A single-domain antibody targeting pyroglutamate aβ and preparation method and application thereof

Abstract

The application relates to a pyroglutamate A beta-targeted single-domain antibody and a preparation method and application thereof, the amino acid sequence of the single-domain antibody is shown in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 5 or SEQ ID NO. 6 or SEQ ID NO. 7 or SEQ ID NO. 8; a striped banded dogfish is taken as an immunization object, PE-A beta is taken as an antigen for immunization, total RNA of peripheral blood cells of the immunized dogfish is extracted, reverse transcription is carried out to obtain cDNA, amplification is carried out by taking the cDNA as a template, a phagemid carrier is constructed by taking the amplification as a template, the phagemid carrier is transferred into a competent cell, the competent cell is cultured, positive clones are screened, and the single-domain antibody is obtained. The single-domain antibody provided by the application can specifically recognize PEA beta, has good binding activity, has a small molecular weight, solves the technical problem that no shark nanobody targeting PEA beta has been found at present, lays an important foundation for subsequent research on the application of the antibody in the detection of PEA beta, and has application prospects in the development of a reagent for detecting PEA beta or a drug for treating PEA beta-related diseases.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a single-domain antibody targeting pyroglutamate Aβ, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease affecting the elderly, clinically manifested as cognitive impairment, memory decline, and behavioral abnormalities, ultimately leading to the loss of independent living abilities and creating enormous healthcare burdens. With the increasing aging of the global population, the incidence of this disease remains high, making it one of the major killers threatening human health, requiring effective interventions.

[0003] Existing research indicates that modified pyroglutamate Aβ (pE-Aβ or pEAβ) possesses extremely strong hydrophobicity and neurotoxicity, and is a core factor in inducing senile plaque formation and neuronal death in the brain. Therefore, accurately identifying and neutralizing pE-Aβ to block its cascade of pathological reactions is currently a core strategy in the development of AD treatments. Shark single-domain antibodies (vNARs), with a molecular weight only one-tenth that of traditional antibodies, a compact structure, and stable physicochemical properties, possess excellent tissue penetration and blood-brain barrier crossing potential, and are considered a next-generation antibody drug development platform.

[0004] However, most of the antibodies currently used in clinical practice are full-length IgG antibodies, which have huge molecular size and often lack specificity for recognizing specific mutation sites of pE-Aβ, making it difficult to achieve precise clearance of pE-Aβ in complex physiological environments. Summary of the Invention

[0005] The purpose of this invention is to provide a single-domain antibody that can specifically recognize pE-Aβ.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes a single-domain antibody targeting pyroglutamate Aβ, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5 or SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO.8.

[0007] The present invention also includes the application of the single-domain antibody in the detection of pE-Aβ.

[0008] The present invention also includes a method for preparing the single-domain antibody, comprising the following steps: S1: Using the striped bamboo shark as the immunization target, PE-Aβ was used as the antigen for immunization. Total RNA was extracted from the peripheral blood cells of the immunized shark, reverse transcribed into cDNA, and amplified using cDNA as a template to obtain the shark-derived single-domain antibody vNAR fragment of the striped bamboo shark. S2: Construct a recombinant phage vector by combining a shark-derived single-domain antibody vNAR fragment with a phage vector, transform the recombinant phage vector into competent cells, and obtain a PEAβ phage antibody library after expansion culture. S3: Screen positive clones from the PEAβ phage antibody library to obtain the single-domain antibody.

[0009] Further, step S3 specifically involves: screening and enriching the PEAβ phage antibody library three times, constructing phage antibodies with helper phages, and screening for positive clones; The three-stage screening and enrichment process includes the following steps: 3.1) PEAβ 3-14 Dissolve in phosphate buffer, transfer to immunotubes, pour in coating solution, and let stand overnight; 3.2) Discard the coating solution, wash three times with phosphate buffer, add bovine serum albumin (BSA) solution, and shake to block; 3.3) Wash three times with phosphate buffer, add the PEAβ phage antibody library obtained in step S2, and incubate with shaking; 3.4) After washing the immunotubes 10 times with phosphate Tween buffer, add glycine hydrochloride to the immunotubes, shake to mix, add Tris-HCl to adjust the pH, then add XL1-Blue bacterial culture and shake to incubate; 3.5) Spread the cultured bacterial solution onto a solid culture medium plate and incubate overnight. Scrape off the bacterial growth on the solid culture medium plate and resuspend it. Then add sterile glycerol and mix thoroughly to obtain the primary antibody library. 3.6) Following the steps in 3.1)-3.5), the primary antibody library is screened and enriched to obtain the secondary antibody library; 3.7) Aβ 1-15 Dissolve in phosphate buffer, transfer to immunotubes, pour in coating solution, and incubate overnight; then incubate the secondary antibody library according to steps 3.2) and 3.3). 3.8) Add XL1-Blue bacterial culture to the incubated secondary antibody library, shake and culture, and repeat the operation steps in 3.5) to obtain the tertiary antibody library.

