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CN114163523B - Single-domain antibody for novel coronavirus and application thereof - Google Patents

Single-domain antibody for novel coronavirus and application thereof Download PDF

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CN114163523B
CN114163523B CN202210050323.6A CN202210050323A CN114163523B CN 114163523 B CN114163523 B CN 114163523B CN 202210050323 A CN202210050323 A CN 202210050323A CN 114163523 B CN114163523 B CN 114163523B
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CN114163523A (en
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杨威
迟晓静
刘秀英
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Beijing Kawin Technology Share Holding Co ltd
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Abstract

The invention discloses a humanized single domain antibody aiming at novel coronavirus SARS-CoV-2 and application thereof. The invention protects the single domain antibody of any one of SEQ ID No. 1-SEQ ID No.5, and the experimental result proves that the single domain antibody provided by the invention has good affinity with RBD antigen and high neutralization activity on SARS-CoV-2 pseudotype virus. The invention has important scientific significance and application prospect for the prevention, clinical treatment and research and development of diagnostic reagents of diseases caused by novel coronavirus SARS-CoV-2.

Description

Single-domain antibody for novel coronavirus and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a humanized single domain antibody aiming at novel coronavirus SARS-CoV-2 and application thereof.
Background
Antibodies are a class of immunoglobulins that specifically bind to an antigen. Antibody drugs are generally known as whole antibody molecules and antibody fragments with therapeutic functions obtained by advanced techniques, and are one of the important means of targeted therapy. Specific targets for antibody drugs include, but are not limited to: cancer cell specific antigens, immune checkpoint molecules, cytokines and their receptors, viral proteins, bacterial toxins, and the like. The main disease types treated by antibody drugs include various cancers, hematopathy, autoimmune diseases such as rheumatoid arthritis, cardiovascular diseases, viral infections, etc. In addition to being a therapeutic means, antibodies are also often used in the fields of prevention, diagnosis and detection of diseases.
Heavy chain antibodies are a naturally occurring type of antibody that was first reported by Hamers doctor at the university of Brussels free Belgium and its team in 1993 (Hamers-Casterman C, atarhouch T, muyldermans Set al. Nature occurring antibodies devoid of light chans. Nature 1993; 363:446-8.). Heavy chain antibodies are unique antibody types that camelids or cartilaginous fish possess, whose antibody domains naturally lack the light chain, consisting of only two heavy chains. The antigen recognition function of heavy chain antibodies is largely determined by the variable region (VHH) of heavy chain antibodies. VHH alone recognizes antigens and is therefore also known as Single-domain antibodies (sdabs). The molecular weight of the single domain antibody is only about 13-15kDa, the diameter is about 2.5nm, and the length is 4nm, so far the smallest antibody fragment with antigen binding function.
Through many years of research, single domain antibodies gradually exhibit very excellent molecular properties and patency. In addition to the antigen binding capacity of conventional antibodies, single domain antibodies have many unique properties, such as good stability, specific epitope accessibility, arbitrary combination of building block patterns, low production cost, etc., compared to conventional antibodies, and thus have broad prospects in both pharmaceutical applications and clinical diagnostics (Muydermans S. Nanobodies: natural single-domain antibodies. Annu Rev biochem.2013;82: 775-97). The conventional method for obtaining single domain antibodies is constituted by numerous steps of immunization of camelids, B lymphocyte isolation, VHH region amplification, phage library construction and screening. With the development of synthetic biology, it becomes possible to construct high-quality randomized large-capacity single domain antibody libraries based on total synthetic humanization.
The coronaviridae family is a family of single-stranded positive-strand RNA viruses that primarily infect vertebrates, whose genomes are relatively large among the known RNA viruses. Coronavirus particles are typically characterized by an electron microscope that exhibits a "imperial crown" like morphology formed by numerous Spike proteins (Spike, S proteins) distributed on the viral envelope. The coronaviridae contains 4 viral genera, respectively: alpha coronavirus genus, beta coronavirus genus, gamma coronavirus genus and delta coronavirus genus. Coronaviruses associated with human infection are mainly alpha and beta coronavirus members. Specific virus members include: 229E, NL63, OC43, HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2. Coronaviruses are commonly infected via the respiratory or fecal route and can cause a variety of diseases such as: common cold, bronchitis, pneumonia, gastroenteritis, heart disease, etc. Certain coronaviruses have a high mortality rate and a high transmission rate, and can cause serious social and public health problems.
