CN114773464B

CN114773464B - Single-domain antibody VHH-2 aiming at new coronavirus omicron strain S protein, coding sequence and application

Info

Publication number: CN114773464B
Application number: CN202210694198.2A
Authority: CN
Inventors: 崔淑娟; 赵佳琛; 刘医萌; 彭晓旻; 卢桂兰; 石伟先; 吴双胜; 张莉; 潘阳; 张代涛; 杨鹏; 王全意
Original assignee: Beijing Center for Disease Prevention and Control
Current assignee: Beijing Center for Disease Prevention and Control
Priority date: 2022-06-20
Filing date: 2022-06-20
Publication date: 2022-08-26
Anticipated expiration: 2042-06-20
Also published as: CN114773464A

Abstract

The invention provides a specific single domain antibody aiming at the S protein of the new coronavirus omicron strain, a gene sequence and a coding sequence of VHH-2, and also discloses that the omicron strain single domain antibody can be efficiently expressed in eukaryotic cells, and can be applied to the research and development of a new coronavirus omicron strain detection reagent and targeted therapy.

Description

Single-domain antibody VHH-2 aiming at S protein of new coronavirus omicron strain, coding sequence and application

Technical Field

The invention relates to the field of biomedical or biotechnological application, in particular to a single domain antibody aiming at the S protein of a new coronavirus omicron strain, a coding sequence and application.

Background

In the early 2020, the global spread of new crown diseases brings serious challenges to human health and economic development, and the continuous variation of new crown viruses causes the new crown viruses to have stronger transmission capability, faster diffusion speed and wider infection range, thus becoming the key and hot areas of global public health attention. The prevention and control of new corona can not be detected and identified in a laboratory, and is more dependent on the effective treatment of new corona cases. At present, the mainstream detection technology of the new coronavirus is a real-time fluorescence PCR method, the specificity and the sensitivity of the real-time fluorescence PCR method are closely related to the design of a primer probe, system optimization, an amplification instrument and the like, and detection personnel also need to be trained strictly. The operation and cost of the antigen detection method are relatively low, but the antibody used for the antigen detection at present mainly aims at the conserved N protein, and the specificity and the sensitivity of the detection cannot meet the market requirement. At present, the omicron strain is a main popular strain of new coronavirus which is abused worldwide, so that the development of a rapid detection kit by utilizing a single-domain antibody aiming at the S protein of the new coronavirus is of great significance, and simultaneously, the single-domain antibody of broad-spectrum targeted therapy can be developed.

The new corona virion is spherical (some is polymorphic), has surface protrusions, and is observed under electron microscope to form virus similar to crown, and its diameter is 75-160 nm. The viral gene is continuous linear single-stranded RNA, the whole genome sequence is about 29000 bp in length, and the total length comprises 14 main Open Reading Frames (ORF) and can encode 27 proteins. The S protein is rich in content in the new coronavirus, is relatively conservative, has high immunogenicity, is a target protein of a neutralizing antibody after infection, and is also a target for detection and a key point of treatment and vaccine design.

Single domain antibodies contain only the variable region of the heavy chain (VHH), which is of small molecular weight and can be isolated and screened from camelids by molecular cloning. The single domain antibody not only has the specificity and reactivity of the traditional antibody, but also has higher stability, solubility, permeability and the like.

However, there is currently a lack of satisfactory single domain antibodies against the S protein of the novel coronavirus. Therefore, there is an urgent need in the art to develop a new specific single domain antibody against the S protein, which is effective for establishing a highly efficient detection method, and provides experimental basis and candidate materials for developing anti-new coronavirus single domain antibody drugs.

Disclosure of Invention

The invention aims to provide a single domain antibody aiming at the S protein of the novel coronavirus omicron strain, and also provides a coding sequence of the single domain antibody and application of the single domain antibody in detection and treatment.

The technical scheme adopted by the invention is as follows:

the first aspect of the invention provides 1 VHH chain of single domain antibody of S protein of the novel coronavirus omicron strain named VHH-2, characterized in that the VHH-2 comprises CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 12. In a specific embodiment, the amino acid sequence of VHH-2 is a VHH chain comprising the above 3 CDRs, and has the same length as SEQ ID NO. 5 and more than 95% homology; in another specific embodiment, the amino acid sequence of the VHH is as shown in SEQ ID NO 5.

