CN115260307A - High-affinity fully human monoclonal antibody for resisting rabies virus and application thereof - Google Patents

High-affinity fully human monoclonal antibody for resisting rabies virus and application thereof Download PDF

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CN115260307A
CN115260307A CN202210649220.1A CN202210649220A CN115260307A CN 115260307 A CN115260307 A CN 115260307A CN 202210649220 A CN202210649220 A CN 202210649220A CN 115260307 A CN115260307 A CN 115260307A
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胡萍
辛洪波
王纯艳
刘苏俊
张文华
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Nanchang University
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Abstract

The invention relates to a fully human monoclonal antibody of high affinity anti-rabies virus, belonging to the field of biological immunology. The heavy chain variable region of the monoclonal antibody or the binding fragment thereof has a nucleotide sequence of the heavy chain variable region shown as SEQ ID NO. 11; and the variable region of the light chain nucleotide sequence shown in SEQ ID NO 12. The invention also provides a coding gene, an expression vector, application and a composition of the antibody. Rabies virus Glycoprotein (GP) is the only protective antigenic protein on the surface of the virus. The invention successfully constructs a prokaryotic expression vector of rabies virus specific antigen protein, and expresses and purifies the prokaryotic expression vector by utilizing an escherichia coli prokaryotic system, and the antigen protein is successfully used for screening antibodies. And 1 strain of specific fully human monoclonal antibody sequence aiming at rabies virus GP fragment is successfully screened by using the screening platform, which lays a foundation for clinically developing antibody medicaments for treating rabies.

Description

High-affinity rabies virus-resistant fully-humanized monoclonal antibody and application thereof
Technical Field
The invention relates to the field of biological immunology, in particular to a fully human monoclonal antibody of high-affinity anti-rabies virus and application thereof.
Background
Rabies (Rabies) is an acute infectious disease causing severe damage to the central nervous system, mainly caused by Rabies virus (RABV). The virus infects the body mainly through damaged skin, mucous membrane or respiratory tract, and saliva is one of the main infection routes. Almost all warm mammalian species, such as raccoon, skunk, bat, fox, are RABV hosts, with the primary source of infection being in diseased or virus-carrying canines[7]. Most of human rabies is mainly caused by bite of sick animals, and some patients are caused by scratching or contamination of wounds, mucous membranes and the like, and there are few cases of attack caused by transplantation of organs or tissues donated by the patients. RABV acts primarily in the central nervous system and is abundant in the cerebrospinal fluid of susceptible animals. Human rabies is predominantly manifested as acute progressive encephalomyelitis, which usually occurs in two forms: manic rabies accompanied by indirect excitement and hallucination, accompanied by symptoms such as wind fear, water fear and vocal fear, usually causes death within 7 days, and paralytic rabies characterized by muscle weakness usually causes death within 14 days. The latency period of the disease is generally 3 weeks to 3 months, and reports of less than 10 days or up to 1 year and more are very rare[9]。
Over 1500 million people worldwide receive vaccination after rabies exposure every year, causing economic losses of up to $ 40 million.
Since the first antibody drug Orthoclone was marketed in 1992, 63 antibody drugs were marketed in 2016, and an average of 2.5 antibody drugs were marketed every year, with increasing rates. Antibody drugs have long been reported for the treatment of viral infectious diseases, and the case of antisera for the treatment of SARS and severe H5N1 hepatitis C virus infected persons has demonstrated the important role played by antibodies in the treatment of viral infections. The market vacancy of the wild dog monoclonal antibody at home and abroad is large at present, a recombinant humanized wild dog antibody NM57S/NC08 injection developed by China North China pharmacy enters a stage III clinical test stage in 2018, a stage III clinical test report is obtained in 4 months in 2020, and the wild dog monoclonal antibody is a combined preparation obtained by combining two wild dog monoclonal antibodies NM57S and NC08 and needs to be combined with a vaccine for use. In foreign markets, mabs RabiMabs developed by Zydus cadila qualified by the U.S. Food and Drug Administration (FDA) for rabies prevention in 2019, month 5, and were approved by the indian Drug Administration General of India (DCGI) on day 3 of the same year, month 9, for use in combination with vaccines for post-rabies exposure prevention. The recombinant anti-RABV mab injection, rabishield, co-developed by indian serum institute and Mass Biologics, was approved for marketing in india at 2016, 12 months. The fully humanized monoclonal antibody for preventing and treating rabies is not available in the global market, once the product is marketed and produced in a large scale, the cost for preventing and treating rabies is greatly reduced, and the fully humanized monoclonal antibody has competitive advantages which are incomparable with the traditional blood products and vaccines. Therefore, we hope to screen one or more broad-spectrum anti-rabies fully humanized monoclonal antibodies with therapeutic effect by a specific screening method after in vitro activation of the special human memory B cells in the laboratory, and improve the current situation of rabies prevention and treatment difficulty.