The early onset of new coronavirus (SARS-CoV-2), also known as 2019-nCoV, a member of the genus beta coronavirus, a new pathogen, has a homology of about 80% to SARS-CoV, mainly causing human new coronavirus pneumonia (COVID-19). SARS-CoV-2 virus can infect a variety of cells in vitro, including Vero-E6, huh7, calu-3, and differentiated human airway epithelial cells, among others. SARS-CoV-2 virus is a positive strand RNA virus with an envelope. The virus particle diameter is about 80-140 nm. The envelope is a lipid bilayer and contains 3 kinds of envelope glycoproteins: s protein, E protein and M protein. The S protein is one of the major proteins forming the viral "corona" morphology, mediating SARS-CoV-2 entry into the cell. The S protein of SARS-CoV-2 consists of 1273 amino acids and structurally belongs to type I membrane fusion protein, and is divided into two regions of S1 and S2. The S1 region mainly comprises the receptor binding region (receptor binding domain, RBD), while the S2 region is essential for membrane fusion. A potential receptor for SARS-CoV-2 is human angiotensin converting enzyme 2 (human angiotensin converting enzyme, ACE 2). The RBD structure of SARS-CoV-2 virus determines its binding efficiency to the potential receptor ACE2 and the species specificity of infection, and is an important neutralizing antibody recognition and development target.
The SARS-CoV-2 virus is potentially originated from bat SARS-like coronavirus, and the process of infecting human is completed through the transition of intermediate hosts, so that it is known that the SARS-CoV-2 has stronger transmission power between people and higher pathogenicity than coronavirus causing common cold. Because SARS-CoV-2 is a new pathogen, antibodies are commonly lacking in the population, and thus the population is generally susceptible to SARS-CoV-2. Through gene sequence comparison analysis, the RBD region of SARS-CoV-2 has extremely low homology with common cold coronavirus and MERS-CoV, and even if compared with the RBD region of SARS-CoV, a large number of mutations exist, so that the known neutralizing antibodies against SARS-CoV and MERS-CoV generally lack cross protection effect with SARS-CoV-2 new pathogens. In view of the above, development of specific antibodies against the SARS-CoV-2 spike protein RBD region has important scientific significance and application prospect for clinical treatment of diseases and development of diagnostic reagents.
Disclosure of Invention
The invention aims to provide a humanized single domain antibody aiming at novel coronavirus SARS-CoV-2 and application thereof.
In a first aspect, the present invention provides a single domain antibody comprising complementarity determining regions CDR1, CDR2 and CDR3;
the single domain antibody is any one of the following (a 1) to (a 5):
(a1) A single domain antibody comprising CDR1 shown in positions 26 to 34, CDR2 shown in positions 51 to 58 and CDR3 shown in positions 97 to 114 of SEQ ID No.1 from the N-terminus, or a functionally active variant thereof;
(a2) A single domain antibody comprising CDR1 shown in positions 26 to 34, CDR2 shown in positions 51 to 58 and CDR3 shown in positions 97 to 114 of SEQ ID No.2 from the N-terminus, or a functionally active variant thereof;
(a3) A single domain antibody comprising CDR1 shown in positions 26 to 34, CDR2 shown in positions 51 to 58 and CDR3 shown in positions 97 to 114 of SEQ ID No.3 from the N-terminus, or a functionally active variant thereof;
(a4) A single domain antibody comprising CDR1 shown in positions 26 to 34, CDR2 shown in positions 51 to 58 and CDR3 shown in positions 97 to 114 of SEQ ID No.4 from the N-terminus, or a functionally active variant thereof;
(a5) A single domain antibody comprising CDR1 shown in positions 26 to 32, CDR2 shown in positions 49 to 56 and CDR3 shown in positions 95 to 112 of SEQ ID No.5 from the N-terminus, or a functionally active variant thereof.
