A shark-derived single-domain antibody binding sars-cov-2 rbd, and preparation method and application thereof

Abstract

The application discloses a shark-derived single-domain antibody binding SARS-CoV-2 RBD, the amino acid sequence of the single-domain antibody is shown as SEQ ID NO: 2 or SEQ ID NO: 4; nucleotides encoding the above-mentioned shark-derived single-domain antibody, characterized in that the nucleotide sequence is shown as SEQ ID NO: 1 or SEQ ID NO: 3; the application also provides a preparation method of the shark-derived single-domain antibody binding SARS-CoV-2 RBD, and the shark-derived single-domain antibody is obtained by sequentially performing immunization, constructing a phage antibody library, screening positive clones from the phage antibody library, finally adding an inducer for expression after large-scale culture, and collecting and purifying antibody proteins; the shark-derived single-domain antibody prepared by the application has good application prospects in the development of a new coronavirus disinfecting spray and a new coronavirus antigen detection kit.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a shark-derived single-domain antibody binding to SARS-CoV-2 RBD, and a preparation method and application thereof. BACKGROUND

[0002] Coronaviruses belong to the family of Coronaviridae and the genus of Coronavirus, and are a class of single-stranded positive-sense RNA viruses. SARS-CoV-2 belongs to the coronavirus. The coronavirus genome encodes spike protein, envelope protein, membrane protein and nucleocapsid protein in turn. Among them, the spike protein is the most important surface membrane protein of the coronavirus, which contains two subunits S1 and S2. Among them, S1 mainly contains a receptor binding domain (RBD) responsible for recognizing cell receptors; SARS-CoV-2 enters the cell by combining with angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells through the receptor binding domain (RBD) of its surface spike protein (spike), and completes the infection.

[0003] Shark-derived single-domain antibody vNAR (variable New Antigen Receptor) is derived from a special immunoglobulin in its body, IgNAR (Ig New Antigen Receptor), which is the smallest antibody fragment capable of binding to antigens, with a molecular weight of about 12-18 kDa. The shark-derived single-domain antibody can be used as a benchmark for traditional monoclonal antibodies in terms of affinity and specificity, and has the advantages of high physicochemical stability, good tissue permeability, strong resistance, high stability, and easy modification. At the same time, in terms of production, vNAR prokaryotic cell or yeast expression system can express a large amount of vNAR, which not only has a short production cycle, but also has low production cost. At present, there is no shark-derived single-domain antibody binding to SARS-CoV-2 RBD for the detection or prevention of COVID-19 infection. SUMMARY

[0004] In order to solve some problems or at least alleviate some problems in the prior art, the present application provides a preparation method of a shark-derived single-domain antibody binding to SARS-CoV-2 RBD, which is used for the detection or prevention of SARS-CoV-2 virus.

[0005] The technical scheme of the present application is as follows:

[0006] The present application provides a shark-derived single-domain antibody binding to SARS-CoV-2 RBD, the amino acid sequence of which is shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0007] In addition, the present application also provides a nucleotide encoding the above-mentioned single-domain antibody, the nucleotide sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0008] The present application also provides a preparation method of a shark-derived single-domain antibody binding to SARS-CoV-2 RBD, which specifically comprises the following steps:

[0009] S1, taking striped spiny dogfish as the immunization object, and using the recombinant protein of SARS-CoV-2 RBD as the antigen for immunization; after completing multiple immunizations, the total RNA of the peripheral blood lymphocytes of the immunized shark is extracted, and the vNAR gene fragment is amplified by taking the cDNA as the template;

[0010] S2, the purified vNAR gene fragment is inserted into the phagemid vector pComb3XSS to construct a recombinant phagemid vector, and the recombinant phagemid vector is transformed into E coli TG-1 cells, and after being cultured, the phage antibody library is obtained by infection of the helper phage VCS-M13;

[0011] S3, the phage antibody library is subjected to screening, and positive clones are obtained, which are respectively named as S-RBD-55 and S-RBD-61, and the gene sequences thereof are subjected to sequencing analysis;

[0012] S4, the vNAR gene fragments contained in S-RBD-55 and S-RBD-61 are amplified, and are cloned into the expression vector PET30a to construct a recombinant expression vector, and the recombinant expression vector is transformed into E coli Shuffle T7 cells, and after screening out the positive strains, the antibody protein is collected and purified after being induced to express by adding an inducer, that is, the single-domain antibody of SARS-CoV-2 RBD.