[0010] Furthermore, the construction of the phage antibody includes the following steps: activating the strains of the tertiary antibody library, adding helper phages, culturing with shaking and centrifuging to discard the supernatant, continuing to culture with shaking, centrifuging to collect the supernatant, and completing the preparation of the phage antibody.

[0011] Furthermore, the screening of positive clones includes the following steps: using PEAβ 3-14 As the antigen, the OD value is read using an ELISA reader. 450 Binding activity was detected by nm value, with the experimental group OD 450 The criterion for judging positive clones is that the reading at nm is more than 3 times higher than that of the negative control group.

[0012] Furthermore, the OD of the XL1-Blue bacterial culture... 600 It is 0.6.

[0013] Furthermore, the step S1, which uses the striped bamboo shark as the immunization target and PE-Aβ as the antigen for immunization, specifically involves subcutaneous injection of the striped bamboo shark into the immunization target, followed by nine immunizations, with a 14-day interval between adjacent immunizations.

[0014] Furthermore, in step 3.4), Tris-HCl is added to adjust the pH to 7-8.

[0015] Compared with existing technologies, this invention provides a single-domain antibody targeting pyroglutamate Aβ, based on a novel antigen receptor (IgNAR) in the shark immune system and obtained through genetic engineering. This antibody specifically recognizes PEAβ, exhibits good binding activity, and has a small molecular weight (approximately 12 kDa). This invention solves the technical problem of the lack of shark nanobodies targeting PEAβ, laying an important foundation for subsequent research on the application of this antibody in the detection of PEAβ. It also has the potential to develop reagents for detecting PEAβ or drugs for treating PEAβ-related diseases. Attached Figure Description

[0016] Figure 1 SDS-PAGE gel electrophoresis image of colony PCR products from the PEAβ phage antibody library – recombination rate used for identification.

[0017] Figure 2 The images show the polyclonal phage ELISA results after the first, second, and third rounds of screening of the PEAβ phage antibody library, used to evaluate the enrichment effect of specific clones.

[0018] Figure 3 This is a representative result of positive antigen targeting PEAβ-positive clones screened by ELISA.

[0019] Figure 4 This is a representative result of the reverse antigen targeting PEAβ-positive clones screened by ELISA. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. Example 1:

[0021] Construction of the PEAβ phage antibody library: (1) Using 5x10 6 PEAβ doses of copies / kg, for striped bamboo shark ( Chiloscyllium plagiosum Multiple subcutaneous injections are performed, which is considered as one immunization. A total of nine immunizations are performed, with a 14-day interval between two consecutive immunizations.

[0022] (2) After completing the immunization in step (1), peripheral blood lymphocytes of the immunized shark were collected. The total RNA of the immunized shark peripheral blood lymphocytes was extracted using the QIAGEN RNA extraction kit and the TAKRA PrimeScript RNA extraction kit was used. TM The II 1st Strand cDNA Synthesis Kit reverse transcribed the total RNA from the immune shark peripheral blood lymphocytes into cDNA.

[0023] (3) Using SEQ ID NO.9 as the upstream primer for gene amplification, SEQ ID NO.10 as the downstream primer for gene amplification, and the cDNA obtained in step (2) as a template, the vNAR fragment was obtained by PCR amplification. The obtained vNAR fragment and the phage vector pComb3XSS were then used separately. Sfi I. After digestion with restriction endonucleases, the ligation was performed to construct a recombinant phage vector.

[0024] (4) The recombinant phage vector constructed in step (3) was transformed into XL1-Blue competent cells by electroporation. XL1-Blue cells can ensure stable replication of high-copy plasmids, thereby obtaining a primary antibody library. Simultaneously, 1 μL of the primary antibody library was serially diluted and plated, and the recombination rate was identified by colony PCR. The results are shown in […]. Figure 1 All visible bands are concentrated in the same molecular weight range, indicating that the amplified products are of uniform size, which is consistent with the expected length of the nanobody insertion fragment in the recombinant phage vector.