The single domain antibody further comprises framework regions FR1, FR2, FR3 and FR4;
the FR1 is shown as 1-25 th bit of SEQ ID No.1 from N end or 1-25 th bit of SEQ ID No.2 from N end or 1-25 th bit of SEQ ID No.3 from N end or 1-25 th bit of SEQ ID No.4 from N end or 1-25 th bit of SEQ ID No.5 from N end;
the FR2 is shown as 35 to 50 bits of SEQ ID No.1 from the N end or 35 to 50 bits of SEQ ID No.2 from the N end or 35 to 50 bits of SEQ ID No.3 from the N end or 35 to 50 bits of SEQ ID No.4 from the N end or 33 to 48 bits of SEQ ID No.5 from the N end;
the FR3 is shown as 59 th to 96 th positions of SEQ ID No.1 from the N end or 59 th to 96 th positions of SEQ ID No.2 from the N end or 59 th to 96 th positions of SEQ ID No.3 from the N end or 59 th to 96 th positions of SEQ ID No.4 from the N end or 57 th to 94 th positions of SEQ ID No.5 from the N end;
the FR4 is shown as 115-125 th bit of SEQ ID No.1 from N terminal or 115-125 th bit of SEQ ID No.2 from N terminal or 115-125 th bit of SEQ ID No.3 from N terminal or 115-125 th bit of SEQ ID No.4 from N terminal or 113-123 th bit of SEQ ID No.5 from N terminal.
The single domain antibody is any one of the following (b 1) - (b 5):
(b1) A functionally active variant of the single domain antibody shown in SEQ ID No. 1;
(b2) A functionally active variant of the single domain antibody shown in SEQ ID No. 2;
(b3) A functionally active variant of the single domain antibody shown in SEQ ID No. 3;
(b4) A functionally active variant of the single domain antibody shown in SEQ ID No. 4;
(b5) The single domain antibody shown in SEQ ID No.5, a functionally active variant thereof.
The functionally active variant is (a) or (b) or (c) as follows:
(a) A polypeptide having the same function and obtained by substituting and/or deleting and/or adding 1 or 2 or 3 amino acid residues to the amino acid sequence of any one of the single domain antibodies;
(b) A polypeptide having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the amino acid sequence of any of the above single domain antibodies and having the same function;
(c) And (3) connecting the N end and/or the C end of any single domain antibody with a label to obtain the fusion protein.
The tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
In a second aspect, the invention also provides a nucleic acid molecule encoding a single domain antibody as described in any one of the preceding.
The nucleic acid molecule is as described in any one of (1) to (7) below;
(1) A DNA molecule shown in SEQ ID No. 1;
(2) A DNA molecule shown in SEQ ID No. 2;
(3) A DNA molecule shown in SEQ ID No. 3;
(4) A DNA molecule shown in SEQ ID No. 4;
(5) A DNA molecule shown in SEQ ID No. 5;
(6) A DNA molecule which hybridizes under stringent conditions to a DNA molecule as defined in any one of (1) to (5) and which encodes an antibody as described above;
(7) A DNA molecule having 80% or more or 90% or more homology with the DNA molecule defined in any one of (1) to (5) and encoding the antibody described above.
The stringent conditions are hybridization and washing of the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing of the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time.
In a third aspect, the invention also provides an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising a nucleic acid molecule as described hereinbefore.
In a fourth aspect, the invention provides the use of a single domain antibody or nucleic acid molecule or expression cassette, recombinant vector, recombinant bacterium or transgenic cell line as described in any of the preceding paragraphs for the preparation of a product; the use of the product is as follows (c 1) and/or (c 2):
(c1) Preventing and/or treating diseases caused by SARS-CoV-2 infection of the novel coronavirus;
(c2) Inhibit SARS-CoV-2 infection of coronavirus.
The disease caused by SARS-CoV-2 infection is specifically human coronavirus pneumonia (COVID-19).
In a fifth aspect, the invention provides the use of a single domain antibody or nucleic acid molecule or expression cassette, recombinant vector, recombinant bacterium or transgenic cell line as described in any of the preceding paragraphs for the preparation of a product; the use of the product is any one of the following (d 1) - (d 4):
(d1) Binding to the novel coronavirus SARS-CoV-2;
(d2) Detecting the binding of novel coronavirus SARS-CoV-2;
(d3) An S protein combined with a novel coronavirus SARS-CoV-2;
(d4) The S protein of the novel coronavirus SARS-CoV-2 is detected.
In a sixth aspect, the invention provides a protective product comprising a single domain antibody as described in any one of the preceding claims; the use of the product is as follows (c 1) and/or (c 2):
(c1) Preventing and/or treating diseases caused by SARS-CoV-2 infection of the novel coronavirus;
(c2) Inhibit SARS-CoV-2 infection of coronavirus.
The disease caused by SARS-CoV-2 infection is specifically human coronavirus pneumonia (COVID-19).