[0013] The present application further provides the use of the shark-derived single-domain antibody binding to SARS-CoV-2 RBD in the preparation of a kit or a medicine for preventing, treating and / or diagnosing SARS-CoV-2 infection.

[0014] The present application further provides the use of the shark-derived single-domain antibody binding to SARS-CoV-2 RBD in the preparation of a spray for preventing SARS-CoV-2 infection.

[0015] The present application still further provides a spray for preventing SARS-CoV-2 infection, which comprises the following components in percentage by mass:

[0016]

[0017]

[0018] Further, the antibacterial agent is ethanol or silver ion bacteriostatic agent; the stabilizer is sodium polyphosphate, sodium percarbonate or sodium sulfite; and the dispersing agent is sodium dodecyl benzene sulfonate.

[0019] Compared with the prior art, the application has the beneficial effects that:

[0020] 1. The application provides shark-derived single-domain antibodies that can bind to SARS-CoV-2 RBD proteins, and the prepared two strains of single-domain antibodies have good affinity for SARS-CoV-2 RBD protein binding, as identified by ELISA, and the IC 50 values thereof are 1.826±0.03 nM and 2.107±0.045 nM, respectively.

[0021] 2. The shark-derived single-domain antibodies prepared by the application have good stability between room temperature and 50 DEG C, as identified by storage stability experiments, and the half-solubility temperatures thereof are all above 75 DEG C, as identified by circular dichroism, which makes the single-domain antibodies of the application free from cold-chain transportation and effectively reduce transportation and storage costs; therefore, the shark-derived single-domain antibodies prepared by the application have good application prospects in the development of new coronavirus disinfecting sprays and new coronavirus antigen detection kits. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 SDS-PAGE gel electrophoresis results of the shark-derived single-domain antibodies prepared by the application;

[0023] Figure 2 Stability detection results of the shark-derived single-domain antibodies prepared by the application stored at 37 DEG C for 14 days;

[0024] Figure 3 Circular dichroism detection results of the shark-derived single-domain antibodies prepared by the application;

[0025] Figure 4 Western blot detection results of the shark-derived single-domain antibodies prepared by the application for specific recognition of SARS-CoV-2 RBD;

[0026] Figure 5 ELISA identification results of the shark-derived single-domain antibodies prepared by the application for binding to SARS-CoV-2 RBD proteins; DETAILED DESCRIPTION

[0027] In order to make the content described in the application more convenient to understand, the technical solutions described in the application are further described below in combination with specific embodiments and drawings, but the application is not limited thereto.

[0028] The striped bonnet shark (Chiloscyllium plagiosum) in the following examples is a small shark, not more than 1 meter in length after adulthood, which is convenient for experimental operation, and is the most commonly used research model for single domain antibody development at present.

[0029] Example 1 Preparation method of single domain antibody binding to SARS-CoV-2 RBD

[0030] 1. Construction of phage antibody library:

[0031] 1) Adult striped bonnet sharks were used as immunization objects, and SARS-CoV-2 RBD recombinant protein was used as an antigen for immunization, and the specific method was as follows: according to the dose of 5 nM / kg, after emulsifying the antigen with Freund's adjuvant, multi-point subcutaneous injection was used for immunization. A total of 6 immunizations were performed, and the immunization interval was 14 days;

[0032] 2) 14 days after the completion of the 6th immunization, the blood of the immunized striped bonnet shark was collected, and the peripheral blood mononuclear cells were separated by density gradient centrifugation. The total RNA was extracted by TRIZOL method, and was reversely transcribed into cDNA;

[0033] 3) The designed striped bonnet shark vNAR gene amplification specific primers were used to amplify the vNAR gene fragment, and the purified vNAR gene fragment was cloned into the phagemid vector pComb3XSS to construct a recombinant phagemid vector, wherein the primer sequences are as follows:

[0034] Upstream primer: CGTGGCCCAGGCGGCCGGGCCCCCTGGTTACCAAATGT;

[0035] Downstream primer: CGTGGCCCAGGCGGCCGGGCCCTTTGCCAGGTTTCACAGTCAG;