[0025] (5) Take the primary antibody library obtained in step (4) and inoculate it into 100 mL of 2×YTG medium (containing 2% glucose, 100 μg / mL ampicillin and 50 μg / mL kanamycin), and culture at 37℃ with shaking at 250 rpm until the bacterial density OD reaches 100%. 600 Reaching 0.5, OD 600It uses the absorbance of bacteria at 600nm to measure the concentration of bacterial culture medium; Add 10 12 PFU VCSM13 helper phage was incubated at room temperature for 15 min, then cultured at 37°C with shaking at 150 rpm for 1 h; the cells were collected by centrifugation at 4000 rpm for 20 min. Use 200 mL of 2×YTG amp+tet+kana The culture medium (containing 2% glucose, 100 μg / mL tetracycline, 50 μg / mL ampicillin and 50 μg / mL kanamycin) was resuspended and cultured at 37℃ with shaking at 250 rpm for 16 h. Transfer the cultured bacterial solution to a sterile centrifuge tube and centrifuge at 10,000 rpm and 4°C for 20 min. Collect the supernatant and add polyethylene glycol / sodium chloride solution to the supernatant. The volume ratio of polyethylene glycol / sodium chloride solution to supernatant is 1:5. Incubate at 4°C on ice for 1 h to precipitate the phage. Centrifuge at 10,000 rpm and 4°C for 20 min and collect the precipitate. Dissolve the precipitate in 10 mL of phosphate buffer. This is the PEAβ phage antibody library. Example 2:

[0026] The PEAβ phage antibody library prepared in Example 1 was subjected to three rounds of screening and enrichment: 1. The steps of the first round of screening and enrichment are as follows: (1) Take 1 μg of PEAβ 3-14 Dissolve in 2 mL of phosphate buffer, transfer to an immunotherapy tube, pour in coating solution, and incubate overnight at 4°C; (2) Discard the coating solution, wash 3 times with phosphate buffer, add 3 mL of 2% bovine serum albumin (BSA) solution, and slowly shake at room temperature for 2 h to block. (3) Wash the immunotubes three times with phosphate buffer, add 1 mL of the PEAβ phage antibody library prepared in Example 1, and incubate at room temperature with gentle shaking for 2 h; (4) After washing the immunoassay tube 10 times with phosphate-Tween buffered PBST, add 2 mL of Glycine-HCl (pH=2.0) to the immunoassay tube, shake slowly at room temperature for 15 minutes, then add 0.2 mL of Tris-HCl (pH=9.0) to adjust the pH to approximately 7.5, and then add 5 mL of XL1-Blue bacterial culture (OD200). 600 =0.6), transferred to a 50 mL centrifuge tube, and incubated at 37 ℃ with shaking for 1 h; (5) Spread the cultured bacterial solution onto 2XYT amp Incubate overnight at 37°C on solid culture plates. The next day, use 2XYT... ampThe bacterial growth on the culture plate was scraped off and resuspended, then an equal volume of 50% sterile glycerol was added and mixed thoroughly to obtain a primary antibody library, which was then frozen and stored at -20°C.

[0027] 2. Subsequently, the primary antibody library was subjected to a second round of screening and enrichment, following the same steps as the first round of screening and enrichment, to obtain the secondary antibody library.

[0028] 3. Finally, the secondary antibody library undergoes a third round of screening and enrichment: 10 μg of Aβ 1-15 Dissolve in 2 mL phosphate buffer, transfer to an immunotherapy tube, pour in coating buffer, and incubate overnight at 4 °C; discard coating buffer, wash 3 times with phosphate buffer, add 3 mL 2% bovine serum albumin (BSA) solution, and incubate slowly at room temperature for 2 h; wash immunotherapy tube 3 times with phosphate buffer, add 1 mL of secondary antibody library, and incubate slowly at room temperature for 2 h. Add 5 mL of XL1-Blue bacterial culture (OD) to the incubated phage antibody. 600 =0.6), transferred to a 50 mL centrifuge tube, and incubated at 37 ℃ with shaking for 1 h; the cultured bacterial solution was then spread on 2XYT amp Incubate overnight at 37°C on solid culture plates. The next day, use 2XYT... amp The bacterial growth on the culture plate was scraped off and resuspended, and then an equal volume of 50% sterile glycerol was added and mixed thoroughly to obtain a tertiary antibody library.

