CN114605555A - Dual-specificity neutralizing antibody for resisting novel coronavirus SARS-CoV-2 and application thereof - Google Patents

Dual-specificity neutralizing antibody for resisting novel coronavirus SARS-CoV-2 and application thereof Download PDF

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CN114605555A
CN114605555A CN202210315128.1A CN202210315128A CN114605555A CN 114605555 A CN114605555 A CN 114605555A CN 202210315128 A CN202210315128 A CN 202210315128A CN 114605555 A CN114605555 A CN 114605555A
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CN114605555B (en
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王健伟
赵振东
任丽丽
黄鹤
朱悦
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Institute of Pathogen Biology of CAMS
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C12N2800/00—Nucleic acids vectors
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    • C12N2800/106—Plasmid DNA for vertebrates
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Abstract

The invention discloses a dual-specificity neutralizing antibody for resisting novel coronavirus SARS-CoV-2 and application thereof, belonging to the technical field of biological medicines. It is bispecific antibody A4-A7 or bispecific antibody A7-A4, both of which comprise Nanobody A4 and Nanobody A7, Nanobody A4 and Nanobody A7 both of which comprise variable regions, each of which has 3 complementarity determining regionsThe regions CDR1, CDR2 and CDR 3. The invention also discloses a nucleotide molecule for coding the anti-novel coronavirus SARS-CoV-2 bispecific neutralizing antibody, a recombinant plasmid, a recombinant bacterium and a transgenic cell line containing the nucleotide molecule and application. The bispecific neutralizing antibody against the novel coronavirus SARS-CoV-2 of the present invention has neutralizing ability, IC, against both SARS-CoV-2-related variant and concerned variant pseudotype virus50Reaching sub-nanomolar level and having good broad-spectrum neutralization activity.

Description

Dual-specificity neutralizing antibody for resisting novel coronavirus SARS-CoV-2 and application thereof
Technical Field
The invention relates to a dual-specificity neutralizing antibody for resisting novel coronavirus SARS-CoV-2 and application thereof, belonging to the technical field of biological medicines.
Background
The novel coronavirus SARS-CoV-2 is a pathogen that causes a novel coronavirus pneumonia pandemic. With the continuous expansion of the infected people, the evolution mutation of the original strain of the new coronavirus begins to appear, and the world health organization classifies the Variants into concerned Variants according to the propagation easiness of the Variants and the severity of related diseases (VOC): alpha, Beta, Gamma, Delta, Omicron; variants of interest (Variants of interest VOI): lambda, Mu, etc.
Neutralizing antibodies are antibodies produced by B cells that recognize and bind to pathogen surface antigens, preventing the pathogen from infecting the cells. Monoclonal antibodies against SARS-CoV-2 have been granted immediate authorization for FBA for treatment of mild to moderate SARS-CoV-2 infection.
A heavy chain antibody naturally occurs in camelids or cartilaginous fish and its antibody domain consists of only two heavy chains. Its antigen recognition function is mainly determined by the variable region (VHH) of the heavy chain antibody. VHH alone can recognize antigen with molecular weight of only 13-15KDa, diameter of about 2.5nm and length of 4nm, and is therefore also called nanobody (nanobody). The CDR3 region on the VHH is longer than the CDR3 region of conventional antibodies, so that the VHH can recognize epitopes that conventional antibodies typically cannot recognize.
At present, SARS-CoV-2 appears to be a plurality of variants, and the neutralizing activity of the existing neutralizing antibody against different variants appears to be reduced to a certain extent. In view of the above, there is a need to provide a neutralizing antibody with broad spectrum neutralizing activity in order to recognize and neutralize multiple SARS-CoV-2 mutant viruses and reduce the risk of immune escape of SARS-CoV-2 virus.
Disclosure of Invention
It is an object of the present invention to provide a bispecific neutralizing antibody against SARS-CoV-2, a novel coronavirus.
The technical scheme for solving the technical problems is as follows: a bispecific neutralizing antibody against novel coronavirus SARS-CoV-2, which is bispecific antibody a4-a7 or bispecific antibody a7-a4, each comprising nanobody a4 and nanobody a7, each of said nanobody a4 and said nanobody a7 comprising a variable region, each of said variable regions having 3 complementarity determining regions CDR1, CDR2 and CDR 3; the amino acid sequence of CDR1 of the nano antibody A4 is shown in 31 st to 37 th positions of SEQ ID No. 3; the amino acid sequence of CDR2 of the nano antibody A4 is shown in the 56 th to 61 th positions of SEQ ID No. 3; the amino acid sequence of the CDR3 of the nano antibody A4 is shown as the 101-109 position of SEQ ID No. 3;
the amino acid sequence of the CDR1 of the nanobody A7 is shown in the 31 st to 37 th positions of SEQ ID No.4, and the amino acid sequence of the CDR2 of the nanobody A7 is shown in the 56 th to 62 th positions of SEQ ID No. 4; the amino acid sequence of the CDR3 of the nano-antibody A7 is shown as the 102 nd-117 th position of SEQ ID No. 4.