[0036] 4) The constructed recombinant phagemid vector was transformed into TG-1 competent cells by electroporation to obtain a primary antibody gene library (the library capacity is at least 10 8 pfu / mL or more);

[0037] 5) After expanding the primary antibody gene library, VCS-M13 helper phage was added to construct a phage antibody library;

[0038] 2. Affinity screening of phage antibodies against SARS-CoV-2 RBD

[0039] According to the combination, washing, elution and amplification, the phage primary antibody library is subjected to three rounds of affinity screening and enrichment, and the method of the first round of affinity screening and enrichment is as follows:

[0040] 1) 10 μg of SARS-CoV-2 RBD recombinant protein is dissolved in PBS buffer to coat the immunotube, and the coating is carried out at 4°C overnight;

[0041] 2) The next day, the coating solution is poured out, and PBS is washed for 3 times. Then, 2% BSA solution is added, and the plate is shaken and sealed at room temperature for 2 hours;

[0042] 3) After the blocking solution is discarded, PBS is washed, and the phage antibody obtained in step 1 is added, and the plate is slowly shaken and incubated at room temperature for 2 hours;

[0043] 4) After the antibody solution is discarded and PBST is washed, Glycine-HCl (pH = 2.0) is added to the immunotube for elution. After being slowly shaken at room temperature for 15 minutes, 0.2 mL of Tris-HCl (pH = 9.0) is added for neutralization, so that the pH is adjusted to about 7.5. Then, TG-1 bacterial solution (OD600 = 0.5) is added, mixed, and then transferred to a 50 mL centrifuge tube. The tube is cultured at 37°C and 150 rpm for 1 hour;

[0044] 5) The tube is centrifuged at 4000 rpm for 5 minutes. After the precipitate is resuspended, SOC plates containing ampicillin and tetracycline are coated. The next day, the bacteria on the plate are scraped, and glycerol is used for preservation for the next round of screening. The affinity screening is carried out for 3 rounds, and the steps are the same as the first round. The coating amount of the antigen is gradually reduced, and the coating amounts are 5 μg and 2.5 μg, respectively. After three rounds of screening, the enrichment amount and recovery rate of the phage antibody specifically targeting SARS-CoV-2 RBD are gradually improved;

[0045] 3. Screening of positive recombinant antibodies

[0046] The antigen-positive recombinant antibody is screened by Phage ELISA, and the specific method is as follows:

[0047] 1) Take a 96-well culture plate, and add 2xYTG amp+tet+kana culture medium to each well;

[0048] 2) Randomly pick single colonies of antibodies from the SOCamp+tet plate coated with the third library (i.e., the SARS-CoV-2 RBD phage antibody library obtained after three rounds of screening), and inoculate them into the above culture plate. Label the plate as Master Plate, and culture it at 250 rpm and 37°C overnight;

[0049] 3) Take another 96-well culture plate, and take 0.4 mL of 2xYTG containing 1x 1010pfu of VCSM13 helper phageamp +tet+kana Culture medium to each well; 50 μL of culture solution from the Master Plate in step 2) was added to the corresponding well, marked as P1 plate, and cultured at 37°C with 150 rpm shaking for 2h; centrifuged at 4000 rpm for 20 min, and 0.4 mL of 2xYT culture medium was added to each well amp+tet+kana Culture solution, 37°C, 250 rpm shaking overnight; the next day, centrifuged at 4000 rpm for 20 min, and the supernatant was taken and stored at 4°C for standby, thus completing the preparation of phage recombinant antibodies;

[0050] 4) The SARS-CoV-2 RBD recombinant protein was diluted with PBS to 1 μg / mL, 100 μL / well coated on a 96-well enzyme-labeled plate, and the coating solution was poured in, and coated at 4°C overnight; the next day, the coating solution was poured out, and after PBS washing, 2% BSA solution was added, and incubated at room temperature for 2h; the blocking solution was discarded, and the phage recombinant antibody prepared in step 3) was added, and incubated at room temperature for 2h with slow shaking; the recombinant antibody solution was discarded, and after PBST washing, enzyme-labeled secondary antibody Anti-M13-HRP (1:5000 dilution ratio) was added, and incubated at room temperature for 1h with slow shaking; the enzyme-labeled secondary antibody solution was discarded, and after PBST washing, 100 μL of TBM color developing solution was added to each well, and after 10 min of dark incubation, 100 μL of 2M H2SO4 was added to each well to terminate the color development, and the OD 450 value was read using an enzyme-labeled instrument; the OD 450 value of the experimental group was more than 2 times higher than that of the negative control group, and the corresponding positive clones were screened out and subjected to sequencing analysis, and after excluding duplicates, the final positive clones were named S-RBD-55 and S-RBD-61;