In a seventh aspect, the invention provides a protective product comprising a single domain antibody as described in any one of the preceding claims; the use of the product is any one of the following (d 1) - (d 4):
(d1) Binding to the novel coronavirus SARS-CoV-2;
(d2) Detecting the binding of novel coronavirus SARS-CoV-2;
(d3) An S protein combined with a novel coronavirus SARS-CoV-2;
(d4) The S protein of the novel coronavirus SARS-CoV-2 is detected.
Any of the above products may be specifically a drug.
In an eighth aspect, the pharmaceutical composition of the invention comprises any of the single domain antibodies described above and a pharmaceutically acceptable excipient, diluent or carrier.
The humanized single domain antibody for SARS-CoV-2 is obtained through phage library screening, and the experiment result proves that the single domain antibody provided by the invention has good affinity with RBD antigen and high neutralization activity for SARS-CoV-2 pseudotype virus. The invention has important scientific significance and application prospect for the prevention, clinical treatment and research and development of diagnostic reagents of diseases caused by novel coronavirus SARS-CoV-2.
Drawings
FIG. 1 is a statistical result of the activity of the single domain antibody 1E2 in inhibiting SARS-CoV-2 pseudotyped virus invading cells.
FIG. 2 is a graph showing the statistical results of the activity of the single domain antibody 2F2 in inhibiting SARS-CoV-2 pseudotyped virus invading cells.
FIG. 3 is a statistical result of the activity of the single domain antibody 3F11 in inhibiting SARS-CoV-2 pseudotyped virus invading cells.
FIG. 4 is a statistical result of the activity of the single domain antibody 4D8 in inhibiting SARS-CoV-2 pseudotyped virus invading cells.
FIG. 5 is a statistical result of the activity of the single domain antibody 5F8 in inhibiting SARS-CoV-2 pseudotyped virus invading cells.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Example 1 screening and preparation of Single-Domain antibodies recognizing the RBD region of SARS-CoV-2 spike protein
1. Phage library screening
Basic principles and basic protocols of phage library screening reference Shao Ningsheng et al, "biological library technology-phage display and SELEX technology", briefly described below: firstly, 50 mug SARS-CoV-2 spike protein RBD region recombinant protein (product number: 40592-V08H; hereinafter RBD protein) is commercialized by Simer Feishier technology Co., ltd TM The Sulfo NHS-LC-LC-Biotin was labeled with Biotin and desalted and purified (desalting column was purchased from Sesameiser technologies). The first round of screening was a liquid phase selection by first combining 12 μg of biotin-labeled RBD protein with 1mL (10 12 -10 13 ) Incubating the phage library mixture at room temperature for 1 hour; subsequently, 100. Mu.L of streptomycin affinity beads (GE life sciences) were added, and the turntable was rotated and incubated at room temperature for 30 minutes; then washing the magnetic beads with PBS-T and PBS for 10-15 times, and adding 400 mu L of eluent into the magnetic beads for eluting for 5min; 200. Mu.L of the eluent was added to 5mL of a logarithmic growth phase TG1 E.coli culture and incubated at 37℃for 1 hour; after infection of the helper phage CM13 with the above culture, the culture was expanded overnight and the phage in the supernatant was purified by PEG6000 (Sigma Co.) on the next day for the next round of screening. The second round of screening was solid phase selection by first coating 50. Mu.g of RBD protein at 4℃overnight in a 5mL immunoadsorption tube followed byThe immune tubes were then washed 3 times with PBS and 1mL of the VHH library 10 harvested by the first round of screening was added 12 Phage, rotating the turntable for 30 minutes at room temperature, and standing for incubation for 1.5 hours; then PBS-T and PBS are used for washing the immune tube for 15-20 times, and eluent is added for eluting; half of the eluent was added to 5mL of a TG1 E.coli culture in the logarithmic growth phase and incubated at 37℃for 1 hour; after infection of the helper phage CM13 with the above culture, the culture was expanded overnight and the phage in the supernatant was purified with PEG the next day for the next round of screening. A third round of screening, which is a liquid phase screening, and a fourth round of screening, which is a solid phase screening, were performed using a similar method. After the last round of screening, 1000 individual clones were picked for culture and phage rescue for subsequent identification of positive clones.
