Camel-derived single-domain antibody GA4 that recognizes a linear B cell antigen epitope in Clostridium difficile glutamate dehydrogenase and its application

Abstract

The present invention discloses a camel-derived single-domain antibody GA4 that recognizes a linear B-cell antigen epitope in Clostridium difficile glutamate dehydrogenase and its application. The nucleotide sequence of the camel-derived single-domain antibody GA4 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The camel-derived single-domain antibody GA4 of the present invention has the advantages of strong specificity, high sensitivity, and good accuracy. It has been demonstrated that it can be used for detection of recombinantly expressed CD-GDH and endogenous CD-GDH by methods such as enzyme-linked immunosorbent assay (ELISA) and western blot hybridization (WB).

Description

Technical Field

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

[0002] Clostridium difficile (CD) glutamate dehydrogenase (GDH) is a stable and highly expressed metabolic enzyme in C. difficile cells. It catalyzes the unidirectional conversion of L-glutamate to ketoglutarate. This enzyme is present in toxigenic, attenuated, and non-toxigenic strains and is highly conserved across all CD strains. GDH is present in fecal samples from CD-infected patients and has been shown to exist as a homotrimer in the cytoplasm of in vitro cultured CD and to be secreted into the culture supernatant. Therefore, GDH has become a specific protein antigen for C. difficile and a preferred target for rapid detection using enzyme immunoassays and other methods. Currently, rapid immunological tests for GDH are used in clinical practice as initial screening, with positive specimens further tested for C. difficile toxins or toxin genes. However, currently available rapid immunological tests for C. difficile GDH are all manufactured by foreign companies, and the antibodies they use are traditional antibodies, such as classic mouse monoclonal antibodies, that target GDH.

[0003] Camelids and other animals naturally lack light chains and are composed solely of heavy chain homodimers. These antibodies contain only one heavy chain variable region, VHH. Cloning and recombinant expression of this heavy chain variable region can produce single-domain antibodies (SdAbs), the smallest units currently available that can bind to target antigens. Because their molecular weight is approximately 1 / 10 that of conventional antibodies, and their crystal size is 2.5 nm wide and 4 nm long, they are called nanobodies. Compared to traditional IgG antibodies, these single-domain / nanobodies have multiple advantages, including high antigen binding capacity, high tolerance, high stability, high expression, and a short production cycle, making them widely used in diagnosis and treatment. Screening for single-domain / nanobodies involves antibody library construction and in vitro display. One efficient and rapid method is phage display of camel-derived natural nanoantibody libraries, from which positive phage clones can be screened using target antigens, such as recombinant CD-GDH, as bait. The determined antibody gene sequences are then recombinantly expressed and purified to produce the corresponding nanoantibodies. Summary of the Invention

[0004] The purpose of the present invention is to address the shortcomings of the existing technology and the current lack of nanoantibodies targeting Clostridium difficile glutamate dehydrogenase (CD-GDH) in the domestic and foreign 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 objectives, the technical solution of the present invention is: to provide a camel-derived single-domain antibody that recognizes a 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 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 instrument and can also be used in immunochromatographic test strips to detect Clostridium difficile glutamate dehydrogenase (CD-GDH), which is beneficial for 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 SDS-PAGE analysis of the recombinant expression and purification results of the 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 detection of recombinantly expressed CD-GDH.

[0018] Figure 4 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 the camel-derived single-domain antibody GA4 in Example 5 to detect recombinant and endogenous CD-GDH by WB. DETAILED DESCRIPTION

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

[0021] The following are preferred embodiments of the present invention. The examples are intended to illustrate the present invention rather than to limit the present invention. Within the spirit of the present invention and the scope of protection of the claims, any modifications and changes made by a person skilled in the art based on the present invention fall within the scope of protection of the present invention. Experimental methods in the examples of the present invention where specific conditions are not specified are generally based on conventional conditions; unless otherwise specified, all reagents and consumables used are commercially available products.

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

[0023] Based on the CD-GDH protein sequence from GenBank (P27346), a codon-optimized DNA fragment was synthesized. An Nco I restriction site (including the start codon) was introduced at the N-terminus, followed by a His tag, a stop codon, and an Xho I restriction site at the C-terminus. After double digestion, the fragment was cloned into the Nco I-Xho I sites of the E. coli expression plasmid pET-28a(+). The recombinant plasmids were transformed into competent E. coli HB101 cells, and positive clones were screened and isolated. Recombinant plasmids were then digested with restriction enzymes and the nucleotide sequence of the inserted fragment was determined for confirmation.

