CN116444665A - Monoclonal antibody aiming at SARS-CoV-2 glycoprotein RBD region of novel coronavirus and application thereof - Google Patents
Monoclonal antibody aiming at SARS-CoV-2 glycoprotein RBD region of novel coronavirus and application thereof Download PDFInfo
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Abstract
The invention discloses 4 monoclonal antibodies XA326.7, XA326.8, XA326.18, XA326.49 and light chains and heavy chains thereof of anti-novel coronavirus glycoprotein. The monoclonal antibody has high neutralizing activity, and has the light chain and heavy chain variable region with the amino acid sequence shown in SEQ ID No.1 and the heavy chain variable region with the amino acid sequence shown in SEQ ID No. 2. Through conventional hybridoma technology and the preparation of monoclonal antibodies of mouse anti-new coronavirus glycoprotein, 4 hybridoma cells XA326.7, XA326.8, XA326.18 and XA326.49 which can stably secrete high neutralizing activity antibodies are screened, and mAbs obtained by preparing ascites can be applied to blocking the infection of the ACE2 positive cells by the new coronavirus. Identifying the uniqueness of the gene sequence and corresponding protein sequence and CDR sequences thereof; provides technical support for developing and developing anti-diagnosis and therapeutic agents.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a monoclonal antibody aiming at a novel coronavirus SARS-CoV-2 glycoprotein RBD region and application thereof.
Background
In addition to clinical diagnosis and treatment aid measures, preventive vaccine research and development and epidemiological control means, the novel coronavirus SARS-CoV-2 causes acute viral pneumonia COVID-19, which also enhances the monitoring of host adaptability, virus evolution, infectivity, pathogenicity, especially the research and development of neutralizing active antibody preparations, and the like, and the relevant technical means reserves have important roles.
SARS-CoV-2 and SARS-CoV causing atypical pneumonia in 2003 and MERS-CoV causing eastern respiratory syndrome in 2012 belong to the genus β of the family coronaviridae, and the genome is continuous linear single-stranded RNA encoding 4 structural proteins such as spinous process (spike), envelope (envelope), membrane (membrane) and nucleus (nucleopetin). The S protein is the most important surface protein of coronaviruses, and the host range and infection specificity of the virus are determined by the binding of the RBD region to host cells expressing ACE2 molecules. Thus, specific epitopes on the S protein become the core targets for therapeutic neutralizing antibody development, while the N protein has strong immunogenicity, and the specific antibodies can be used in virology, pathology and clinical diagnosis.
Antibodies are the core component of the humoral immune response of the body and are the molecular basis for protecting vaccines. In the past medical practice, by utilizing the high specificity and the neutralization effect of the antibody, various therapeutic agents for infectious diseases and toxin poisoning are developed, and the neutralizing activity of the antibody against pathogenic components such as tetanus toxin, snake venom and the like is successfully applied to the treatment of clinical patients. For the new coronaviruses, scientists such as Xie Xiaoliang and Chen Wei clone and obtain neutralizing activity human antibodies from peripheral blood of convalescence patients, and a cocktail neutralizing activity antibody preparation of American regeneration company is clinically used. The monoclonal antibody has higher affinity, diversified selection and genetic engineering potential obtained by the standard controllable immune process, and is still an important means for developing antibody biological agents.
The SARS-CoV-2 virus has a large molecular weight, the proportion of neutralizing antibodies in all the antibodies prepared is relatively small, the international research on therapeutic monoclonal antibodies is underway, and the number of antibodies with neutralizing activity prepared is small. The antibody monomer molecule has a tetrapeptide chain structure formed by connecting two identical heavy chains (H chains) and two identical light chains (L chains) through inter-chain disulfide bonds. The H and L chains comprise an amino (N) terminal and a carboxyl (C) terminal, and the variable region near the N terminal (V region) consists of a hypervariable region/complementarity determining region (HVR/CDR) and a Framework Region (FR); the constant region (C region) is near the C-terminus. The protein folding formed by the heavy chain variable region (VH) and the light chain variable region (VL) is an antigen binding site, wherein the CDR/HVR is the site of complementary binding of the antibody to an epitope, and the C region initiates a reaction upon antigen-antibody recognition.
Although human antibodies derived from peripheral blood B lymphocytes of patients in the new coronary convalescence phase exist at present, the affinity, neutralization activity and binding site of antibodies from different sources are different, and the therapeutic effect which can be produced is different. In response to acute infectious diseases with severe risk of new crowns, development of new antibody preparations is continually desired.