Disclosure of Invention
The inventor obtains a fully human monoclonal antibody with broad-spectrum combination with rabies virus through deep research, the monoclonal antibody is fully human, and the variable regions and the constant regions of the heavy chain and the light chain of the monoclonal antibody are all derived from human genes, so the monoclonal antibody has the characteristics of low immunogenicity and high safety.
The 1861 rabies virus gene was first reported to be a single-stranded non-segmented negative-strand RNA virus that is bullet-like in shape, about 75nm in diameter and 100-300 nm in length, about 12kb. Viral RNA encodes 5 proteins, in turn, the nucleocapsid protein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), and the polymerase large protein (L). The invention selects G protein as antigen epitope, the G protein is composed of 1575 nucleotides, is the only protein located on the surface of virus, can combine with intracellular receptor acetylcholine receptor to make the virus have neurotropic property, can induce host to generate neutralizing antibody, and has protective effect.
One aspect of the present invention is to provide a fully human monoclonal antibody against rabies virus, which has a heavy chain variable region and a light chain variable region:
(I) The amino acid sequence of the heavy chain variable region has the nucleotide sequence of the heavy chain variable region shown in any one of SEQ ID NOs 11, 13, 15, 17 and 19; or has an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in any one of SEQ ID NO 11, 13, 15, 17 and 19 and has the same function;
(II) the amino acid sequence of the light chain variable region has the nucleotide sequence of the light chain variable region as shown in any one of SEQ ID NOs 12, 14, 16, 18 and 20; or an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in any one of SEQ ID NO 12, 14, 16, 18 and 20 and has the same function.
Preferably, the monoclonal antibody has any one of the following groups of heavy chain variable region and light chain variable region:
(i) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11 (1H)10) (ii) a The amino acid sequence of the light chain variable region is shown as SEQ ID NO:12 (3 lambda)5)；
(ii) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 13 (1H)16) (ii) a The amino acid sequence of the light chain variable region is shown as SEQ ID NO:14 (3 lambda)7)；
(iii) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 15 (1H)17) (ii) a The amino acid sequence of the light chain variable region is shown as SEQ ID NO:16 (3 lambda)8)；
(iv) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17 (1H)3) (ii) a The amino acid sequence of the light chain variable region is shown as SEQ ID NO. 18 (3 lambda)1)；
(v) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19 (1H)18) (ii) a The amino acid sequence of the light chain variable region is shown as SEQ ID NO:20 (3 lambda)10)。
The monoclonal antibody provided by the invention can be specifically combined with rabies virus G protein.
In another aspect of the present invention, the present invention also provides a nucleic acid molecule encoding the monoclonal antibody described above.
Preferably, the nucleic acid molecule has the nucleotide sequence of the heavy chain variable region as set forth in any one of SEQ ID NOs 1, 3, 5, 7 and 9; and the nucleotide sequence of the variable region of the light chain as set forth in any one of SEQ ID NOs 2, 4, 6, 8 and 10.
More preferably, the nucleic acid molecule has any one of the following sets of heavy chain variable region and light chain variable region:
(i) The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 1 (1H)10) (ii) a The variable region of the light chain has the nucleotide sequence shown in SEQ ID NO:2 (3 lambda)5)；
(ii) The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 3 (1H)16) (ii) a The variable region of the light chain has the nucleotide sequence shown in SEQ ID NO:4 (3 lambda)7)；
(iii) The variable region of the heavy chain has the nucleotide sequence shown in SEQ ID NO:5 (1H)17) (ii) a The variable region of the light chain has the nucleotide sequence shown in SEQ ID NO. 6 (3 lambda)8)；
(iv) The variable region of the heavy chain has the nucleotide sequence shown in SEQ ID NO:7 (1H)3) (ii) a The variable region of the light chain has the nucleotide sequence shown in SEQ ID NO. 8 (3 lambda)1)；
(v) The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO:9 (1H)18) (ii) a Nucleotide sequence of light chain variable regionShown in SEQ ID NO:10 (3. Lambda.)10)。
In another aspect of the invention, there is provided an expression vector comprising the nucleic acid molecule described above.