2. Phage ELISA assays identified positive clones that bound to RBD protein.
Basic principles and basic procedures of enzyme-linked immunosorbent assays are described in immunological techniques and applications by Cao Xuetao et al. Briefly described as follows: RBD protein 25 ng/well was coated overnight in 96 well immunoplates. After blocking the multiwell plates with BSA, 100. Mu.L/well of phage was rescued in the procedure described above and incubated at room temperature for 1 hour. After washing the plate 5 times with PBS-T, anti-phage antibody Anti-M13 Anti-body [ B62-FE2] (HRP) (Abcam company #ab50370) was added, and incubated at room temperature for 1 hour. After washing the plate 6 times with PBS-T, HRP-labeled secondary antibody was added and incubated at room temperature for 0.5 hours. After washing the plate with PBS-T6 times to remove unbound antibody, 100. Mu.L of TMB substrate (Beijing Soy Bao Co.) was added to each well, incubated at 37℃in the dark for 5-30 minutes, stop solution (50. Mu.L) was added to each well to terminate the reaction, the experimental results were measured within 20 minutes, and absorbance was read at 450 nm. A group of negative control measurement holes are arranged in the experiment, and positive results can be judged when the ratio of the sample holes to the control holes is greater than 5.
Through the method, more than 200 positive clones are obtained through screening, and sequencing and homology comparison analysis are carried out on all positive clones, so that the results show that all positive clones can be summarized into 5 independent clones, and representative clones are respectively: 1E2, 2F2, 3F11, 4D8 and 5F8.
3. Obtaining anti-RBD Single-Domain antibodies
And screening, namely amplifying the complete reading frame of the single-domain antibody by the positive clone through high-fidelity PCR to obtain the encoding nucleic acid molecule of the RBD-resistant single-domain antibody and the encoded amino acid sequence thereof.
Five single-domain antibodies are obtained by co-screening, and the single-domain antibodies sequentially consist of a framework region FR1, a complementarity determining region CDR1, a framework region FR2, a complementarity determining region CDR2, a framework region FR3, a complementarity determining region CDR3 and a framework region FR4, wherein specific information is as follows:
the amino acid sequence of the single domain antibody 1E2 is shown as SEQ ID No. 1. SEQ ID No.1 shows the framework region FR1 at positions 1-25, the complementarity determining region CDR1 at positions 26-34, the framework region FR2 at positions 35-50, the complementarity determining region CDR2 at positions 51-58, the framework region FR3 at positions 59-96, the complementarity determining region CDR3 at positions 97-114 and the framework region FR4 at positions 115-125 from the N-terminus. The nucleic acid molecule encoding the single domain antibody 1E2 is shown in SEQ ID No. 6.
The amino acid sequence of the single domain antibody 2F2 is shown as SEQ ID No. 2. SEQ ID No.2 shows the framework region FR1 at positions 1-25, the complementarity determining region CDR1 at positions 26-34, the framework region FR2 at positions 35-50, the complementarity determining region CDR2 at positions 51-58, the framework region FR3 at positions 59-96, the complementarity determining region CDR3 at positions 97-114 and the framework region FR4 at positions 115-125 from the N-terminus. The nucleic acid molecule encoding the single domain antibody 2F2 is shown in SEQ ID No. 7.
The amino acid sequence of the single domain antibody 3F11 is shown as SEQ ID No. 3. SEQ ID No.3 shows the framework region FR1 at positions 1-25, the complementarity determining region CDR1 at positions 26-34, the framework region FR2 at positions 35-50, the complementarity determining region CDR2 at positions 51-58, the framework region FR3 at positions 59-96, the complementarity determining region CDR3 at positions 97-114 and the framework region FR4 at positions 115-125 from the N-terminus. The nucleic acid molecule encoding the single domain antibody 3F11 is shown in SEQ ID No. 8.
The amino acid sequence of the single domain antibody 4D8 is shown as SEQ ID No. 4. SEQ ID No.4 shows the framework region FR1 at positions 1-25, the complementarity determining region CDR1 at positions 26-34, the framework region FR2 at positions 35-50, the complementarity determining region CDR2 at positions 51-58, the framework region FR3 at positions 59-96, the complementarity determining region CDR3 at positions 97-114 and the framework region FR4 at positions 115-125 from the N-terminus. The nucleic acid molecule encoding the single domain antibody 4D8 is shown in SEQ ID No. 9.
