amplification were diluted with PBS, with the dilution multiple increasing by 3 times, and the initial concentration was 1x10 12 pfu / ml. Add 100 μL of phage solution to each well, incubate at 32°C for 1 hour, and then wash 3 times with 300 μL PBST. Add 100 μL of HRP-labeled anti-phage M13 antibody dilution to each well, incubate at 30°C for 1 hour, and then wash 3 times with 300 μL PBST. Add 100 μL of color development solution TMB to develop color for 5 minutes in the dark, add 50 μL 2M HCl to terminate the reaction, and measure the OD value (450 nm) with an enzyme reader.

[0028] Monoclonal ELISA test verification: Select the phages eluted in the fourth round, dilute them to the appropriate concentration, infect Escherichia coli TG1 in the logarithmic phase, and culture them overnight. The next day, pick 192 monoclones from the plate, inoculate them into 96 deep-well plates, shake and culture them at 37°C and 250rpm until the bacterial solution OD = 0.4-0.6, add the helper phage, let it stand at 37°C for 30min, and then shake and culture it at 37°C and 250rpm for 1h. Centrifuge the 96 deep-well plate at 4000rpm for 5min, discard the supernatant, resuspend the bacterial solution in 2YT-Amp-Kan medium in each well, and shake and culture it at 30°C and 250rpm overnight. The next day, centrifuge the 96 deep-well plate at 4000rpm for 10-15min, and take the supernatant phage solution for ELISA experiment. The immunoplate is coated with two groups of 4μg / mL purified recombinant CD-GDH and control NC-His proteins, 100μL per well, and other experimental conditions are the same as above. Finally, 6 positive phage clones were obtained.

[0029] WB: Take 100ng of purified recombinant CD-GDH for SDS-PAGE, then transfer to PVDF membrane by electrophoresis, wash the membrane with TBST once, block it with 5% milk / TBST at room temperature for 1h, and rinse it with TBST for 2min. Block the membrane with a hybridization bag, add the phage supernatant (i.e., primary antibody) diluted with 1% BSA / PBST, and store it in a refrigerator at 4°C overnight. Wash the membrane with PBST 3 times, 10min each time. Then put it in HRP-labeled anti-phage M13 antibody (i.e., secondary antibody) diluted with 5% milk / PBST, and incubate it on a shaker at room temperature for 1h. Wash the membrane with PBST 3 times, 10min each time. Add the luminescent reagent, develop the color for 2min, and observe the results with a gel imaging system. Five of the six positive phage clones obtained by ELISA screening in the previous step were positive for WB reaction, indicating that the antigen epitopes they each recognized were linear B cell antigen epitopes.

[0030] Sequencing: The phage clones with positive results in monoclonal ELISA were subjected to nucleotide sequence determination, and the genes of positive clones VHH were determined by comparison analysis. After comprehensive consideration, the present invention selected one of the clones with positive WB reaction, GA4, for further recombinant expression and purification and verified it as a primary antibody for ELISA and WB detection of CD-GDH.

[0031] Example 3: Recombinant expression and purification of camel-derived single-domain antibody GA4 in Escherichia coli

[0032] Construction of prokaryotic expression plasmid of camel-derived single-domain antibody GA4: According to the above sequencing results, the DNA fragment was artificially synthesized after codon optimization, the Nco I restriction site (including the start codon) was introduced at the N-terminus, and the His tag, Myc tag, stop codon and Xho I restriction site were introduced at the C-terminus in sequence, and cloned into the E. coli expression plasmid pET-28a (+) after double enzyme digestion. The above recombinant plasmids were transformed into E. coli HB101 competent cells, positive clones were screened and recombinant plasmids were extracted, and restriction endonuclease digestion identification and nucleotide sequence determination of the inserted fragment were performed to confirm that they were correct.

[0033] Recombinant expression, purification and identification of camel-derived single-domain antibody GA4: The correctly identified recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, inoculated onto a plate containing kanamycin (5 mg / L), and cultured overnight at 37 degrees Celsius. Pick a single colony for liquid culture, culture at 37°C at 200rpm until OD600 reaches about 0.6, add IPTG with a final concentration of 1mM to induce the expression of the recombinant protein, and collect the Escherichia coli cells by low-temperature centrifugation at 37°C at 200rpm for 3h. Add 10 times the volume of PBS containing lysozyme and protease inhibitors, resuspend and ultrasonically disrupt the bacteria, collect the supernatant and precipitate after low-temperature centrifugation at 11000rpm for 15mins, and analyze the expression form of the recombinant single-domain antibody by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, nickel column affinity purification was used to purify the recombinant nanoantibody GA4 from the lysate supernatant mixture. The results of SDS-PAGE electrophoresis are as follows: Figure 2 As shown, the molecular weight of GA4 was consistent with the expected value (15.3 kD).