[0024] The correctly identified recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, 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 culture at 37°C and 200rpm for 3h, the E. 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 Cameloid Single Domain Antibody Phage-Positive Clones Recognizing CD-GDH

[0026] Panning: 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 three times with 5 mL of PBST, blocked with 5 mL of 5% milk / PBST at 30°C for 1 hour, and washed once with 5 mL of PBS. 12 pfu, 50 μg / mL NC-His protein and 1% skim milk / PBST were added, and the total volume was 1 mL with PBS. After incubation at room temperature for 0.5 h for negative screening, the cells were added to immunotubes coated with CD-GDH. The cells were washed four times with 5 mL of PBST. The phage were eluted with 1 mL of Gly-HCl (pH 2.2) and incubated at room temperature with shaking for approximately 6-8 min. Tris-HCl (pH 9.6) was added to neutralize the solution to pH 7.0-8.0. The eluted phage were diluted and amplified by infecting logarithmic-phase Escherichia coli TG1 cells. The titer was then plated and determined. The amplified phage were harvested and added to immunotubes coated with CD-GDH. The washing, elution, neutralization, dilution, and infection amplification procedures were repeated four times. The phage harvested in each round were validated by polyclonal ELISA.

[0027] Polyclonal ELISA assay validation: Purified recombinant CD-GDH was coated onto immunoplates at a concentration of 4 μg / mL, with 100 μL per well, overnight at 4°C. Simultaneously, 4 μg / mL NC-His protein and PBS were coated onto the immunoplates as negative controls. After washing three times with 300 μL PBST, 300 μL of 5% skim milk was added and the plates were blocked at 37°C for 2 hours. Phages were diluted with PBS after each round of amplification in 3-fold increments, with an initial concentration of 1x10 12 pfu / ml. Add 100 μL of phage solution to each well, incubate at 32°C for 1 hour, and wash three times with 300 μL of PBST. Add 100 μL of HRP-labeled anti-phage M13 antibody dilution to each well, incubate at 30°C for 1 hour, and wash three times with 300 μL of PBST. Add 100 μL of TMB colorimetric solution and develop for 5 minutes in the dark. Terminate the reaction by adding 50 μL of 2M HCl, and measure the OD value (450 nm) with a microplate reader.

[0028] Monoclonal ELISA validation: Phage eluted from the fourth round were selected, diluted to an appropriate concentration, and infected with logarithmic-phase Escherichia coli TG1 cells, plated, and cultured overnight. The next day, 192 monoclonal clones were selected from the plate and plated into 96-well plates. The culture was shaken at 37°C, 250 rpm, and the OD value was 0.4-0.6. After adding helper phage, the cells were incubated at 37°C for 30 minutes, followed by shaking at 37°C, 250 rpm, and incubated for 1 hour. The 96-well plates were centrifuged at 4000 rpm for 5 minutes, the supernatant discarded, and the cells resuspended in 2YT-Amp-Kan medium in each well. The cells were shaken at 30°C, 250 rpm, and cultured overnight at 30°C. The next day, the 96-well plates were centrifuged at 4000 rpm for 10-15 minutes, and the supernatant phage fluid was collected for ELISA. The immunoplates were coated with 100 μL per well of 4 μg / mL of purified recombinant CD-GDH and control NC-His proteins, respectively. All other experimental conditions were the same as above. Finally, 6 positive phage clones were obtained.

[0029] Western blot analysis: 100 ng of purified recombinant CD-GDH was subjected to SDS-PAGE and electrotransferred to a PVDF membrane. The membrane was washed once with TBST and blocked with 5% milk / TBST for 1 hour at room temperature. The membrane was then rinsed with TBST for 2 minutes. The membrane was blocked with a hybridization bag and incubated with the supernatant of a phage clone (primary antibody) identified as positive by monoclonal ELISA, diluted in 1% BSA / PBST, and refrigerated at 4°C overnight. The membrane was washed three times with PBST for 10 minutes each. The membrane was then incubated with HRP-conjugated anti-phage M13 antibody (secondary antibody) diluted in 5% milk / PBST and incubated on a shaker at room temperature for 1 hour. The membrane was washed three times with PBST for 10 minutes each. A luminescent reagent was added, and the color was developed for 2 minutes before visualization using a gel imaging system. Five of the six positive phage clones identified in the previous ELISA screening were positive by Western blot analysis, indicating that the epitope they recognized was a linear B cell epitope.