SEQ ID No.3：
ESGGGSVQPGGSLRLSCTASGGSEYTYSYPYAGWFRQAPGQEREAVSAIAGENYEYYADSVKGRFTISRDNSKNTVTLQMNNLRAEDTAIYYCAAWPTQDGYAAWGQGTQVTVSS。
SEQ ID No.4：
EVQLVESGGGSVQPGGSLRLSCAASGGSEYWYSDWTSGWFRQAPGQEREAVAQAGYDGTPNYADSVKGRFTISRDNSKNTVTLQMNNLRAEDTAIYYCAAWYIKMNSDGHVQREWEWGQGTQVTVSS。
The bispecific neutralizing antibody for resisting the novel coronavirus SARS-CoV-2 has the following beneficial effects:
1. the invention recombines the nanometer antibody A4 and the nanometer antibody A7 which can recognize different epitopes on SARS-CoV-2 into the dual specificity neutralizing antibody, can recognize and neutralize a plurality of SARS-CoV-2 mutant viruses, has broad spectrum neutralizing activity, and reduces the risk of SARS-CoV-2 virus immune escape.
2. The invention is anti-SARSThe bispecific neutralizing antibody of CoV-2 has neutralizing ability, IC, against SARS-CoV-2 related variant strain and concerned variant strain pseudotype virus50Reaching sub-nanomolar level and having good broad-spectrum neutralization activity.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the bispecific antibody A4-A7 is connected by a connecting peptide from the C end of the nano antibody A4 to the N end of the nano antibody A7, the nucleotide sequence of the bispecific antibody is shown as SEQ ID NO.5, and the amino acid sequence of the bispecific antibody is shown as SEQ ID NO.1, or the amino acid sequence of a protein with the same function is obtained by replacing, deleting or inserting one or more amino acids;
the bispecific antibody A7-A4 is formed by connecting the C end of a nano antibody A7 with the N end of a nano antibody A4 through a connecting peptide, the nucleotide sequence of the bispecific antibody is shown as SEQ ID NO.6, and the amino acid sequence of the bispecific antibody is shown as SEQ ID NO.2 or the amino acid sequence of a protein with the same function is obtained by replacing, deleting or inserting one or more amino acids; the above-mentioned "amino acid sequence of a protein having the same function by substitution, deletion or insertion of one or more amino acids" refers to amino acid substitutions known to those skilled in the art, and such substitutions are usually made without changing the biological activity of the resulting molecule. In general, it is recognized by those skilled in the art that amino acid substitutions can be made without substantially altering or increasing the biological activity.
The amino acid sequence of the connecting peptide is (GGGGS) n, and n is any integer from 1 to 4;
the nucleotide sequence of the nano antibody A4 is shown in SEQ ID NO. 7;
the nucleotide sequence of the nano antibody A7 is shown in SEQ ID NO. 8.
SEQ ID No.1：
EVQLVESGGGSVQPGGSLRLSCTASGGSEYTYSYPYAGWFRQAPGQEREAVSAIAGENYEYYADSVKGRFTISRDNSKNTVTLQMNNLRAEDTAIYYCAAWPTQDGYAAWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCAASGGSEYWYSDWTSGWFRQAPGQEREAVAQAGYDGTPNYADSVKGRFTISRDNSKNTVTLQMNNLRAEDTAIYYCAAWYIKMNSDGHVQREWEWGQGTQVTVSS。
SEQ ID No.2：
EVQLVESGGGSVQPGGSLRLSCAASGGSEYWYSDWTSGWFRQAPGQEREAVAQAGYDGTPNYADSVKGRFTISRDNSKNTVTLQMNNLRAEDTAIYYCAAWYIKMNSDGHVQREWEWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTASGGSEYTYSYPYAGWFRQAPGQEREAVSAIAGENYEYYADSVKGRFTISRDNSKNTVTLQMNNLRAEDTAIYYCAAWPTQDGYAAWGQGTQVTVSS。
SEQ ID No.5：
gaagttcaattggttgaatctggtggtggttctgttcaacctggtggttctttgagattgtcttgtactgcttctggtggctccgaatatacctactcttacccttacgcaggttggtttagacaagctccaggtcaagaaagagaagctgtttctgctattgctggtgaaaactacgagtactatgctgattctgttaaaggtagattcactatttctcgagataattctaaaaatactgttactttgcaaatgaataatttgagagcagaagatactgctatttattattgcgctgcatggccaacccaggatggttacgcagcatggggtcaaggtactcaagttactgtttcttctggaggaggcggaagcggaggtgggggttcaggcggcggaggcagcgaagttcaactggttgaatctggcggcggttctgttcaaccgggcggtagtctgcgtctgagttgcgcagcaagcggcggtagcgaatattggtatagcgactggacctcaggttggtttcgtcaagcaccgggtcaagaacgcgaagcagttgcgcaagcgggttacgatgggaccccgaattacgcggatagcgttaaaggtcgtttcaccatcagccgcgataacagcaaaaacaccgtcaccctgcaaatgaataacctgcgcgcggaagataccgcaatttattactgcgcggcctggtacatcaaaatgaacagcgacggtcacgttcaacgcgagtgggagtggggtcaaggtacccaagttaccgttagcagt。
SEQ ID No.6：
gaagttcaactggttgaatctggcggcggttctgttcaaccgggcggtagtctgcgtctgagttgcgcagcaagcggcggtagcgaatattggtatagcgactggacctcaggttggtttcgtcaagcaccgggtcaagaacgcgaagcagttgcgcaagcgggttacgatgggaccccgaattacgcggatagcgttaaaggtcgtttcaccatcagccgcgataacagcaaaaacaccgtcaccctgcaaatgaataacctgcgcgcggaagataccgcaatttattactgcgcggcctggtacatcaaaatgaacagcgacggtcacgttcaacgcgagtgggagtggggtcaaggtacccaagttaccgttagcagtggaggaggcggaagcggaggtgggggttcaggcggcggaggcagcgaagttcaattggttgaatctggtggtggttctgttcaacctggtggttctttgagattgtcttgtactgcttctggtggctccgaatatacctactcttacccttacgcaggttggtttagacaagctccaggtcaagaaagagaagctgtttctgctattgctggtgaaaactacgagtactatgctgattctgttaaaggtagattcactatttctcgag。