The expression vector may contain, in addition to the nucleic acid molecule described above, a suitable promoter or control sequence. The vector may be used to transform an appropriate host cell so that it can express the protein.
In another aspect of the present invention, there is provided a host cell comprising the above-described expression vector.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: bacterial cells such as E.coli, streptomyces; salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, NSO or Bowes melanoma cells, etc. Particularly suitable host cells for use in the present invention are eukaryotic host cells, especially mammalian cells, such as 293 cells.
In another aspect of the present invention, there is provided a method for preparing the monoclonal antibody, comprising the steps of:
(1) Memory B cell sorting: separating peripheral blood mononuclear cells from blood samples of volunteers infected with rabies viruses, and sorting the peripheral blood mononuclear cells to obtain memory B cells;
(2) In vitro activation culture and positive well screening of memory B cells: activating the memory B cells into plasma cells, detecting secretory IgG antibodies of the memory B cells after the memory B cells are activated into the plasma cells in vitro by ELISA, and screening to obtain positive holes;
(3) Constructing and screening an antibody variable region gene library: carrying out reverse transcription on the positive hole cells to obtain cDNA, and amplifying genes of heavy chain variable regions and light chain variable regions of the antibodies;
(4) Constructing antibody gene heavy chain variable region and light chain variable region libraries: respectively connecting the amplified genes of the heavy chain variable region and the light chain variable region of the antibody to an expression vector;
(5) Screening of antibody heavy chain variable region and light chain variable region genes: the recombinant plasmid was co-transfected into HEK293T cells for expression.
In another aspect of the invention, the monoclonal antibody or the fragment thereof is provided for use in the preparation of a medicament for detecting, treating and preventing rabies virus infection.
In another aspect of the present invention, there is provided a composition comprising an antibody mixture of one or more of said monoclonal antibodies in a therapeutically effective amount, and a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable" as used herein means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human. As used herein, a "pharmaceutically acceptable carrier" should be compatible with, i.e., capable of being blended with, the monoclonal antibody of the invention or fragment thereof without substantially reducing the effectiveness of the composition as is often the case.
Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tableting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like.
The compositions of the present invention may be formulated into various dosage forms as desired, and may be administered by a physician in light of such factors as the type, age, weight and general condition of the patient, the mode of administration, and the like, of the dose to be administered to the patient. The administration may be, for example, by injection or other therapeutic means.
The pharmaceutical composition may comprise two or more monoclonal antibodies or fragments thereof having binding activity to rabies virus.
In another aspect of the present invention, there is provided a kit for detecting rabies virus, comprising said monoclonal antibody or fragment thereof.
Based on the monoclonal antibody or the fragment thereof, the kit for conveniently, quickly and accurately detecting the rabies virus can be prepared. As a detection method of the present invention, an indirect ELISA method is used, in which an antigen to be detected is coated on a solid phase carrier, and detection is performed using the monoclonal antibody or the fragment thereof of the present invention. In a preferred embodiment of the present invention, the monoclonal antibody or a fragment thereof is an antibody, and is detected according to the principle of double antibody sandwich method. The double antibody sandwich method is conventionally performed by immobilizing a primary antibody (e.g., the monoclonal antibody of the present invention) on a carrier, reacting the primary antibody with an antigen, washing, reacting with a secondary antibody (the secondary antibody carries a detectable signal or can bind to a substance carrying a detectable signal), and detecting the signal by chemiluminescence or enzyme-linked color reaction. The double antibody sandwich method is particularly suitable for the detection of antigens having two or more epitopes.
For convenience in detection, the kit may further comprise other detection reagents or auxiliary reagents besides the monoclonal antibody or fragment thereof of the present invention, such as reagents conventionally used in ELISA kits, the properties of which and their formulation methods are well known to those skilled in the art, such as color-developing agents, labels, secondary antibodies, anti-antibodies, sensitizers, etc. It will be understood by those skilled in the art that various modifications of the detection kit are encompassed by the present invention as long as the monoclonal antibody or fragment thereof of the present invention is utilized therein as a reagent for recognizing rabies virus.
In addition, instructions for use may be included in the kit to instruct the method of use of the reagents loaded therein.