[0030] Sequencing: Nucleotide sequencing was performed on each of the phage clones that tested positive by ELISA. Comparative analysis confirmed the VHH gene of the positive clone. After comprehensive consideration, the present invention selected one of the WB-positive clones, GA4, for further recombinant expression and purification, and validated its use 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 a prokaryotic expression plasmid for the camel-derived single-domain antibody GA4: Based on the above sequencing results, a DNA fragment was synthesized after codon optimization. An Nco I restriction site (including the start codon) was introduced at the N-terminus, followed by a His tag, a Myc tag, a stop codon, and an Xho I restriction site at the C-terminus. After double enzyme digestion, the fragment was cloned into the E. coli expression plasmid pET-28a(+). The recombinant plasmids were transformed into E. coli HB101 competent cells, positive clones were screened, and the recombinant plasmids were extracted. Restriction enzyme digestion and nucleotide sequence analysis of the inserted fragment were performed to confirm the fragment's accuracy.

[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 and culture at 37°C and 200rpm until OD600 reaches about 0.6. Add IPTG with a final concentration of 1mM to induce the expression of the recombinant protein. Centrifuge at 37°C and 200rpm for 3 hours to collect the E. coli cells. Add 10 times the volume of PBS containing lysozyme and protease inhibitors, resuspend and ultrasonically break the bacteria. Centrifuge at 11000rpm for 15 minutes to collect the supernatant and precipitate. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the expression form of the recombinant single-domain antibody. Finally, nickel column affinity purification was used to purify the recombinant nanoantibody GA4 from the lysis 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: Application of camel-derived single-domain antibody GA4 in indirect enzyme-linked immunosorbent assay (ELISA) for detection of recombinantly expressed CD-GDH

[0035] Recombinant CD-GDH protein was coated as a capture antigen at 10 μg / mL in 96-well plates at 100 μL per well and incubated overnight at 4°C. The plates were washed three times with PBST, followed by addition of 1% BSA at 200 μL per well and blocking at 37°C for 1 hour. After washing the plates three times with PBST, an appropriately diluted purified camel-derived single-domain antibody, GA4, was added as the primary antibody and incubated at 37°C for 1 hour. After washing the plates five times with PBST, a 1:500 dilution of HRP-labeled mouse anti-myc monoclonal antibody was added as the secondary antibody and incubated at 37°C for 1 hour. After washing the plates five times with PBST, TMB chromogenic substrate was added at 100 μL / well and the reaction was continued for 15 minutes. The reaction was terminated by adding 50 μL of TMB chromogenic stop solution, and the OD value of each well was read at 450 nm using a microplate reader. The results showed that the OD value of the recombinant CD-GDH protein coated wells (0.815) was significantly higher than the OD value of the control PBS coated wells (0.061), which was 2.1 times higher than that of the control PBS coated wells. 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-fold 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, with an EC50 value of 3.44±0.20 nM ( Figure 4).

[0037] Example 5: Application of camelid single-domain antibody GA4 in Western blotting (WB) to detect recombinant and endogenous CD-GDH

[0038] Cultures of Clostridium difficile strain 1870 and Escherichia coli strain BL21(DE3) were centrifuged, the supernatant discarded, and the pellet resuspended in PBS buffer containing lysozyme and protease inhibitors. After sonication and cold centrifugation, the supernatant was collected for protein determination. 2 μg of recombinant CD-GDH, 10 μg of supernatant from C. difficile lysates, and 10 μg of supernatant from E. coli lysates were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The samples were then transferred to PVDF membranes and blocked with 5% skim milk powder for 2 hours on a shaker. Cameloid single-domain antibody GA4 (1:1000 dilution) was used as the primary antibody, incubated overnight at 4°C, washed 3 times with TBST, 10 minutes each time, and incubated for 2 hours with HRP-labeled anti-myc tag mouse monoclonal antibody (1:10000 dilution) as the secondary antibody. The membrane was washed 3 times with TBST, 10 minutes each time; the substrate of FDbio-Femto ECL Kit was added, and the results were observed and photographed using BIORAD chemiluminescence imager. The results showed that only the recombinant CD-GDH protein and the lysis product of Clostridium difficile 1870 strain showed positive signal bands at the corresponding expected molecular weight (46.8kD), while the lysis product of Escherichia coli BL21 (DE3) strain as the control showed 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 according to claim 1.

3. The gene according to claim 2, wherein 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 according to claim 1 in the preparation of a kit for detecting Clostridium difficile glutamate dehydrogenase.

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

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

Images