SEQ ID No.7：
gaagttcaattggttgaatctggtggtggttctgttcaacctggtggttctttgagattgtcttgtactgcttctggtggctccgaatatacctactcttacccttacgcaggttggtttagacaagctccaggtcaagaaagagaagctgtttctgctattgctggtgaaaactacgagtactatgctgattctgttaaaggtagattcactatttctcgagataattctaaaaatactgttactttgcaaatgaataatttgagagcagaagatactgctatttattattgcgctgcatggccaacccaggatggttacgcagcatggggtcaaggtactcaagttactgtttcttct。
SEQ ID No.8：
gaagttcaactggttgaatctggcggcggttctgttcaaccgggcggtagtctgcgtctgagttgcgcagcaagcggcggtagcgaatattggtatagcgactggacctcaggttggtttcgtcaagcaccgggtcaagaacgcgaagcagttgcgcaagcgggttacgatgggaccccgaattacgcggatagcgttaaaggtcgtttcaccatcagccgcgataacagcaaaaacaccgtcaccctgcaaatgaataacctgcgcgcggaagataccgcaatttattactgcgcggcctggtacatcaaaatgaacagcgacggtcacgttcaacgcgagtgggagtggggtcaaggtacccaagttaccgttagcagt。
The adoption of the further beneficial effects is as follows: the invention provides two bispecific antibodies obtained by the transformation of the genetic engineering method, and researches show that the bispecific antibodies with the two structures have good technical effects, have neutralizing capacity on SARS-CoV-2 related variant strains and concerned variant strain pseudotyped viruses, and have IC50Reaching sub-nanomolar level and having good broad-spectrum neutralization activity. IC of bispecific antibody A4-A7 on Omicron pseudotype virus50Up to 0.391 nM; IC of bispecific antibody A7-A4 on Delta pseudotyped viruses50Up to 0.268 nM.
Further, the linker peptide is (GGGGS)3The amino acid sequence is shown in SEQ ID No. 18.
SEQ ID No.18：GGGGSGGGGSGGGGS。
The further beneficial effects of the adoption are as follows: the nano antibody A4 and the nano antibody A7 are connected and recombined into two kinds of bi-specific neutralizing antibodies through the connecting peptide.
Further, each of the nanobody a4 and the nanobody a7 further comprises framework regions FR1, FR2, FR3 and FR4, wherein the FR1 is represented by SEQ ID No.3 from position 1 to 30 of N terminus or SEQ ID No.4 from position 1 to 30 of N terminus;
the FR2 is shown as SEQ ID No.3 from the 38 th to 55 th positions of the N end or SEQ ID No.4 from the 38 th to 55 th positions of the N end;
the FR3 is shown as SEQ ID No.3 from the 63 th to the 101 th position of the N end or SEQ ID No.4 from the 62 th to the 100 th position of the N end;
the FR4 is shown as SEQ ID No.3 from the 108 th-118 th position of the N-terminal or SEQ ID No.4 from the 110 th-120 th position of the N-terminal.
The adoption of the further beneficial effects is as follows: the above antibodies may retain the neutralizing activity against the novel coronavirus SARS-CoV-2, or even have a stronger neutralizing activity.
The method for constructing the dual-specificity neutralizing antibody for resisting the novel coronavirus SARS-CoV-2 comprises the following steps:
recombining the nucleotide sequences of the nano antibody A4 shown in SEQ ID NO.7 and the nano antibody A7 shown in SEQ ID NO.8 to obtain recombinant DNA; respectively constructing recombinant expression plasmids containing a bispecific antibody A4-A7 and a bispecific antibody A7-A4; respectively transforming the recombinant expression plasmids into escherichia coli to be cultured to obtain recombinant bacteria; respectively extracting recombinant plasmids, and respectively transfecting the recombinant expression vectors into host cells to respectively obtain the host cells stably expressing the bispecific antibody A4-A7 and the bispecific antibody A7-A4; culturing host cell, separating and purifying culture supernatant to obtain bispecific antibody A4-A7 and bispecific antibody A7-A4.
Another object of the present invention is to provide a nucleotide molecule.
The technical scheme for solving the technical problems is as follows: nucleotide molecules encoding the above-described bispecific, neutralizing antibody against the novel coronavirus SARS-CoV-2.
The nucleotide molecules of the invention have the beneficial effects that:
the nucleotide molecule of the invention can code the dual-specific neutralizing antibody against the novel coronavirus SARS-CoV-2 described in any one of the above. Based on the codon encoding rules and the degeneracy and preference of codons, one skilled in the art can design the encoding gene based on the amino acid sequence of the above-mentioned bispecific antibody.