The amino acid sequence of the single domain antibody 5F8 is shown as SEQ ID No. 5. SEQ ID No.5 shows the framework region FR1 at positions 1-25, the complementarity determining region CDR1 at positions 26-32, the framework region FR2 at positions 33-48, the complementarity determining region CDR2 at positions 49-56, the framework region FR3 at positions 57-94, the complementarity determining region CDR3 at positions 95-112 and the framework region FR4 at positions 113-123 from the N-terminus. The nucleic acid molecule encoding the single domain antibody 5F8 is shown in SEQ ID No. 10.
4. Prokaryotic expression vector construction for expressing anti-RBD single domain antibody
1. Prokaryotic expression vector for expressing single domain antibody 1E2
The DNA molecule 1 was used to replace the DNA molecule between NcoI and XhoI of the prokaryotic expression vector pET-28b (Novagen, cat# 69865-3) to obtain a prokaryotic expression vector expressing the single domain antibody 1E2 (fused with a histidine tag). DNA molecule 1 was obtained by adding XhoI cleavage site and 6 histidine-tagged coding sequence (ctcgagcaccaccaccaccaccac) to the 3' -end of SEQ ID No. 6.
2. Prokaryotic expression vector for expressing single domain antibody 2F2
And (3) replacing the DNA molecule between NcoI and XhoI of the prokaryotic expression vector pET-28b by using the DNA molecule 2 to obtain the prokaryotic expression vector for expressing the single-domain antibody 2F2 (fused with a histidine tag). DNA molecule 2 was obtained by adding XhoI cleavage site and 6 histidine-tagged coding sequence (ctcgagcaccaccaccaccaccac) to the 3' -end of SEQ ID No. 7.
3. Prokaryotic expression vector for expressing single domain antibody 3F11
The DNA molecule 3 is adopted to replace the DNA molecule between NcoI and XhoI of the prokaryotic expression vector pET-28b, so as to obtain the prokaryotic expression vector for expressing the single-domain antibody 3F11 (fused with a histidine tag). DNA molecule 3 was obtained by adding XhoI cleavage site and 6 histidine-tagged coding sequence (ctcgagcaccaccaccaccaccac) to the 3' -end of SEQ ID No. 8.
4. Prokaryotic expression vector for expressing single domain antibody 4D8
The DNA molecule 4 is used for replacing the DNA molecule between NcoI and XhoI of the prokaryotic expression vector pET-28b, so as to obtain the prokaryotic expression vector for expressing the single-domain antibody 4D8 (fused with a histidine tag). DNA molecule 4 was obtained by adding XhoI cleavage site and 6 histidine-tagged coding sequence (ctcgagcaccaccaccaccaccac) to the 3' -end of SEQ ID No. 9.
5. Prokaryotic expression vector for expressing single domain antibody 5F8
The DNA molecule 5 is used for replacing the DNA molecule between NcoI and XhoI of the prokaryotic expression vector pET-28b, so as to obtain the prokaryotic expression vector for expressing the single-domain antibody 5F8 (fused with a histidine tag). DNA molecule 5 was obtained by adding XhoI cleavage site and 6 histidine-tagged coding sequence (ctcgagcaccaccaccaccaccac) to the 3' -end of SEQ ID No. 10.
5. Expression and purification of anti-RBD single domain antibodies
1. Expression of anti-RBD single domain antibodies
And (3) respectively converting the five prokaryotic expression vectors obtained in the step four into BL21 (DE 3) escherichia coli competent cells (purchased from Beijing full-scale gold company) to obtain recombinant bacteria. Inoculating the recombinant strain into LB liquid medium, culturing at 37deg.C and 230rpm to OD 600 =0.5, then IPTG was added to the culture system at a final concentration of 0.3mM and the induction culture was continued at 18 ℃ at 230rpm for 16 hours, after which the cells were harvested by centrifugation and stored at-80 ℃ for use.
2. Purification of anti-RBD single domain antibodies
(1) Lysing bacteria
The cells obtained in step 1 were resuspended in lysis buffer (50 mM Tris-Cl pH8.0, 100mM NaCl,5mMImidazole), sonicated, and centrifuged at 12000 Xg for 15 minutes, and the supernatant was transferred to a new tube.
(2) Affinity chromatography
The supernatant obtained in the above step (1) was passed through a Ni-NTA agarose column (GE life sciences) on an AKTA Purifier (GE life sciences), unbound protein was washed off with a washing solution (50 mM Tris-Cl pH8.0, 300mM NaCl,10mMImidazole) of 20 column volumes, and finally, 0-500mM imidazole was eluted linearly (Sigma Co.) and protein peaks were collected in fractions by using the apparatus self-contained program, 1 tube was collected for each 1mL of eluted protein, and the purified anti-RBD single domain antibody was obtained by SDS-PAGE identification.