The second aspect of the present invention provides a nucleic acid encoding the single domain antibody VHH-2 described above; in a specific embodiment, the sequence of said nucleic acid has more than 95% homology with the nucleic acid molecule as shown in SEQ ID NO. 2 and is capable of encoding the polypeptide molecule as shown in SEQ ID NO. 5; in another embodiment, the sequence of said nucleic acid has more than 99% homology with the nucleic acid molecule as shown in SEQ ID NO. 2 and is capable of encoding the polypeptide molecule as shown in SEQ ID NO. 5; in another specific embodiment, the nucleic acid has the sequence shown in SEQ ID NO 2.

The third aspect of the invention provides a method for preparing the new coronavirus omicron strain S protein single domain antibody VHH-2, which comprises the steps of inserting nucleic acid for coding the VHH-2 chain into an expression vector, transferring the expression vector into host cells, culturing the host cells, and purifying to obtain the single domain antibody. In one specific embodiment, the expression vector is a baculovirus expression vector pFastBac, and the host cell is sf9 insect cell; in one specific example, the method is to insert the sequence shown in SEQ ID NO. 2 into a vector, and then transfect a host cell for expression and purification.

The fourth aspect of the invention provides the application of the single domain antibody VHH-2 of the S protein of the novel coronavirus omicron strain in the preparation of products for diagnosing the novel coronavirus omicron strain. The product includes but is not limited to a kit, a test strip and the like.

The fifth aspect of the invention provides the application of the S protein single domain antibody VHH-2 of the new coronavirus omicron strain of the invention in the preparation of products for treating the new coronavirus omicron strain; in a specific embodiment, the therapeutic product includes, but is not limited to, oral, injectable, or nasal spray.

The sixth aspect of the present invention provides a pharmaceutical composition comprising the novel coronavirus omicron strain S protein single domain antibody VHH-2 of the present invention described in the present invention and a pharmaceutically acceptable carrier.

The invention has the beneficial effects that:

the method comprises the steps of utilizing a natural single domain antibody phage display library established by established alpaca peripheral blood lymphocytes, using biotinylated S protein as an antigen, carrying out 3 rounds of immune panning, coating an ELISA plate with the S protein, using an antibody expressed by a panned monoclonal colony as a primary antibody, screening positive monoclonal colonies, amplifying corresponding bacterial liquid, sequencing, and finally obtaining a gene sequence of a single domain antibody VHH-2 chain specific to the S protein. Specific single-domain antibodies against the S protein, which were efficiently expressed in insect sf9 cells, were obtained by amplification cloning of the specific single-domain antibodies into baculovirus expression vectors. Through WB test identification, the antibody has a good immunodetection effect, and a key foundation is laid for establishing a rapid and accurate antigen method for detecting the new coronavirus. And the single-domain antibody is identified to have strong specificity by using western blot. Through virus neutralization test, the neutralizing titer of the antibody on the new crown omicron strain reaches 1: 64. the sensitivity is high, and a research direction is provided for the targeted therapy of the new coronavirus.

Drawings

FIG. 1 shows that S protein single domain antibody of the novel coronavirus omicron strain is expressed and purified in Sf9 cells and is identified by Western blot specificity; wherein, 1A is the expression purification and identification of the S protein single domain antibody in Sf9 insect cells, and 1B is the specificity identification of the S protein single domain antibody;

FIG. 2 identification of neutralizing activity of S protein single domain antibody of the novel coronavirus omicron strain (neutralizing titer 1: 32).

Detailed Description

The invention is further described with reference to specific examples and the accompanying drawings, but it should be understood that the scope of the invention is not limited to the following examples.