The third object of the present invention is to provide a recombinant plasmid.
The technical scheme for solving the technical problems is as follows: a recombinant plasmid containing the nucleic acid molecule.
The recombinant plasmid of the invention has the beneficial effects that:
the recombinant plasmid of the present invention contains the above-mentioned nucleic acid molecule and can encode the above-mentioned bispecific neutralizing antibody against novel coronavirus SARS-CoV-2.
The fourth object of the present invention is to provide a recombinant bacterium.
The technical scheme for solving the technical problems is as follows: a recombinant bacterium containing the nucleic acid molecule.
The recombinant bacterium has the beneficial effects that:
the recombinant bacterium of the present invention contains the nucleic acid molecule and may be used in prokaryotic system to express the double-specificity neutralizing antibody for resisting the new coronavirus SARS-CoV-2.
In another aspect, the present invention provides a transgenic cell line.
The technical scheme for solving the technical problems is as follows: a transgenic cell line containing the nucleic acid molecule.
The transgenic cell line has the beneficial effects that:
the transgenic cell line of the invention contains the nucleic acid molecule and can be used for expressing the bispecific neutralizing antibody against the novel coronavirus SARS-CoV-2 in a eukaryotic system.
The sixth object of the present invention is to provide the bispecific neutralizing antibody against the novel coronavirus SARS-CoV-2, the nucleic acid molecule, the recombinant plasmid, the recombinant bacterium, and the transgenic cell line, and the use of the bispecific neutralizing antibody, the nucleic acid molecule, the recombinant plasmid, the recombinant bacterium, and the transgenic cell line in the preparation of a medicament for preventing and/or treating the novel coronavirus SARS-CoV-2 pneumonia.
The technical scheme for solving the technical problems is as follows: the bispecific neutralizing antibody against the novel coronavirus SARS-CoV-2, the nucleic acid molecule, the recombinant plasmid, the recombinant bacteria and the transgenic cell line are applied to the preparation of medicaments for preventing and/or treating the novel coronavirus SARS-CoV-2 pneumonia.
The application of the invention has the beneficial effects that:
the dual-specificity neutralizing antibody for resisting the novel coronavirus SARS-CoV-2, the nucleic acid molecule, the recombinant plasmid, the recombinant bacterium and the transgenic cell line can inhibit the infection of SARS-CoV-2 to cells, and can be used for preparing medicaments for preventing and/or treating the novel coronavirus SARS-CoV-2 and mutant pneumonia thereof.
The seventh object of the present invention is to provide the neutralizing nanobody against the novel coronavirus SARS-CoV-2, the nucleic acid molecule, the recombinant plasmid, the recombinant bacterium, and the transgenic cell line, and the application thereof in preparing a detection reagent or a detection kit for the novel coronavirus SARS-CoV-2.
The technical scheme for solving the technical problems is as follows: the neutralizing nano antibody for resisting the novel coronavirus SARS-CoV-2, the nucleic acid molecule, the recombinant plasmid, the recombinant bacterium and the transgenic cell line are applied to preparing a detection reagent or a detection kit for the novel coronavirus SARS-CoV-2.
The application of the invention has the beneficial effects that:
the dual-differential neutralizing antibody for resisting the novel coronavirus SARS-CoV-2, the nucleic acid molecule, the recombinant plasmid, the recombinant bacterium and the transgenic cell line can be used for preparing a detection reagent or a detection kit for the novel coronavirus SARS-CoV-2 and a mutant strain thereof, and can be used for the following specific purposes: s protein combined with novel coronavirus SARS-CoV-2 and mutant strain thereof; detecting S protein of novel coronavirus SARS-CoV-2 and mutant strain thereof.
Drawings
FIG. 1 is a graph showing the results of the neutralization experiment of SARS-CoV-2 pseudotype virus with nanobody A4 in example 2 of the present invention;
FIG. 2 is a graph showing the results of the neutralization experiment of SARS-CoV-2 pseudotype virus with nanobody A7 in example 2 of the present invention;
FIG. 3 is a schematic diagram of the construction of bispecific antibody in example 3 of the present invention;
FIG. 4 is a graph showing the results of the neutralization experiment of SARS-CoV-2 pseudotype virus with the bispecific neutralizing antibody A4-A7 in example 3 of the present invention;
FIG. 5 is a graph showing the results of the neutralization experiment of SARS-CoV-2 pseudotype virus with the bispecific neutralizing antibody A7-A4 in example 3 of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with the following detailed drawings, which are given by way of illustration only and are not intended to limit the scope of the invention.