And (3) loading the purified anti-RBD single-domain antibody into a dialysis bag, dialyzing overnight to replace a PBS buffer system, and completing the steps of concentration and the like to obtain five single-domain antibody solutions.
Example 2 affinity assay for anti-RBD Single-Domain antibodies
Antibody to be tested: five single domain antibodies prepared in example 1.
The affinity determination of antibodies was performed using a BIAcore T-200 biomolecular interaction apparatus (GE life sciences), which was developed based on surface plasmon resonance SPR technology, and allows label-free, rapid, real-time, automated manipulation and detection of interactions between biological macromolecules.
When the interaction between RBD protein and corresponding single-domain antibody is measured, the RBD protein is coated on a sensor chip, and then the single-domain antibody is used as a mobile phase to measure the binding constant, dissociation constant and affinity constant.
1. RBD protein coupled CM5 chip: the RBD protein coupling temperature is 25 ℃, and the buffer is HBS-P (10mM HEPES,150mM NaCl,3mM EDTA and 0.05%P20,pH 7.4). The program template is Immobilisation, CM5 chip channel 2 amino coupling is selected, ligand is RBD protein of 5 mug/ml, protein buffer system is sodium acetate (NaAC5.5) of pH5.5, target coupling amount is 300RU, eluent is 50mM NaOH. Chip activator 50mmol/L N-hydroxysuccinimide (NHS) and 200 mmol/L3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) activated chip, and blocking agent 1mol/L ethanolamine hydrochloride.
2. RBD single domain antibodies with RBD affinity and kinetic assay: a multi-cycle kinetic template was selected, the assay temperature was 25 ℃, the buffer was HBS-P, the sample flow path was 2-1, the sample binding time was 180s, the flow rate was 30ul/min, the dissociation time was 300s, the regeneration eluent was Glycine-HCL2.5, the regeneration liquid binding time was 30s, the flow rate was 30ul/min, the stabilization time was 0s, and the single domain antibody concentrations were serially diluted (3.125, 6.25, 12.5, 25, 50 nM). The resulting data were analyzed by Biacore Evaluation Software software to calculate the binding constant (ka), dissociation constant (KD) and affinity constant (KD).
The chips, reagents and buffers used in the Biacore assays described above were all purchased from GE life sciences.
The results are shown in Table 1. The results show that the affinity constant of the obtained single domain antibody of the invention with SARS-CoV-2RBD protein is between 0.71nMol/L and 33.9 nMol/L. The lower KD value indicates stronger affinity, and the obtained single domain antibodies have ideal affinity with RBD proteins as a whole. From the results, the antibodies 2F2 and 5F8 are preferably 3F11 times, and 1E2 and 4D8 are again used.
Table 1 affinity parameters of Single Domain antibodies interaction with SARS-CoV-2RBD protein
Antibody numbering KD(M) ka(1/Ms) kd(1/s)
1E2 2.113E-8 2.451E+5 0.005179
2F2 8.461E-10 3.701E+5 3.131E-4
3F11 3.154E-9 2.141E+6 0.006751
4D8 3.397E-8 1.804E+5 0.006128
5F8 7.155E-10 2.954E+5 2.114E-4
EXAMPLE 3 determination of SARS-CoV-2 pseudotype Virus neutralization Activity of anti-RBD Single-Domain antibodies
Antibody to be tested: five single domain antibodies prepared in example 1.
A novel virus particle is formed by assembling the replication core element of a retrovirus with the envelope spike glycoprotein (S protein) of SARS-CoV-2 virus. The ability of pseudoviruses to infect cells depends on the nature and character of the glycoprotein it coats and is an ideal tool for studying the neutralizing antibody inhibition efficiency, receptor utilization and invasion infection mechanism of SARS-CoV-2.
1. Packaging preparation of SARS-CoV-2 pseudotype virus
293T cells (from basic medical institute of China medical sciences) were inoculated into 10 cm dishes, cultured to 80% cell confluence with DMEM medium (from Simer Feishmania) containing 10% fetal bovine serum (from Gibco Co.), and co-transfected with 15. Mu.g SARS-CoV-2S gene expression plasmid (product number: VG 40589-UT; beijing Yinqiao Shenzhou science and technology Co.) and 15. Mu.g PNL4.3-Luc-R - E - Plasmid (Biovector NTCC), DMEM medium containing 2% fetal bovine serum was replaced 36 hours after transfection, culture was continued for 12 hours and culture supernatants containing SARS-CoV-2 pseudotype virus were harvested, sub-packaged and frozen for long term storage at-80 ℃.