Disclosure of Invention
In order to overcome the above-mentioned drawbacks of the prior art, the present invention aims to provide a monoclonal antibody directed against the SARS-CoV-2 glycoprotein RBD region of a novel coronavirus and the use thereof.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
the invention discloses 4 monoclonal antibodies aiming at a novel coronavirus SARS-CoV-2 glycoprotein RBD region, which comprises the following components:
designated XA326.8, comprises a light chain with the amino acid sequence shown in seq id No.1 and a heavy chain with the amino acid sequence shown in seq id No. 2.
Designated XA326.7, comprises a light chain with the amino acid sequence shown in seq id No.3 and a heavy chain with the amino acid sequence shown in seq id No. 4.
Designated XA326.18, comprises a light chain with the amino acid sequence shown in seq id No.5 and a heavy chain with the amino acid sequence shown in seq id No. 6.
Designated XA326.49, comprises a light chain with the amino acid sequence shown in seq id No.7 and a heavy chain with the amino acid sequence shown in seq id No. 8.
The invention also includes isolated nucleic acid molecules capable of corresponding to encode the monoclonal antibodies described above.
The invention also discloses an expression vector based on the nucleic acid molecule, and the expression vector comprises an expression control sequence which is operatively connected with the sequence of the nucleic acid molecule besides the nucleic acid molecule.
Wherein the expression vector is a nucleic acid vehicle capable of expressing the genetic material elements carried by the host cell by transformation, transduction or transfection of the host cell. The types of vectors include bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
The invention also discloses a host cell containing the nucleic acid molecule or the expression vector. The host cell may be a prokaryotic cell (e.g., bacterial cell), a lower eukaryotic cell (yeast cell), a higher eukaryotic cell (mammalian cell).
The invention also discloses a detection reagent or a detection kit containing the monoclonal antibody, nucleic acid molecule, expression vector or host cell aiming at the SARS-CoV-2 glycoprotein RBD region of the novel coronavirus.
The invention also discloses the application of the monoclonal antibody, nucleic acid molecule, expression vector or host cell aiming at the novel coronavirus SARS-CoV-2 glycoprotein RBD region in preparing novel coronavirus SARS-CoV-2 detection products.
The invention also discloses the application of the monoclonal antibody, nucleic acid molecule, expression vector or host cell aiming at the novel coronavirus SARS-CoV-2 glycoprotein RBD region in preparing medicaments for inhibiting the novel coronavirus SARS-CoV-2.
The present invention also discloses the application of the monoclonal antibody, nucleic acid molecule, expression vector or host cell to the SARS-CoV-2 glycoprotein RBD region of new coronavirus in preparing medicine preparation for preventing and treating pneumonia caused by new coronavirus SARS-CoV-2.
Compared with the prior art, the invention has the following beneficial effects:
according to the virus infection pathogenic characteristics, the research uses recombinant S1 and S1-RBD as immunogens and screening antigens, adopts a mature monoclonal antibody preparation technology, screens out 4 neutralizing antibodies from a large number of positive clones, is used for measuring the subsequent neutralizing protection activity, provides great convenience for the development and development of genetic engineering antibodies, and has the specific advantages that:
1. the 4 strain of monoclonal antibody resisting novel coronavirus SARS-CoV-2 glycoprotein RBD region provided by the invention is a monoclonal antibody resisting novel coronavirus glycoprotein with high neutralization activity: can block the infection of ACE2 positive cells by new coronavirus pseudovirus, and can be further used for the research of in vivo experiments.
2. The invention clones the light chain and heavy chain variable region amino acid sequence of monoclonal antibody of SARS-CoV-2 glycoprotein RBD region, and the sequence analysis proves the uniqueness of the antibody sequence.
3. The amino acid sequences of the light chain and heavy chain variable regions are obtained by analysis, and the support is provided for constructing the chimeric or humanized genetic engineering antibody of the SARS-CoV-2 glycoprotein RBD region of the anti-novel coronavirus with high specificity and high affinity.