Example 1: preparation of nano antibody for recognizing SARS-CoV-2RBD
Step 1: display nano antibody library for rescuing phage surface
The fully synthetic nano antibody library is stored in a host bacterium in a phagemid form, and the library is rescued to become a phage display antibody library before the panning process is started. The specific method comprises the following steps:
take 1mL (OD)600100) Glycerol antoalgate, inoculated into 5 bottles of 200mL of 2TY-CARB medium (OD)6000.1), 37 ℃, 250rpm shake to OD600About 0.5; adding 1.6X 10 bacteria solution into each bottle12PFU M13KO7, 37 ℃ standing for 30 minutes, 37 ℃, 200rpm bacteria shaking for 30 minutes; transferring the bacterial liquid into a centrifugal bottle, centrifuging for 15 minutes at 2200g, re-suspending the bacterial precipitate by 400mL of 2TY-CARB-KAN, and shaking the bacterial at 30 ℃ and 250rpm for 14-16 hours; the bacterial liquid is respectively filled into centrifugal bottles (200 mL per bottle), 10000g is centrifugated for 30 minutes at 4 ℃, the supernatant is collected, 50mL (1/4 volume) of PEG/NaCl is added, the mixture is kept stand for 1 hour on ice, 7700g is centrifugated for 15 minutes at 4 ℃; the pellet was resuspended in 10mL PBS and a total of 100mL phage were collected, filtered through a 0.22 μm filter and stored at 4 ℃. Titrating bacteriophage titer by colony method, preparing TG1 in logarithmic phase, infecting the bacteriophage (10-fold dilution), mixing 200 μ L bacterial solution and 200 μ L bacteriophage, standing at 37 deg.C for 30 min, coating 100 μ L2 TY-GLU-CARB agar plate, and collecting 10 μ L-10，10-11，10-12Each was coated in 2 blocks and counted the next day.
Step 2: phage panning
mu.L of Streptavidin T1 magnetic beads (purchased from Seimer Feishale) were added to 1mL of PBS, allowed to stand on a magnetic stand for 1 minute, and the supernatant was aspirated off twice. Adding 10 into magnetic beads13PFU/mL phage 1mL, reverse spin incubation for 2 hours at room temperature, magnetic shelf standing for 1 min, retention of supernatant. Another 10. mu.L of Streptavidin T1 magnetic beads was added to biotinylated SASR-CoV spike protein RBD antigen (available from Beijing Yiqiao Shenzhou science Co., Ltd., cat # 40592-V08H-B), incubated for 30 minutes at room temperature by inversion and washed 4-5 times with PBS (0.1% BSA); adding the supernatant obtained in the step 1, performing rotary incubation for 2 hours at room temperature in a reversed manner, washing the supernatant for 10 times by using PBS and 0.1% Tween-20, and washing the supernatant for 1 time by using PBS; 1mL of eluent (100mM triethylamine, 70. mu.L TEA +5mL H)2O) eluting at room temperature for 10 minutes, and adding 1mL of 1M Tris-HCl (pH 7.5) for neutralization; preparation of OD600TG115mL of 0.4, 1mL TG1 added to the eluted magnetic beads, left to stand at 37 ℃ for 30 minutes, 1.5mL eluted product added to 13.5mL TG1, left to stand at 37 ℃ for 30 minutes; the bacterial solution was centrifuged at 4000g for 15 min, and resuspended and precipitated to 15mL of 2TY-GLU-CARB, 37 ℃ and shaken at 200rpm overnight.
And step 3: amplification of post-panning phage
Inoculating 200mL of 2TY-GLU-CARB into 15mL of bacterial liquid obtained in the step 2, shaking the bacterial liquid at 37 ℃ and 250rpm to OD600About 0.5; adding 1.6X 1012PFU M13KO7, 37 ℃ standing for 30 minutes, 37 ℃, 200rpm bacteria shaking for 30 minutes; transferring the bacterial liquid into a centrifuge bottle, centrifuging for 15 minutes at 2200g, re-suspending the bacterial precipitate by using 400mL of 2TY-CARB-KAN, and shaking the bacteria at 30 ℃ and 250rpm for 14-16 hours; respectively filling the bacterial liquid into centrifugal bottles (200 mL per bottle), centrifuging at 4 ℃ and 10000g for 15 minutes, taking supernate, adding 50mL (1/4 volume) of PEG/NaCl, standing for 1 hour on ice, and centrifuging at 4 ℃ and 5000g for 15 minutes; each 200mL of the corresponding pellet was resuspended in 2mL of PBS, the supernatant was collected by centrifugation at 4000rpm for 5 minutes at 4 ℃ and filtered through a 0.22 μm filter, and the titer of phage was titrated by the colony method.
And 4, step 4: positive clones were identified by Phage enzyme-linked immunosorbent assay (Phage ELISA)
Preparing a round-bottom deep-hole 96-well plate, wherein each well contains 600 mu L of 2TY-CARB culture medium, picking a single colony from the bacterial plate subjected to the 4 th round of elutriation into the 96-well plate, shaking the bacteria at 37 ℃ and 150rpm overnight; preparing a new round-bottom 96-well plate, wherein each well contains 90 mu L of 2TY-CARB culture medium, taking 10 mu L of bacterial liquid from the overnight-cultured 96-well plate into the new 96-well plate, and shaking the bacteria at 37 ℃ and 150rpm for 1 hour; 25 μ L M13KO7 (predilution to 10) was added9PFU/mL), standing at 37 ℃ for 30 minutes, shaking at 37 ℃ for 30 minutes at 200rpm, centrifuging at 4000rpm for 15 minutes, discarding the supernatant, and adding 100 partsAfter being resuspended, the mu L of 2TY-CARB-KAN is transferred to a new deep-hole 96-well plate, 900 mu L of 2TY-CARB-KAN is additionally added to each well, and the bacteria are shaken at 30 ℃ and 250rpm overnight; ELISA plate with 1 u g/mL RBD antigen coating, each hole 100 u L, 4 degrees overnight; washing each hole of the ELISA plate once with 200 mu L PBS + 0.1% Tween-20, and then blocking each hole with 200 mu L PBS + 0.1% Tween-20+ 5% BSA at room temperature for 1 hour; 200 u L PBS + 0.1% Tween-20 wash 5 times, each hole adding 20 u L PBS + 5% BSA; centrifuging a 96-hole deep-hole plate at 4 ℃ and 4000rpm for 15 minutes, adding 80 mu L of phase into an ELISA plate, mixing with PBS + 5% BSA, uniformly mixing by using an oscillator, and standing at room temperature for 1 hour; 200 μ L PBS + 0.1% Tween-20 was washed 5 times, 100 μ L HRP-anti-M13 anti-body (purchased from Beijing Yi Qiao Shenzhou science Co., Ltd., product number: 11973-MM05T-H) (PBS + 0.1% Tween-20+ 5% BSA diluted) was added to each well, and the mixture was allowed to stand at room temperature for 1 hour; washing 3 times with 200 μ L PBS + 0.1% Tween-20, washing 3 times with 200 μ L PBS, adding 100 μ L TMB into each well, shading at room temperature, developing to appropriate degree, adding 50 μ L0.5M H into each well2SO4The reaction was stopped and OD was measured450The absorbance value of (a). Phage with a ratio of experimental group readings to negative control readings greater than 5 were identified as positive clones. The positive clones were sequenced with the primers shown in SEQ ID No. 9.