2. Pseudovirus invasion inhibition assay
The SARS-CoV-2 pseudotyped virus prepared in the first step was mixed with different dilutions of single domain antibodies (the final concentration of the single domain antibodies contained was 0, 0.000832, 0.00416, 0.0208, 0.104, 0.52, 2.6, 13. Mu.g/mL, respectively) and added to a pre-inoculated 96-well plate containing Calu-3 cells (purchased from basic medical institute of China medical sciences) and incubated for 48 hours. SARS-CoV-2 pseudotype virus contains luciferase reporter gene, and the pseudotype virus has the capability of infecting target cell of interest, and can be used for measuring infectivity and level of pseudotype virus by means of detecting reporter gene, etc. Cells were lysed according to the product instructions and the reporter activity in the cell lysate was detected using the luciferase reporter assay kit from Promega company (cat# E4550), and the raw readouts of luciferase were converted into percentage data and plotted and IC was calculated 50 。
The results are shown in FIGS. 1 to 5.
The result shows that the single domain antibody obtained by the invention has excellent capability of inhibiting SARS-CoV-2 pseudotyped virus infection, and the antiviral effect is as follows from high to low: 5F8 (IC) 50 ＝0.00338μg/mL),3F11(IC 50 ＝0.0038μg/mL),2F2(IC 50 ＝0.0235μg/mL),4D8(IC 50 ＝0.247μg/mL),1E2(IC 50 ＝0.31μg/mL)。
Sequence listing
<110> Beijing Kain science and technology Co., ltd
<120> a single domain antibody against novel coronavirus and use thereof
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Ile Gly Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val
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aacgacttcg tttggcaggg ttactcttct gactattggg gccaggggac ccaagtaaca 360
gttagcagt 369

Claims (8)

1. A single domain antibody that specifically binds to a novel coronavirus SARS-CoV-2, comprising complementarity determining regions CDR1, CDR2, and CDR3;
the single domain antibody comprises a single domain antibody of CDR1 shown in 26 th to 34 th positions, CDR2 shown in 51 th to 58 th positions and CDR3 shown in 97 th to 114 th positions of the N end of SEQ ID No. 1.
2. The single domain antibody of claim 1, wherein:
the single domain antibody further comprises framework regions FR1, FR2, FR3 and FR4;
the FR1 is shown in the 1 st to 25 th positions of the N end of SEQ ID No. 1;
the FR2 is shown in 35 th to 50 th positions of the N end of SEQ ID No. 1;
FR3 is shown in SEQ ID No.1 from 59 th to 96 th positions of the N end;
FR4 is shown as SEQ ID No.1 from the 115 th to 125 th ends of the N terminal.
3. The single domain antibody of claim 1 or 2, wherein:
the single domain antibody is shown in SEQ ID No. 1.
4. A nucleic acid molecule encoding the single domain antibody of any one of claims 1 to 3.
5. An expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of claim 4.
6. Use of the single domain antibody of any one of claims 1 to 3 or the nucleic acid molecule of claim 4 or the expression cassette, recombinant vector, recombinant bacterium or transgenic cell line of claim 5 in the preparation of a product; the use of the product is as follows (c 1) and/or (c 2):
(c1) Treating diseases caused by SARS-CoV-2 infection of the novel coronavirus;
(c2) Inhibit SARS-CoV-2 infection of coronavirus.
7. Use of the single domain antibody of any one of claims 1 to 3 or the nucleic acid molecule of claim 4 or the expression cassette, recombinant vector, recombinant bacterium or transgenic cell line of claim 5 in the preparation of a product; the use of the product is any one of the following (d 1) - (d 4):
(d1) Binding to the novel coronavirus SARS-CoV-2;
(d2) Detecting the binding of novel coronavirus SARS-CoV-2;
(d3) An S protein combined with a novel coronavirus SARS-CoV-2;
(d4) The S protein of the novel coronavirus SARS-CoV-2 is detected.
8. A pharmaceutical composition comprising the single domain antibody of any one of claims 1 to 3 and a pharmaceutically acceptable excipient, diluent or carrier.
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