The invention will be further illustrated with reference to specific examples:

example 1: immunopanning process for natural single domain antibodies to S protein

(1) Amplifying the established natural single-domain antibody phage library: adding a 2 XYT culture medium into a 100 mu L glycerol bacterial library, adding 20 MOI auxiliary phage when OD600=0.5, standing for 30min, resuspending the precipitate with the 2 XYT culture medium after centrifugation, culturing for 1h, adding antibiotics, culturing for 16h, centrifuging, precipitating the supernatant with precooled PEG-NaCl (1/4 volume), and resuspending with 1mL PBS to obtain the amplified single domain antibody library;

(2) immune tube panning: coating an immune tube with 50 micrograms/tube of S protein (omicron strain S gene ORF full-length expression, biotin labeling and purification) overnight, removing coating liquid, washing for 3 times, sealing for 2h with 2mL BSA (1%), washing for 3 times with PBST, adding 100 muL of the single-domain antibody library amplified in the step 1 as a primary antibody, acting for 2h at 37 ℃, washing for 3 times with PBST, eluting with Glycine-HCI (PH2.2), adjusting the eluent with Tris-HCI to PH 7.4, and obtaining a first round of natural single-domain antibody library after elutriation;

(3) amplifying the 1 st round of natural single-domain antibody library obtained in the step (2) according to the step (1), obtaining a 1 st round of natural single-domain antibody heavy suspension library, then repeating the step (2) of immune tube panning, only adding 100 mu L of amplified 1 st round of natural single-domain antibody heavy suspension library as primary antibody, and finally obtaining a panned 2 nd round of natural single-domain antibody library;

(4) amplifying the 2 nd round natural single-domain antibody library obtained in the step (3) according to the step (1), obtaining a 2 nd round natural single-domain antibody resuspension library, then repeating the step (2) of immune tube panning, only adding 100 mu L of amplified 2 nd round natural single-domain antibody resuspension library as a primary antibody, and finally obtaining a panned 3 rd round natural single-domain antibody library.

Example 2: ELISA identification of Individual clones

(1) Panning for single positive clonal expansion of the shake bacteria for antibody expression: the natural single domain antibody library of round 3 selected in example 1 was inoculated into 2 XYT medium, OD _600nm If =0.5, adding 20 MOI helper phage, standing for 30min, centrifuging, then resuspending the precipitate with 2 XYT medium, culturing for 1h, then coating on 2 XYT plates containing antibiotics, culturing overnight, selecting 40 single colonies to inoculate into 2 XYT medium the next day, if OD600=0.5, then adding 20 MOI helper phage, standing for 30min, centrifuging, then resuspending the precipitate with 2 XYT medium, culturing again, and adding IPTG to induce expression for 8 h.

(2) And (3) ELISA identification: coating an ELISA plate with an S protein (an omicron strain S gene ORF full-length expression, biotin labeling and purification, concentration of 1 ng/muL) 100 muL/hole overnight, washing 3 times after removing a coating solution, sealing for 2h by using 200 muL/hole BSA (3%), washing for 3 times by using PBST, adding a single domain antibody library which is respectively amplified in the step of 1 muL/hole as a primary antibody (a library construction carrier with M13), acting at 37 ℃ for 2h, washing for 3 times by using PBST, adding M13-HRP into a secondary antibody, detecting an OD450nm value after termination, and judging the result: the test piece was determined to be positive by 3 times higher than the control group OD450nm value (see Table 1 for the results).

Table 1: screening ELISA experiment to obtain positive clone

Example 3: sequencing identification of Positive clones

The clones which were detected as positive in example 2 by ELISA were extracted with plasmids from the corresponding bacterial solutions and sequenced using the universal primers for the plasmid vectors. Analyzing the gene sequence of each clone according to the sequence alignment software MEGA 6.0, regarding the strains with the same CDR1, CDR2 and CDR3 sequences as the same clone, and regarding the strains with different sequences as different clones, and finally obtaining 3 strains of S protein specific single domain antibody sequences. The nucleotide sequence of the antibody is SEQ ID NO: 1-3, the amino acid sequence is SEQ ID NO: 4-6 comprising the CDR1-CDR2-CDR3 regions as shown, constitute the VHH chain of the subject.