SEQ ID No.9：5′-acgacaggtttcccgactg-3′。
Analysis after sequencing showed that all positive clones could be summarized as clones of two different sequences, numbered a4 and a7, in preparation for subsequent construction of recombinant expression vectors.
And 5: construction of recombinant expression plasmids
The positive clone gene is amplified by adopting an upstream primer shown as SEQ ID No.10 and a downstream primer shown as SEQ ID No. 11.
SEQ ID No.10：5′-ggcgctagccaagttcaattggttgaat-3′；
SEQ ID No.11：
5′-tgagcctccactgaattcagaagaaacagtaacttgagtacct-3′。
Obtaining the nucleic acid molecule for coding the anti-RBD nano antibody. The antibody gene is subjected to double enzyme digestion by NheI and EcoRI, inserted into an antibody expression vector pCDNA4-Fc, and transformed into DH5a Escherichia coli competent cells (purchased from Takara Shuzo Co., Ltd.) to obtain recombinant bacteria. The recombinant strain was inoculated in a liquid LB-Amp medium, cultured overnight at 37 ℃ and plasmid was extracted with a plasmid extraction kit (QIGEN). Obtaining the nano antibody recombinant plasmid. pCDNA4-Fc was modified from pCDNA4/myc-HisA (available from Seimer Feishol), and interferon secretion signal peptide and human IgG1 Fc gene were inserted into the HindIII and AgeI cleavage sites of the vector.
Step 6: expression of fusion proteins
2×107HEK293T cells (from the institute of basic medicine, national institute of medicine and technology, cell resources) were seeded in a 15cm culture dish, and culture medium (DMEM high-sugar medium (purchased from Saimer Feishal) containing 10% fetal bovine serum (purchased from Saimer Feishal) was used, and after 12 hours, 36. mu.g of recombinant plasmid was transfected, and 12 hours after transfection, the medium was changed to serum-free medium (purchased from Saimer Feishal), and culture supernatant was collected at 72 hours. Protein purification was performed by Protein A-agarose (available from Beijing Yiqiao Hibiscus technologies, Inc.). Filtering cell debris by using a 0.45 mu m filter, and then passing through a column, wherein the flow rate is controlled to be 1 mL/min; the column was washed with 5 bed volumes of binding buffer (50mM Tirs, 100mM NaCl, pH 8.0); eluting with 2 bed volumes of elution buffer (100mM glycine, 10mM NaCl, pH 3.0), collecting eluate, and neutralizing with 1/5 bed volumes of neutralizing solution (1M Tirs, pH 9.4). Finally, further purifying by using a molecular sieve.
Example 2: determination of neutralizing Activity of SARS-CoV-2 pseudotype Virus
104293T stably-transformed ACE2 cells (from cell resource center of basic medicine institute of Chinese academy of medicine) were inoculated into a 96-well plate, SARS-CoV-2 and its mutant pseudotype virus were mixed with different dilutions of neutralizing nanobodies, incubated at room temperature for 30 minutes, added to the cells, cultured for 6 hours, replaced with 2% FBS DMEM medium, and cultured for another 48 hours. SARS-CoV-2 and its mutant pseudotype virus contain luciferase reporter gene, luciferase activity in cells was detected using luciferase reporter detection kit (Promega, cat # E4550), IC was calculated by fitting a curve to GraphPad Prism 850The value is obtained.
The results of experiments on the inhibition of SARS-CoV-2 pseudotyped virus invasion by Nanobody A4 and Nanobody A7 are shown in Table 1.
TABLE 1 inhibition rate of nano-antibodies against SARS-CoV-2 pseudotyped virus invasion
Figure BDA0003568791290000121
The neutralization experiment result of SARS-CoV-2 pseudotype virus of nano antibody A4 is shown in figure 1;
the results of the neutralization experiment of SARS-CoV-2 pseudotype virus with the nano antibody A7 are shown in FIG. 2.