Drawings
FIG. 1 is a diagram showing the result of DNA electrophoresis of heavy chain (H) and light chain (K) after reverse transcription and PCR reaction of mRNA obtained from 4 monoclonal antibodies of the present invention;
FIG. 2 shows the result of measurement of the neutralization half-inhibitory concentration (IC 50) of the 4-strain antibody of the present invention, and shows the specific IC50 value;
FIG. 3 is a simulated cartoon of the structure of the variable region of the antibody of the present invention;
wherein A is an XA326.7 variable region structure simulation cartoon;
wherein B is an XA326.8 variable region structure simulation cartoon;
wherein C is the analog cartoon diagram of the XA326.49 variable region structure.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention uses recombinant expressed novel coronavirus SARS-CoV-2 glycoprotein molecules to immunize Babl/c mice, screens out monoclonal antibody XA326.7, XA326.8, XA326.18 and XA326.49 mAb hybridoma cell strains capable of stably secreting high specificity anti-novel coronavirus SARS-CoV-2 glycoprotein RBD region mAb, prepares ascites, and obtains high specificity anti-novel coronavirus SARS-CoV-2 glycoprotein RBD region mAb; and confirm the uniqueness of the protein sequence. The invention is described in detail below with reference to methods for preparing specific monoclonal antibodies, antibody specificity and activity assays, and sequence assays and determination of uniqueness.
1. Preparation of anti-novel coronavirus SARS-CoV-2 glycoprotein RBD region monoclonal antibody and specific identification thereof
1.1 preparation and purification of monoclonal antibodies
CD226KO mice (50. Mu.g/mouse per mouse, 3 mice total) were immunized with the novel coronavirus SARS-CoV-2 glycoprotein antigen according to monoclonal antibody preparation method (cell and molecular immunology assay technique (first edition), P9-P17). The primary immunization uses Freund's complete adjuvant, the subsequent immunization uses Freund's incomplete adjuvant, and each time is divided into 3 weeks, and the subcutaneous multipoint injections are carried out for 4 times. And taking blood from the tail tip after the last immunization for 7-10 days, measuring the serum antibody titer, and detecting the immune effect. One mouse with the highest serum titer is taken to be subjected to boosting immunity again by intraperitoneal injection of antigen, the mouse is sacrificed after 3 days, and spleen cells are separated for cell fusion.
Cell fusion procedures were as follows: log-growing mouse myeloma cells SP2/0 were counted while preparing a suspension of splenocytes from immunized mice. Myeloma cells and spleen cells were mixed in a ratio of 1:10, and PEG was added for cell fusion. The cell suspension after fusion was added to a 96-well plate containing feeder cells (normal Balb/c mouse peritoneal macrophages), 37℃and 5% CO 2 Incubator culture. After the clone appears, the new coronavirus SARS-CoV-2 glycoprotein antigen is coated on a polystyrene ELISA plate, and the cell supernatant is detected by indirect ELISA, and positive reaction clone is selected. The cells containing positive cloning holes are cloned by adopting a limiting dilution method, and after 4 times of cloning, 53 strains of monoclonal hybridoma cells capable of stably secreting positive reaction antibodies are obtained, and different clones are respectively numbered as XA326.1-XA326.53 according to running water.
Induction of ascites and antibody purification: after obtaining a hybridoma cell line capable of stably secreting an antibody, ascites was prepared according to a conventional technique (Experimental techniques for cell and molecular immunology (first edition), P9-P17). The host is a Babl/c mouse, ascites fluid adopts Protein A/G affinity chromatography to purify the antibody, and the purity of the purified mAb reaches 95 percent. The identification of the IgG subtype of the antibody was performed according to Sigma detection kit instructions. Ig subclass assay was performed on antibodies secreted therefrom, and the identification was IgG1 subclass and kappa light chain.
1.2 potency determination of monoclonal antibody against New coronavirus SARS-CoV-2 glycoprotein RBD region
The relative affinities of the purified mAbs were determined by indirect ELISA. Wherein the coating antigen is recombinant expressed new coronavirus SARS-CoV-2 glycoprotein immunogen, the sample to be detected is serial diluted ascites and purified mAb, the detection antibody is goat anti-mouse-HRP enzyme labeled antibody, and the substrate uses ABTS. The ascites titer of the screened 53 monoclonal antibodies is 1 multiplied by 10 -6 。
1.3 identification of neutralizing Activity of the anti-New coronavirus SARS-CoV-2 glycoprotein RBD mab
The SARS-CoV-2 pseudovirus uses HIV virus as skeleton, and is coated with Spike protein of SARS-Cov-2, so that it can infect the cells over-expressing human ACE 2. Can be used for testing neutralizing antibodies or other drugs which block virus invasion. The pseudovirus can only be infected once, cannot be replicated, has high safety and can be used in a P2 class laboratory. Pseudoviruses carry both Firefly Luc and EGFP markers, and infection efficiency can be detected by Firefly Luc and EGFP.