[0029] PEAβ at a concentration of 1 μg / μl 3-14 Add the coating buffer to a 96-well microplate, pour in the coating buffer, and incubate overnight at 4°C. Discard the coating buffer, wash three times with PBS, add 2% bovine serum albumin (BSA) solution, and incubate with gentle shaking at room temperature for 2 hours. Add the corresponding PEAβ phage antibody libraries from the first, second, and third rounds, and incubate with gentle shaking at room temperature for 2 hours. Wash six times with PBST, add the enzyme-labeled secondary antibody Anti-M13-HRP (diluted with PBST at a ratio of 1:5000), and incubate with gentle shaking at room temperature for 1 hour. Wash six times with PBST, add 100 μL of TMB chromogenic buffer to each well, incubate in the dark for 10 minutes, and then add 100 μL of 2M H2SO4 to each well to stop the chromogenic reaction. Measure the chromogenic reaction using a microplate reader at OD500. 450 The reading at nm is shown in the results. Figure 2 It can be observed that the value of positive results gradually increases with the increase of screening rounds. Example 3:

[0030] The tertiary antibody library obtained in Example 2 was combined with helper phages to construct phage antibodies, and positive clones were screened: (1) Take a 72-well culture plate and add 0.4 mL of 2×YTG to each well.amp+tet+kana Culture medium; (2) Take the third-level antibody library and spread it on solid culture medium. Then, randomly pick a single colony from the solid culture medium and inoculate it into the above culture plate, label it Master Plate, and incubate it overnight with shaking at 250 rpm and 37°C. (3) Take another 72-well culture plate and take 0.4 mL of a solution containing 1 x 10⁻⁶ mg / L. 10 2×YTG of pfu VCSM13 helper phage amp +tet+kana Add culture medium to each well; take 50 μL of culture medium from each well of the Master Plate in step (2) and add it to the corresponding well, label it P1 plate, and incubate with shaking at 150 rpm and 37℃ for 2 h; centrifuge at 4000 rpm for 20 min, discard the supernatant, and add 0.4 mL of 2×YT to each well. amp+tet+kana Culture medium, incubate overnight at 37℃ and 250 rpm with shaking; after centrifugation at 4000 rpm for 20 min, collect the supernatant and store at 4℃ for later use to complete the preparation of phage antibodies; (4) PEAβ at a concentration of 1 μg / ml 3-14 Add to a 96-well microplate, pour in coating buffer, and incubate overnight at 4°C; discard coating buffer, wash 3 times with PBS, add 2% bovine serum albumin (BSA) solution, and slowly shake to block for 2 hours at room temperature; add the phage antibody prepared in step (3) accordingly, and slowly shake to incubate for 2 hours at room temperature; wash 6 times with PBST, add enzyme-labeled secondary antibody Anti-M13-HRP (diluted with PBST at a ratio of 1:5000), and slowly shake to incubate for 1 hour at room temperature; wash 6 times with PBST, add 100 μL of TMB colorimetric solution to each well, incubate for 10 minutes in the dark, and then add 100 μL of 2M H2SO4 to each well to stop color development. Use a microplate reader to measure the color at OD 450 The reading at nm; with the experimental group OD 450 A value more than 3 times higher than the negative control group (without phage antibody added as the negative control) is used as the judgment criterion. A positive antigen detection result is obtained, and corresponding positive clones are screened out. Results are shown below. Figure 3 The obtained positive clones were named P1-P8. Forward antigen detection results showed that antibodies against P1-P8 could interact with PEAβ. 3-14 Specific binding occurred, indicating a positive clone targeting PEAβ. Sequencing analysis was performed on P1-P8, and the amino acid sequences of P1-P8 are shown in SEQ ID NO.1-SEQ ID NO.8.

[0031] Replace the coated antigen with Aβ 1-15 Then repeat experiment (4) to obtain the reverse antigen detection results, the results of which are shown in [the table below]. Figure 4 OD 450Values ​​P1-P8 showed no significant difference from the negative control group. The standard for detection was a 0.1-fold increase in the positive antigen result compared to the reverse antigen result. (See [reference needed]). Figure 3 and Figure 4 The results for the positive antigen of P1-P8 were all 0.1 higher than those for the reverse antigen, indicating that P1-P8 does not interact with Aβ. 1-15 Specific binding occurs, to PEAβ 3-14 It has specific recognition capabilities.