The above results show that: IC of nano antibody A4 for SARS-CoV-2 pseudotype virus such as WT, Alpha, Beta and Omicron50Reach sub-nanomolar level, have good ability to neutralize SARS-CoV-2 pseudotyped virus, but have no neutralizing effect to Delta and other pseudotyped viruses;
IC of nano antibody A7 for SARS-CoV-2 pseudotype virus such as WT, Alpha, Delta and the like50Reach subnanomolar level, has good capability of neutralizing SARS-CoV-2 pseudotype virus, but has no neutralizing effect on the pseudotype viruses such as Omicron and the like;
the two neutralizing nano antibodies have neutralizing activity on partial SARS-CoV-2 pseudotype virus, and can not neutralize all SARS-CoV-2 related variant strains and concerned variant strain pseudotype virus.
Example 3: preparation of bispecific neutralizing antibodies
Step 1: amplification and recombination of gene sequences
The nanobody a4 and the nanobody a7 recombinant plasmid obtained in example 1 were used as templates and amplified, respectively. Using the recombinant plasmid A4 as a template, carrying out amplification by using a primer F and a primer 4R to obtain a gene segment A4-1, and carrying out amplification by using a primer 4F and a primer R to obtain a gene segment A4-2; the recombinant plasmid A7 is used as a template, a gene fragment A7-1 is obtained by amplification with a primer F and a primer 7R, and a gene fragment A7-2 is obtained by amplification with the primer 7F and the primer R. The primers used are shown below.
F：5′-caagtcaagctgctctgtg-3′(SEQ ID No.12)；
4F：
5′-agcggaggtgggggttcaggcggcggaggcagcgaagttcaattggttgaatc-3′(SEQ ID No.13)；
4R：5′-tgaacccccacctccgcttccgcctcctccagaagaaacagtaacttgag-3′(SEQ ID No.14)；
7F：
5′-agcggaggtgggggttcaggcggcggaggcagcgaagttcaactggttgaatc-3′(SEQ ID No.15)；
7R：5′-tgaacccccacctccgcttccgcctcctccactgctaacggtaacttggg-3′(SEQ ID No.16)；
R：5′-tgggctctgagcctccactgaatt-3′(SEQ ID No.17)。
A4-1 and A7-2 are taken as templates, primers F and R are used for amplification to obtain a bispecific neutralizing antibody A4-A7 gene, the nucleotide sequence of A4-A7 is shown as SEQ ID No.5, wherein the 1-360 th position is A4, the 1-393 th position is A4-1, the 375-786 th position is A7-2, and the 406-786 th position is A7;
a7-1 and A4-2 are taken as templates, primers F and R are used for amplification to obtain a bi-specific neutralizing antibody A4-A7 gene, the nucleotide sequence of A7-A4 is shown as SEQ ID No.6, wherein the 1-381 st position is A7, the 1-411 st position is A7-1, the 394-786 th position is A4-2, and the 427-786 th position is A4.
Step 2: construction of recombinant expression plasmids
The gene of the bispecific neutralizing antibody was digested with NheI and EcoRI, inserted into the antibody expression vector pCDNA4-Fc of example 1, and transformed into DH5a E.coli competent cells (purchased from Okinawa Co., Ltd.) to obtain a recombinant strain. The recombinant strain was inoculated in a liquid LB-Amp medium, cultured overnight at 37 ℃ and plasmid was extracted with a plasmid extraction kit (QIGEN). The recombinant plasmid of the bispecific neutralizing antibody is obtained.
The schematic of the construction of recombinant expression plasmids for bispecific neutralizing antibodies is shown in FIG. 3.
And 3, step 3: expression of bispecific neutralizing antibodies
Expression of the bispecific neutralizing antibody the expression procedure of the fusion protein of example 1 was followed.
The structure of the bi-specific neutralizing antibody is shown in FIG. 3.
And 4, step 4: SARS-CoV-2 pseudotype virus invasion inhibition experiment
The same experiment as that for inhibiting invasion of SARS-CoV-2 pseudotype virus in example 2 was performed.
The results of the experiments for inhibiting SARS-CoV-2 pseudotyped virus invasion by the bispecific neutralizing antibody are shown in Table 2.
TABLE 2 inhibition rate of bispecific neutralizing antibody against SARS-CoV-2 pseudotype virus invasion
Figure BDA0003568791290000141
The results of the neutralization experiments with SARS-CoV-2 pseudotype virus that bi-specifically neutralizes antibody A4-A7 are shown in FIG. 4;
the results of the neutralization experiments for SARS-CoV-2 pseudotype virus with the bi-specific neutralizing antibody A7-A4 are shown in FIG. 5.
The above results show that: the dual-differential neutralizing antibody A4-A7 and the dual-differential neutralizing antibody A7-A4 provided by the invention have neutralizing capacity and IC for SARS-CoV-2 related variant strains and concerned variant strain pseudotyped viruses50Reaching sub-nanomolar level and having good broad-spectrum neutralization activity.