[0034] Example 4: Cameloid single domain antibody GA4 is used for indirect enzyme-linked immunosorbent assay (ELISA) to detect recombinantly expressed CD-GDH

[0035] Recombinant CD-GDH protein was coated as capture antigen in 96-well plates at 10 μg / mL and 100 μL per well, respectively, and incubated at 4°C overnight. After washing the plate 3 times with PBST, 1% BSA was added, 200 μL per well, and blocked at 37°C for 1 hour. After washing the plate 3 times with PBST, a purified camel-derived single-domain antibody GA4 with an appropriate dilution was added as the primary antibody and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, a 1:500 diluted HRP-labeled mouse anti-myc monoclonal antibody was added as the secondary antibody and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, TMB colorimetric substrate was added at 100 μL / well for 15 minutes, and 50 μL of TMB colorimetric stop solution was added to stop the reaction, and the OD value of each well was read at 450 nm using an enzyme reader. The results showed that the OD value of the wells coated with recombinant CD-GDH protein (0.815) was significantly higher than the OD value of the wells coated with control PBS (0.061), which was 2.1 times higher than that of the wells coated with control PBS (0.061). This indicated that the recombinant camel-derived single-domain antibody GA4 could be used for indirect enzyme-linked immunosorbent assay (ELISA) to specifically detect recombinantly expressed CD-GDH ( Figure 3 ).

[0036] ELISA was used to detect the affinity of GA4 binding to recombinant CD-GDH protein: the initial concentration of GA4 was 2 mg / mL, and it was diluted 10 times and adjusted to a uniform volume of 100 μL before being added to each well as the primary antibody. The experiment was repeated 3 times for each dilution. The results showed that GA4 had a good affinity for binding to CD-GDH, and the EC50 value was 3.44±0.20 nM ( Figure 4).

[0037] Example 5: Cameloid single domain antibody GA4 was used for Western blotting (WB) to detect recombinant and endogenous CD-GDH

[0038] The cultures of Clostridium difficile 1870 and Escherichia coli BL21 (DE3) were centrifuged, the supernatant was discarded, the precipitate was resuspended in PBS buffer containing lysozyme and protease inhibitors, the bacteria were broken by ultrasound and centrifuged at low temperature, and the supernatant was taken to determine the protein concentration. Then 2μg of recombinant CD-GDH, 10μg of supernatant of Clostridium difficile lysate and 10μg of supernatant of Escherichia coli lysate were loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation, and then transferred to PVDF membrane and blocked with 5% skim milk powder on a shaker for 2h. Cameloid single-domain antibody GA4 (1:1000 dilution) was used as the primary antibody, incubated at 4°C overnight, washed with TBST 3 times, 10 minutes each time, and incubated with HRP-labeled anti-myc tag mouse monoclonal antibody (1:10000 dilution) as the secondary antibody for 2 hours, washed with TBST 3 times, 10 minutes each time; added the substrate of FDbio-Femto ECL Kit, and observed the results and took pictures using BIORAD chemiluminescence imager. The results showed that only the recombinant CD-GDH protein and the cleavage product of Clostridium difficile 1870 strain showed positive signal bands at the corresponding expected molecular weight (46.8kD), while the cleavage product of Escherichia coli BL21 (DE3) strain as the control had no positive signal bands, indicating that GA4 can specifically target and recognize CD-GDH protein ( Figure 5 ).

Claims

1. A camel-derived single domain antibody that recognizes a linear B cell antigen epitope in glutamate dehydrogenase of Clostridium difficile, characterized in that: The amino acid sequence of the camel-derived single domain antibody is shown in SEQ ID NO:

2.

2. A gene encoding the camel-derived single-domain antibody as claimed in claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

4. A carrier, characterized in that Comprising the gene according to claim 2 or 3.

5. Use of the camel-derived single-domain antibody as claimed in claim 1 in the preparation of a kit for detecting glutamate dehydrogenase of Clostridium difficile.

6. The use according to claim 5, characterized in that Detection methods include enzyme-linked immunosorbent assay and protein immunoblotting hybridization.

7. A kit for detecting Clostridium difficile glutamate dehydrogenase, characterized in that: Comprising the camel-derived single-domain antibody according to claim 1.

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