The experimental steps are as follows:
1) The first day, 96-well plates were seeded with 5000 Cos7-Hace2 cells per well.
2) The next day, the medium in the plate was removed, pseudoviruses incubated with antibody samples at 37℃for 1 hour, and the wells were added and the plate centrifuged (1200 g. Times.2 Hour,4 ℃).
3) Centrifuging the infected cells, sucking up the medium, and washing once with PBS; 30uL of 0.25% pancreatin was added to each well, incubated at 37℃for 5 minutes, 100uL of complete medium was added, and the culture continued.
4) Culture was continued for 3 days (daily changing) after infection, and luc detection was performed.
The results show that 4 antibodies have good neutralizing activity, can bind to the RBD region of SARS-CoV-2 glycoprotein, and the antibody number is: XA326.7, XA326.8, XA326.18, XA326.49.
2. Light chain and heavy chain variable region sequencing analysis of anti-novel coronavirus SARS-CoV-2 glycoprotein RBD monoclonal antibody
2.1 cultivation of XA326 mAbs hybridoma cells
Culturing of XA326.7, XA326.8, XA326.18, XA326.49 hybridoma cell lines by conventional methods, culturing with RPMI 1640 containing 15% calf serum at 37℃on 5% CO 2 Culturing in incubator to logarithmic phase.
2.2 extraction of Total RNA and Synthesis of first strand cDNA
Extracting total RNA by using TRIZOL Reagent (purchased from GIBCO corporation of America), wherein the specific operation steps are carried out according to instructions; cDNA first Strand Synthesis kit was purchased from GIBCO corporation, USA, and after total RNA was obtained, reverse transcription was performed according to instructions to synthesize cDNA first strand.
2.3RT-PCR amplified VL and VH genes of XA326.7, XA326.8, XA326.18, XA326.49 mAb. The kit for amplifying the VL and VH genes of the hybridoma cell antibody by the one-step method RT-PCR is purchased from TakaRa company and amplified according to the specification of the kit;
the primers were as follows:
primers for amplifying antibody Fd fragment and light chain full-length gene (degenerate bases in brackets)
Mouse heavy chain V region 5' primer:
VH1:5’-GGG GAT ATC CAC CAT GG(AG)ATG(CG)AG CTG(TG)GT(CA)AT(CG)CT CTT-3’
VH2:5’-GGG GAT ATC CAC CAT G(AG)A CTT CGG G(TC)T GAG CT(TG)GGT TTT-3’
VH3:5’-GGG GAT ATC CAC CAT GGC TGT CTT GGG GCT GCT CTT CT-3’
VH4:5’-GGG GAT ATC CAC CAT GAT(AG)GT GTT(AG)AG TCT T(CT)TGT(AG)CCT G 3’
Fd 3':5'-AGG CTT ACT AGT ACA ATC CCT GGG CAC AAT-3'；
mouse light chain V region 5' end primer
VL1:5’-GGG GAT ATC CAC CAT GGA GAC AGA CAC ACT CCT GCT AT-3’
VL2:5’-GGG GAT ATC CAC CAT GGA TTT TCA AGT GCA GAT TTT CAG-3’
VL3:5’-GGG GAT ATC CAC CAT GGA G(AT)C ACA (GT)(AT)C TCG GGTCTT T(GA)T A -3’
VL4:5’-GGG GAT ATC CAC CAT G(GT)C CCC(AT)(AG)C TCA G(CT)TC(CT)C T(TG)G T-3’
VL5:5’-GGG GAT ATC CAC CAT GAA GTT GCC TGT TAG GCT GTT G-3’
Light chain 3' end primer:
MLC-3’:5'-GCG CCG TCT AGA ATT AAC ACT CAT TCC TGT TGA A-3'；
PCR program TOUCHDOWN: the reaction volume was 50. Mu.L, and the reaction conditions were: 98 ℃ for 5min;98℃for 10s,57℃for 30s (+0.7 ℃/cycle, ×15 cycle), 98℃for 10s,57℃for 30s-72℃for 30s (+28 cycle), 72℃for 10min.