Therefore, the dual-specific neutralizing antibody for resisting the novel coronavirus SARS-CoV-2 can be effectively combined with RBD antigen, has high broad-spectrum neutralizing activity on SARS-CoV-2 and mutant strain pseudotype virus thereof, and has important scientific significance and application prospect in the research and development of novel coronavirus SARS-CoV-2 and mutant strain-caused diseases, clinical treatment and diagnostic reagents.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> institute of pathogenic biology of Chinese academy of medical sciences
<120> a dual-specific neutralizing antibody against novel coronavirus SARS-CoV-2 and its application
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ggcggtagcg aatattggta tagcgactgg acctcaggtt ggtttcgtca agcaccgggt 540
caagaacgcg aagcagttgc gcaagcgggt tacgatggga ccccgaatta cgcggatagc 600
gttaaaggtc gtttcaccat cagccgcgat aacagcaaaa acaccgtcac cctgcaaatg 660
aataacctgc gcgcggaaga taccgcaatt tattactgcg cggcctggta catcaaaatg 720
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agcag 785
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gaagttcaac tggttgaatc tggcggcggt tctgttcaac cgggcggtag tctgcgtctg 60
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caagtcaagc tgctctgtg 19
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15

Claims (10)

1. A bispecific neutralizing antibody against novel coronavirus SARS-CoV-2, which is bispecific antibody a4-a7 or bispecific antibody a7-a4, each comprising nanobody a4 and nanobody a7, wherein nanobody a4 and nanobody a7 each comprise a variable region having 3 complementarity determining regions CDR1, CDR2 and CDR 3; the amino acid sequence of CDR1 of the nano antibody A4 is shown in 31 st to 37 th positions of SEQ ID No. 3; the amino acid sequence of CDR2 of the nano antibody A4 is shown in the 56 th to 61 th positions of SEQ ID No. 3; the amino acid sequence of the CDR3 of the nano antibody A4 is shown as the 101-109 position of SEQ ID No. 3;
the amino acid sequence of the CDR1 of the nanobody A7 is shown in the 31 st to 37 th positions of SEQ ID No.4, and the amino acid sequence of the CDR2 of the nanobody A7 is shown in the 56 th to 62 th positions of SEQ ID No. 4; the amino acid sequence of the CDR3 of the nano-antibody A7 is shown as the 102 nd-117 th position of SEQ ID No. 4.
2. The bispecific neutralizing antibody against novel coronavirus SARS-CoV-2 according to claim 1, wherein the bispecific neutralizing antibody A4-A7 is formed by connecting C terminal of Nanobody A4 with N terminal of Nanobody A7 via a connecting peptide, the nucleotide sequence of the bispecific neutralizing antibody is shown as SEQ ID No.5, the amino acid sequence of the bispecific neutralizing antibody is shown as SEQ ID No.1, or the amino acid sequence of a protein with the same function is obtained by replacing, deleting or inserting one or more amino acids;
the bispecific antibody A7-A4 is formed by connecting the C end of a nano antibody A7 with the N end of a nano antibody A4 through a connecting peptide, the nucleotide sequence of the bispecific antibody is shown as SEQ ID NO.6, and the amino acid sequence of the bispecific antibody is shown as SEQ ID NO.2 or the amino acid sequence of a protein with the same function is obtained by replacing, deleting or inserting one or more amino acids;
the amino acid sequence of the connecting peptide is (GGGGS) n, and n is any integer from 1 to 4;
the nucleotide sequence of the nano antibody A4 is shown in SEQ ID NO. 7;
the nucleotide sequence of the nano antibody A7 is shown in SEQ ID NO. 8.
3. The bispecific neutralizing antibody against novel coronavirus SARS-CoV-2 according to claim 2, wherein the linker peptide is (GGGGS)3The amino acid sequence is shown in SEQ ID No. 18.
4. The bispecific neutralizing antibody against novel coronavirus SARS-CoV-2 according to claim 1, wherein each of nanobody A4 and nanobody A7 further comprises framework regions FR1, FR2, FR3 and FR4, wherein FR1 is represented by SEQ ID No.3 from positions 1 to 30 of the N-terminus or SEQ ID No.4 from positions 1 to 30 of the N-terminus;
the FR2 is shown as SEQ ID No.3 from the 38 th to 55 th positions of the N end or SEQ ID No.4 from the 38 th to 55 th positions of the N end;
the FR3 is shown as SEQ ID No.3 from the 63 th to the 101 th position of the N end or SEQ ID No.4 from the 62 th to the 100 th position of the N end;
the FR4 is shown as SEQ ID No.3 from the 108 th-118 th position of the N-terminal or SEQ ID No.4 from the 110 th-120 th position of the N-terminal.
5. A nucleotide molecule encoding the bispecific neutralizing antibody against novel coronavirus SARS-CoV-2 according to any one of claims 1 to 4.
6. A recombinant plasmid comprising the nucleic acid molecule of claim 5.
7. A recombinant bacterium comprising the nucleic acid molecule of claim 5.
8. A transgenic cell line comprising the nucleic acid molecule of claim 5.
9. Use of the bispecific neutralizing antibody against novel coronavirus SARS-CoV-2 according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the recombinant plasmid according to claim 6, the recombinant bacterium according to claim 7 and the transgenic cell line according to claim 8 for the preparation of a medicament for the prophylaxis and/or treatment of novel coronavirus SARS-CoV-2 pneumonia.
10. Use of the bispecific neutralizing antibody against novel coronavirus SARS-CoV-2 according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the recombinant plasmid according to claim 6, the recombinant bacterium according to claim 7 and the transgenic cell line according to claim 8 for the preparation of a detection reagent or a detection kit for novel coronavirus SARS-CoV-2.
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