[0051] The amino acid sequence of S-RBD-55 is shown in SEQ ID NO: 2: MNIFLLSGLLAWLPNVFSQRIEQTPTTTTKEAGESLTINCVLKGSSCAVSSTYRYFTKKGATKKARLSAGGRYWDTKNATSKSFSLRISDLRVEDSGTYHCEGYTGTAGCVLFWKMAIEGGGTILTVKPGK.

[0052] The nucleotide sequence of the positive clone designated S-RBD-55 is set forth in SEQ ID NO: 1 : ATGAATATTTTCTTGCTTTCGGGCCTGTTAGCCTGGTTACCAAATGTCTTTAGTCAACGGATTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTGTGCAGTGAGTAGCACGTACAGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGATTATCAGCTGGCGGAAGATATTGGGACACAAAGAATGCGACATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGGGTATACTGGTACAGCTGGATGTGTGCTGTTCTGGAAAATGGCCATTGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGGCAAA;

[0053] wherein the amino acid sequence of S-RBD-61 is set forth in SEQ ID NO: 4

[0054] MNIFLLSGLLAWLPNVFSQRIEQTPTTTTKEAGESLTINCVLKGSSCAVSSTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYTRTAGCVLFSKLAIEGGGTILTVKPGK;

[0055] The nucleotide sequence of the positive clone named S-RBD-61 is shown in SEQ ID NO: 3: ATGAATATTTTCTTGCTTTCGGGCCTTTTAGCCTGGTTACCAAATGTCTTTAGTCAACGGATTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTGTGCAGTGAGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGCTTATCAACTGGCGGACGATACTCGGACACAAAGAATACGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGCGTATACTCGTACAGCTGGATGTGTACTGTTCTCCAAATTAGCCATTGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGGCAAA;

[0056] 4. Expression and purification of single domain antibody

[0057] According to the above sequencing results, the vNAR gene fragments of the positive clones S-RBD-55 and S-RBD-61 were amplified and cloned into the PET-30a expression vector; the recombinant expression vector plasmid was transformed into E. coli Shuffle T7 competent cells, and after the positive strain was determined, it was inoculated into LB medium containing kanamycin resistance, and cultured at 200 rpm until the OD 600 was between 0.6 and 1.0. The final concentration of the inducer IPTG solution was 0.5 mmol / L, and the expression was induced at 200 rpm and 18°C for 18 h. After the induction expression was completed, the bacterial cells were collected by centrifugation, the bacterial cells were broken by ultrasonic wave, and the supernatant was collected after centrifugation. The antibody protein was purified by conventional His-Tag affinity chromatography method, and the protein purity of S-RBD-55 and S-RBD-61 single domain antibodies was > 90%, and the SDS-PAGE electrophoresis detection results are shown in Figure 1 .

[0058] Example 2 Storage stability test

[0059] The shark-derived single domain antibodies S-RBD-55 and S-RBD-61 prepared according to Example 1 were placed in an incubator and stored at 37°C and 50°C, respectively, and the degradation was detected at 3, 6, 9, 12, and 15 days, respectively, and the results are shown in Figure 2As shown, the remaining amounts of shark-derived single-domain antibodies S-RBD-55 and S-RBD-61 both remained at 100% after being placed at 37°C for 15 days; after being placed at 50°C for 15 days, the remaining amounts of shark-derived single-domain antibodies S-RBD-55 and S-RBD-61 still maintained above 90%, indicating that the shark-derived single-domain antibodies prepared in the present application have good storage stability under the condition of 50°C.

[0060] Example 3 Circular dichroism detection of thermal stability of single-domain antibodies

[0061] The concentrations of shark-derived single-domain antibodies S-RBD-55 and S-RBD-61 prepared according to Example 1 were diluted to 1 mg / mL, and were detected by a circular dichroism instrument, with a temperature change range of 20-85°C, a temperature change rate of 2.5°C / min, and a detection wavelength of 205 nm. The results are shown in Figure 3 As shown, the solubilization temperatures (Tm values) of S-RBD-55 and S-RBD-61 single-domain antibodies were 72°C and 77.5°C, respectively, indicating that the single-domain antibodies S-RBD-55 and S-RBD-61 have good thermal stability.