After obtaining the monoclonal antibody or the fragment thereof provided by the present invention, various immunologically related methods can be used to detect rabies virus Glycoprotein (GP) in the sample, so as to know whether the donor of the sample to be detected is infected with rabies virus, and these methods are all included in the present invention. Preferably, the method is for the purpose of non-disease diagnosis.
In another aspect of the invention, there is provided a method of non-therapeutically inhibiting rabies virus, said method comprising administering to a patient an effective amount of said monoclonal antibody or fragment thereof.
In another aspect of the present invention, there is provided a method for non-therapeutic detection of rabies virus, wherein the monoclonal antibody or fragment thereof is contacted with a test sample, and the presence and amount of rabies virus is detected by detecting the binding of the monoclonal antibody or fragment thereof to the test sample.
As used herein, the term "test sample" encompasses a variety of sample types, including blood and other bodily fluid samples of biological origin, solid tissue samples such as biopsy tissue samples or tissue cultures, or cells derived therefrom or progeny thereof. The term also includes samples that have been treated by any means after they have been obtained, for example by treatment with reagents, solubilization, or enrichment for certain components such as proteins or polynucleotides. The term encompasses various clinical samples obtained from any species, also including cultured cells, cell supernatants and cell lysates.
Has the beneficial effects that: compared with the prior art, the fully human monoclonal antibody for resisting the rabies viruses is successfully prepared, has the characteristic of high affinity, can be specifically combined with the rabies viruses, and can obviously resist the rabies viruses. Compared with a mouse antibody, the gene of the fully human antibody is completely derived from the human gene, has no other species of components, does not generate toxic and side effects such as an anti-mouse antibody and the like in a human body, has better biocompatibility, is more suitable and has more potential to become a macromolecular drug for treating rabies viruses, and provides great guarantee for the standardized production of finished drugs of the antibody.
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Figure 1 positive well cell screen. A: elisa screening memory B cell positive holes, wherein red is positive hole cells for plasmid construction; b: and (5) carrying out agarose electrophoresis detection on the sample, wherein M is a DNA molecular standard.
FIG. 2 shows the result of gene library amplification electrophoresis of heavy chain VH and VH' and light chain VK and Vlambda variable regions of sample antibodies; m is a DNA molecular standard.
FIG. 3 construction of gene library of heavy and light chain variable region of antibody. A: carrying out enzyme digestion electrophoresis on the target plasmid and the carrier plasmid of the sample; b: K. performing secondary enzyme digestion electrophoresis on the IgK; c: transforming the recombinant plasmid TOP 10; d: and (3) carrying out enzyme digestion electrophoresis identification on the recombinant plasmid, wherein M is a DNA molecular standard.
FIG. 4 shows the results of screening the combination of heavy and light chain variable region genes of antibodies.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: cold Spring harbor laboratory Press, 1989), or according to the manufacturer's recommendations.
Example 1: preparation of fully human monoclonal antibody of (1)
1. Sorting of memory B cells
1.1 acquisition of peripheral blood mononuclear cells
Fresh 3.5ml of blood was drawn from the rabies virus infected volunteers and placed in an anticoagulation tube. Diluting fresh anticoagulated whole blood and PBS in equal volume, slowly adding into lymphocyte separation liquid (Solarbio, cat. NO. P8610) for centrifugation, taking out carefully, absorbing the second cloudy mononuclear cell layer, washing in a centrifuge tube filled with PBS, centrifuging, discarding supernatant, washing 1 time with PBS, and collecting the cells as Peripheral Blood Mononuclear Cells (PBMC).
1.2 memory B cell sorting
The supernatant was thoroughly removed by centrifugation of PBMCs of the separated volunteers every 10 th7The individual cells were resuspended in cell pellet with 40. Mu.L buffer. Add 10. Mu.L Biotin-Antibody Cocktail, mix well and incubate in refrigerator at 4 ℃ for 5min. Adding 30. Mu.L buffer solution and 20. Mu.L Anti-Biotin MicroBeads, mixing well, and standing in a refrigerator at 4 ℃ for 10min. The cells were washed with 1-2mL of buffer, centrifuged at 1000rpm for 10min and the supernatant removed. Resuspend the cells with 500. Mu.L buffer. The column was placed on a magnet and the column was washed with 3mL of buffer. The cell suspension is applied to a sorting column to prevent the formation of air bubbles, and the outflowing unlabeled cells are collected. The column was washed 3 times with 500 μ L of buffer and all the eluents were collected, i.e. the suspension containing B cells. The above experiment used the "human memory B cell sorting magnetic bead" kit (Miltenyi, cat. NO.99-130-046-901, germany).