Example 4: expression, purification and identification of S protein single-domain antibody in Sf9 insect cell

(1) Construction of a pFastBac-VHH recombinant plasmid for an S protein single domain antibody: as set forth in SEQ ID NO:2, extracting plasmid as a template, and adopting a primer VHH-F of a VHH chain: ggatccgatg tgcagctgca ggagtctgg (SEQ ID NO: 16) and VHH-R: aagcttgctg gagacggtga cctgggtcc (SEQ ID NO:18), the upstream and downstream primers have BamH I and Hind III sites, and the amplified fragment is about 400 bp. In addition, after the pFastBac vector is subjected to double enzyme digestion by BamH I and Hind III, fragments are recovered, and then the VHH fragment is inserted into the pFastBac vector to form the pFastBac-VHH recombinant plasmid.

(2) Bacmid-VHH Bacmid screening for S-protein single domain antibodies: and after the pFastBac-VHH recombinant plasmid is extracted aseptically, the recombinant plasmid is transformed into DH10Bac competent cells, the competent cells are coated on a Luria Agar selective plate, white spots are selected after 48 hours and inoculated into selective KTG/LB culture solution, the plasmid is extracted after culture, M13 primer is used for amplification and sequencing identification, and the positive clone is Bacmid-VHH Bacmid.

(3) Recombinant viral packaging of S protein single domain antibodies: sf9 cells are cultured to 80% in a 6-well plate by using a complete culture medium of 2 mL/well, the obtained recombinant Bacmid-VHH plasmid is used for transfecting Sf9 cells by using a liposome Cellffectine reagent, the cells are swollen and rounded after 96 h, the refractivity is enhanced, the intercellular tight connection disappears, the cell nucleus is enlarged, vacuoles appear in the nucleus, the cells in the late stage of infection gradually fall off and other typical CPE, and the normal control Sf9 cells are uniform in size, compact in cell nucleus and free of cell lesions, so that the successful packaging of the recombinant baculovirus is indicated.

(4) Secretory expression and identification of S protein single domain antibody: after successful packaging of the recombinant baculovirus, the pFastBac vector (vector with 6 × His tag) was used, so that the expression of the gene could be identified with anti-His antibody. Western blot detection is carried out on the supernatant and cells after the recombinant baculovirus is infected with Sf9 cells, and the result shows that a 15kD band (shown in figure 1A: Marker: protein Marker; Lane 1: protein electrophoresis control; Lane 2: S protein single domain antibody SDS electrophoresis) appears in the supernatant and cells of the Sf9 cells infected with the recombinant virus, and the molecular weight of the band is consistent with that of the single domain antibody.

(5) Purification of single domain antibody of protein S: transferring the High Five cells into a T75 cell bottle, infecting the High Five cells with recombinant baculovirus Bacmid-VHH by MOI = 8 after the cells grow to 70% -80% abundance in an adherent manner, collecting culture supernatant of the infected cells after the High Five cells grow to be large, round and shed and other lesions after infecting for 96 h, centrifuging for 10 min at 4 ℃ and 2000 rpm, collecting the culture supernatant, firstly precipitating with 35% ammonium sulfate, and then passing through Ni ⁺ NTA affinity column purification and quantification of each recovered purified protein using the Qubit 2.0 quantification kit.

Example 5: specificity identification of S protein single domain antibody

Western blot identification specificity of S protein single domain antibody:

protein A electrophoresis: respectively diluting the purified S protein, influenza A HA protein, influenza B HA protein, parainfluenza virus HN protein and respiratory syncytial virus G protein to 60 mu G by using 6 x SDS protein electrophoresis loading buffer solution, boiling for 5min at 100 ℃, pre-staining Marker 2 mu L, carrying out protein electrophoresis concentrated gel 80 v, and separating gel 120 v.

B. Film transfer:placing the gel on a nitrocellulose membrane (NC membrane), placing 3 Whatman 3 mm filter papers on the upper and lower sides respectively, and soaking the above materials in a membrane-transfer electrophoresis buffer for 15min to remove the air bubbles remained on the filter membrane. The electrotransfer device is installed in sequence, 3 pieces of filter paper, gel, NC membrane and 3 pieces of filter paper are sequentially placed on the negative electrode plate, each layer is ensured to be accurately aligned (from bottom to top), bubbles between each layer are removed, the position is marked, and the anode plate is closed. 2 mA/cm ² And (5) rotating the membrane under the constant current condition of 2 h.