[0062] Example 4 Western Blot detection of specific recognition of single-domain antibodies to SARS-CoV-2 RBD protein

[0063] After the SARS-CoV-2 RBD recombinant protein was subjected to SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane by a semi-dry transfer method; 5% skimmed milk was blocked, and incubated at room temperature for 1 h; after TBST washing, purified S-RBD-55 and S-RBD-61 single-domain antibodies (antibody concentration used was 1 μg / mL) were added, and incubated at 37°C for 1 h; after TBST washing, a HRP-conjugated rabbit anti-stripe striped shark IgNAR secondary antibody was added, and incubated at room temperature for 45 min; after TBST washing, a chemiluminescence gel imager was used for color development; the experimental results are shown in Figure 4 As shown: there is a specific band at about 35 kDa, indicating that the single-domain antibodies S-RBD-55 and S-RBD-61 can both specifically recognize SARS-CoV-2 RBD protein.

[0064] Example 5 Determination of binding affinity of single-domain antibodies to SARS-CoV-2 RBD protein

[0065] According to the optimal antigen coating concentration obtained by orthogonal experiment, the SARS-CoV-2 RBD recombinant protein is diluted with PBS to 4 μg / mL, 100 μL of the coating enzyme-labeled plate is added to each well, and the blocking is carried out. The single-domain antibody S-RBD-55 and S-RBD-61 are gradiently diluted at a ratio of 1:2, and are sequentially added to the corresponding wells, and are incubated at 37°C for 1.5 h. After washing, the HRP-conjugated rabbit anti-stripe spotted shark vNAR antibody is added, and is incubated at 37°C for 1 h. After washing, 100 μL of TBM color developing substrate is added to each well, and is placed in the dark for 10 min. After adding 100 μL of 2M H2SO4 to each well to terminate the color development, the OD 450 nm is read by using an enzyme-labeled instrument, and the binding condition is detected. As shown in Figure 5 , the experimental results show that the single-domain antibodies S-RBD-55 and S-RBD-61 have good affinity for the SARS-CoV-2 RBD protein, and the EC 50 values thereof are 1.256 nM ± 0.03 nM and 0.832 ± 0.045 nM, respectively.

[0066] Example 6 Application of shark-derived single-domain antibodies

[0067] The single-domain antibodies S-RBD-55 or S-RBD-61 prepared according to the method of Example 1 are optionally used to prepare a spray for preventing SARS-CoV-2 infection, and the spray comprises the following components in mass percentage:

[0068]

[0069] The preparation method of the above spray is as follows: the triethanolamine, sodium dodecylbenzenesulfonate and sodium polyphosphate in the above formula are added into ethanol, heated at 55°C, continuously and rapidly stirred, uniformly mixed, and then the shark-derived single-domain antibodies S-RBD-55 or S-RBD-61 and flower fragrance oil are added, inactivated by the pasteurization method, and sterilized by filtering with a 0.22 μm membrane, to obtain the spray for preventing SARS-CoV-2 infection.

[0070] Example 7

[0071] The spray for preventing SARS-CoV-2 infection comprises the following components in mass percentage:

[0072]

[0073]

[0074] Example 8

[0075] The spray for preventing SARS-CoV-2 infection comprises the following components in mass percentage:

[0076]

[0077] The above merely illustrates the embodiments of the present application, and is not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process conversion, or direct or indirect application in other related technical fields, made by using the content of the present application, are also included in the patent protection scope of the present application.

Claims

1. A shark-derived single-domain antibody that binds to SARS-CoV-2 RBD, characterized in that, The amino acid sequence of the single-domain antibody is shown in SEQ ID NO:2 or SEQ ID NO:

4.

2. A nucleotide encoding a shark-derived single-domain antibody as described in claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO:1 or SEQ ID NO:

3.

3. The use of a shark-derived single-domain antibody that binds to SARS-CoV-2 RBD as described in claim 1 in the preparation of a kit or drug for diagnosing SARS-CoV-2 infection.

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