[0032] The coating solution used in the above examples was PBS.

[0033] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the invention. Modifications and variations made by those skilled in the art in accordance with the spirit of the invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A single-domain antibody targeting pyroglutamate Aβ, characterized in that: The amino acid sequence of the single-domain antibody is shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5 or SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO.

8.

2. The application of the single-domain antibody according to claim 1 in the detection of pE-Aβ.

3. A method for preparing the single-domain antibody according to claim 1 or 2, characterized in that: Includes the following steps: S1: Using the striped bamboo shark as the immunization target, PE-Aβ was used as the antigen for immunization. Total RNA was extracted from the peripheral blood cells of the immunized shark, reverse transcribed into cDNA, and amplified using cDNA as a template to obtain the shark-derived single-domain antibody vNAR fragment of the striped bamboo shark. S2: Construct a recombinant phage vector by combining a shark-derived single-domain antibody vNAR fragment with a phage vector, transform the recombinant phage vector into competent cells, and obtain a PEAβ phage antibody library after expansion culture. S3: Screen positive clones from the PEAβ phage antibody library to obtain the single-domain antibody.

4. The method for preparing a single-domain antibody according to claim 3, characterized in that: Step S3 specifically involves: performing three screenings and enrichment processes on the PEAβ phage antibody library, constructing phage antibodies with helper phages, and screening for positive clones; The three-stage screening and enrichment process includes the following steps: 3.1) PEAβ 3-14 Dissolve in phosphate buffer, transfer to immunotubes, pour in coating solution, and let stand overnight; 3.2) Discard the coating solution, wash three times with phosphate buffer, add bovine serum albumin (BSA) solution, and shake to block; 3.3) Wash three times with phosphate buffer, add the PEAβ phage antibody library obtained in step S2, and incubate with shaking; 3.4) After washing the immunotubes 10 times with phosphate Tween buffer, add glycine hydrochloride to the immunotubes, shake to mix, add Tris-HCl to adjust the pH, then add XL1-Blue bacterial culture and shake to incubate; 3.5) Spread the cultured bacterial solution onto a solid culture medium plate and incubate overnight. Scrape off the bacterial growth on the solid culture medium plate and resuspend it. Then add sterile glycerol and mix thoroughly to obtain the primary antibody library. 3.6) Following the steps in 3.1)-3.5), the primary antibody library is screened and enriched to obtain the secondary antibody library; 3.7) Aβ 1-15 Dissolve in phosphate buffer, transfer to immunotubes, pour in coating solution, and incubate overnight; then incubate the secondary antibody library according to steps 3.2) and 3.3). 3.8) Add XL1-Blue bacterial culture to the incubated secondary antibody library, shake and culture, and repeat the operation steps in 3.5) to obtain the tertiary antibody library.

5. The method for preparing a single-domain antibody according to claim 4, characterized in that: The preparation of the phage antibody includes the following steps: activating the strains of the tertiary antibody library, adding helper phages, culturing with shaking and centrifuging to discard the supernatant, continuing to culture with shaking, centrifuging to collect the supernatant, and completing the preparation of the phage antibody.

6. The method for preparing a single-domain antibody according to claim 4, characterized in that: The screening for positive clones includes the following steps: using PEAβ 3-14 As the antigen, the OD value is read using an ELISA reader. 450 Binding activity was detected by nm value, with the experimental group OD 450 The criterion for judging positive clones is that the reading at nm is more than 3 times higher than that of the negative control group.

7. The method for preparing a single-domain antibody according to claim 4, characterized in that: The OD of the XL1-Blue bacterial culture 600 It is 0.

6.

8. The method for preparing a single-domain antibody according to claim 3, characterized in that: The step S1, which uses the striped bamboo shark as the immunization target and PE-Aβ as the antigen, specifically involves subcutaneous injection of the striped bamboo shark into the immunization target, followed by nine immunizations, with a 14-day interval between adjacent immunizations.

9. The method for preparing a single-domain antibody according to claim 4, characterized in that: In step 3.4), add Tris-HCl to adjust the pH to 7-8.

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