2.4 cloning and screening of PCR amplified products
PCR products were subjected to 1.5% agarose gel electrophoresis, PCR amplified fragments were recovered, and the fragments were inserted into pMD-18T vector (purchased from TakaRa) using A-tailing according to instructions, and the ligation was transformed into E.coli, and inoculated in Amp-resistant LB agar plates for overnight culture at 37 ℃.
Cloning in LB agar culture plates, shaking in Amp-resistant LB culture medium at 37 ℃ overnight, taking l μl bacterial liquid as a template, and screening recombinant positive E.coli clones by PCR method through the primers designed for light chain and heavy chain variable regions. And (3) cloning the obtained recombinant positive E.coli, shaking the bacteria, and determining the gene sequence, wherein the protein sequence of the variable region of the antibody light chain is shown as SEQ ID NO.1, and the protein sequence of the heavy chain is shown as SEQ ID NO. 2.
3. Homology analysis of the amino acid sequences of the light and heavy chains of XA326.7, XA326.8, XA326.18, XA326.49 mAb
After determining that the sequencing is correct, the variable region gene is translated into an amino acid sequence, and the amino acid sequence analysis is performed. The amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2.
Amino acid sequence homology analysis (Blastp) was performed in the non-redundant Genbank CDS translations +PDB+SwissProt+PIR+PRF protein database.
4. Analysis of three monoclonal antibody variable region structures of XA326.7, XA326.8 and XA326.49 by molecular docking technique
The spatial structure of binding to the novel coronavirus was determined, as shown in FIG. 3, from which the spatial relationship of binding of antibodies to structural proteins of the novel coronavirus can be seen.
The neutralizing active antibody aiming at the specific target point of the virus is derived from an immunized mouse, B cells after the mouse undergoes V-D-J gene recombination in vivo, B cell receptor BCR of the B cells is screened and amplified with advantages under the action of antigen stimulation, and then various antigen epitope specific antibodies with high specificity and high affinity can be produced through links such as somatic cell high-frequency mutation, affinity maturation and the like. By adopting an in vitro experiment method and utilizing an enzyme-linked immunosorbent and pseudovirus infection neutralization screening technology, the high-specificity and high-affinity mouse-origin neutralizing activity antibody against the novel coronavirus can be screened, and the light chain variable region gene and the heavy chain variable region gene of the antibody are cloned from the neutralizing activity antibody, so that the neutralizing activity antibody is a unique and brand-new protein sequence. Identifying the uniqueness of the gene sequence and corresponding protein sequence and CDR sequences thereof; provides important technical support for developing and developing anti-diagnosis and therapeutic agents, can be applied to the research of new coronavirus infection mechanisms, and has important practical application value as an important technical means for diagnosis and immunotherapy.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (8)
1. A monoclonal antibody for SARS-CoV-2 glycoprotein RBD region of novel coronavirus is characterized by being named XA326.8, comprising a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown as SEQ ID No.1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 2.
2. A monoclonal antibody aiming at a novel coronavirus SARS-CoV-2 glycoprotein RBD region is characterized by being named XA326.7 and comprising a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown as SEQ ID No.3, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 4.
3. A monoclonal antibody aiming at a novel coronavirus SARS-CoV-2 glycoprotein RBD region is characterized by being named XA326.18 and comprising a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown as SEQ ID No.5, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 6.
4. A monoclonal antibody aiming at a novel coronavirus SARS-CoV-2 glycoprotein RBD region is characterized by being named XA326.49 and comprising a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown as SEQ ID No.7, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 8.
5. Use of a monoclonal antibody directed against the RBD region of SARS-CoV-2 glycoprotein of a novel coronavirus according to any one of claims 1 to 4 for the preparation of a novel SARS-CoV-2 assay product.
6. Use of a monoclonal antibody directed against the RBD region of SARS-CoV-2 glycoprotein of a novel coronavirus as claimed in any of claims 1 to 4 for the preparation of a medicament for inhibiting SARS-CoV-2 of a novel coronavirus.
7. Use of a monoclonal antibody directed against the RBD region of SARS-CoV-2 glycoprotein of a novel coronavirus as claimed in any of claims 1 to 4 for the preparation of an antibody pharmaceutical formulation for the prevention or treatment of pneumonia caused by SARS-CoV-2 of a novel coronavirus.
8. A test reagent or test kit comprising the monoclonal antibody of any one of claims 1 to 4 directed against the RBD region of SARS-CoV-2 glycoprotein of a novel coronavirus.
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