C. Western blot identification: sealing with 5% skimmed milk powder at 4 deg.C overnight; adding purified His-tagged S protein single-domain antibody (5% skimmed milk diluted at 1: 3000), and incubating the membrane at room temperature for 3 h; washing membrane with PBST (Tween 1 ‰) for three times, each for 10 min; adding Dy800 antibody (diluted with 5% skimmed milk at a ratio of 1: 20000) of goat anti-His, and incubating at room temperature in dark for 40 min; finally, 700 and 800 fluorescence channels of Odyssey are used for scanning identification (see 1B in figure 1, Lane 3: WB identification of S protein loading, Lane 4-Lane 7: WB identification of HA protein of influenza A, HA protein of influenza B, HN protein of parainfluenza virus and G protein of respiratory syncytial virus), which shows that the S protein single domain antibody is specifically combined with the S protein and does not have cross reaction with other 4 respiratory pathogen proteins.

Example 6: neutralization activity and stability identification of S protein single-domain antibody

Validation was performed in the P3 laboratory using a microcytoneutralization assay. The S protein single domain antibody is diluted by 1: 2-1: 128 in a multiple ratio by using normal saline, 1 part of immune serum is used as a positive control, and the dilution is also carried out in a multiple ratio of 1: 2-1: 128, and each dilution is provided with 5 times. 100TCID is added into each hole of the culture plate with the diluted sample to be detected ₅₀ And simultaneously setting a multiple dilution hole for negative serum control, and adding the same amount of diluent. All plates were placed in a 37 ℃ cell incubator and neutralized for 2 h. And adding the Vero cell suspension into each well after neutralization, and placing the mixture in an incubator at 37 ℃ for 5 days. Meanwhile, a normal cell control is set. After 5 days, each culture well was observed and counted for lesions, i.e., to protect 50% of the cells from 100TCID ₅₀ Highest dilution of serum for viral infectionThe antibody titer of the serum was measured, and the whole experiment was repeated 3 times. FIG. 2 is the identification of the neutralizing activity of the S protein single domain antibody of the novel coronavirus omicron strain. The final detection titer of the S protein single-domain antibody is 1:64, and the neutralization titer of the positive control serum is 1:32 (the determination result is positive if ≧ 1: 4). The neutralizing activity test result shows that the S protein single domain antibody has higher neutralizing titer (1: 32) and the coefficient of variation of 3 repeated tests<3%, indicating that the single domain antibody has excellent stability.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

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Claims

1. A single domain antibody VHH-2 specifically binding to the S protein of the novel coronavirus omicron strain, characterized in that the VHH-2 comprises a CDR1 shown in SEQ ID NO. 10, a CDR2 shown in SEQ ID NO. 11 and a CDR3 shown in SEQ ID NO. 12.

2. The single domain antibody VHH-2 that specifically binds to the S protein of the novel coronavirus omicron strain of claim 1, wherein the amino acid sequence of VHH-2 is represented by SEQ ID NO. 5.

3. A nucleic acid molecule encoding the single domain antibody VHH-2 of claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein the sequence of said nucleic acid molecule is set forth in SEQ ID NO. 2.

5. A method for preparing the single domain antibody VHH-2 specifically binding to the S protein of the novel coronavirus omicron strain of claim 1 or 2, which comprises inserting a nucleic acid encoding the VHH-2 into an expression vector, transferring the expression vector into a host cell, culturing the host cell, and purifying to obtain the single domain antibody.

6. The method according to claim 5, wherein the expression vector is a baculovirus expression vector pFastBac and the host cell is an sf9 insect cell.

7. Use of the single domain antibody VHH-2 specifically binding to the S protein of the new coronavirus omicron strain of claim 1 or 2 for the manufacture of a product for the diagnosis of the new coronavirus omicron strain.

8. Use of the single domain antibody VHH-2 specifically binding to the S protein of the novel coronavirus omicron strain of claim 1 or 2 for the preparation of a product for the treatment of the novel coronavirus omicron strain.

9. A pharmaceutical composition comprising the single domain antibody VHH-2 specifically binding to the S protein of the novel coronavirus omicron strain of claim 1 or 2 and a pharmaceutically acceptable carrier thereof.