The cells were counted, centrifuged at 1000rpm for 10min, the supernatant was thoroughly removed, and the cell pellet was resuspended in 80. Mu.L of buffer. Add 20. Mu.L of CD27 MicroBeads to the cells, mix well, incubate for 15min in a refrigerator at 4 ℃. Cells were washed with 1-2mL buffer and centrifuged at 1000rpm for 10min to remove the supernatant completely. Resuspending with 500. Mu.L buffer solution to less than or equal to 108A cell. The post was placed on the magnet. The column was washed with the appropriate volume of buffer. The cell suspension was applied to the column. The eluted unlabeled cells were collected and the column was washed 3 times with the appropriate volume of buffer. Collecting all the eluent, namely the suspension containing the unlabeled cells, and adding the washing liquid after the liquid in the column is drained in the washing process. The column was removed from the separator, placed on a collection tube, the buffer was pipetted onto the column and the plunger was immediately forced to wash out the magnetic bead labeled cells. The cell is a memory B cell. Counting the sorted memory B cells. The above experiment used the "human B cell CD27 sorting magnetic beads" kit (Miltenyi, germany, cat. No. 99-130-051-601).
2. In-vitro activation culture and positive hole screening of memory B cells
The method comprises the steps of collecting 50ml of fresh whole blood of 2 volunteers with high RABV IgG antibody expression in serum, separating memory B cells by a magnetic bead sorting method, culturing the memory B cells and feeder layer cells in a U-shaped 96-well plate together, detecting the expression of the antibody in the supernatant by Elisa after culturing for 10 days, wherein the result is shown in table 1 (figure 1A is a quantization diagram), screening 3 positive cell wells with the highest OD values from the memory B cells, sequentially numbering I, II and III (respectively containing 10, 50 and 100 memory B cells), extracting RNA, carrying out reverse transcription to cDNA, and then verifying by taking GAPDH as a primer, wherein the result is shown in figure 1B, the single normal 3T3 cell has no band, and the samples of the positive wells I, II and III have obvious bands at 250bp positions, which shows that the RNA of the screened positive cell wells secreting the antibody is successfully extracted, and the later-stage plasmid construction can be carried out.
TABLE 1Elisa screening of Positive well cells secreting anti-rabies Virus antibodies
Figure BDA0003685410860000081
Figure BDA0003685410860000091
TABLE 1 detection of B cell supernatant specificity using Enzyme-linked immunosorbent assay (ELISA).
3. Construction and screening of antibody variable region gene library
RNA of cells in each hole is extracted from samples I, II and III, variable regions of heavy chain VH, light chain VK and V lambda of an antibody are amplified by a nested PCR two-wheel amplification method, and the variable regions of the heavy chain are divided into VH and VH' parts for respective amplification due to the large molecular weight and long amino acid sequence. Detecting the amplification result by agarose gel electrophoresis, and recovering gel to obtain the DNA of the amplified antibody heavy chain VH, VH' and light chain VK, vlambda variable region fragments. The electrophoresis results are shown in FIG. 2, and the antibody heavy chain VH and VH' and light chain VK and V lambda variable regions have obvious bands at 400bp, which is expected.
3.4.2 construction of antibody heavy and light chain variable region Gene library
Carrying out enzyme digestion on the amplified heavy chain VH and VH' variable region genes, light chain VK and V lambda variable region genes and constant region vector plasmids IgH, igK and Ig lambda according to the specification of an enzyme digestion kit, wherein the electrophoresis result is shown in figures 3A and 3B, the target gene has an obvious strip at 400bp, the vector plasmid has an obvious strip at 5000bp, and the result accords with the expectation. Subsequently, the objective plasmid and the vector plasmid were subjected to enzyme ligation reaction, and the recombinant plasmid was transformed into TOP10 cells, resulting in fig. 3C, wherein colonies grew on control plates and colonies grew in each experimental group. After the plasmid is extracted slightly, the recombinant plasmid is subjected to enzyme digestion for identification, the result is shown in figure 3D, and the samples of the recombinant plasmids I, II and III screened earlier are subjected to enzyme digestion to obtain two bands with different molecular weights, wherein the heavy chain VH and VH' of the target plasmid and the light chain VK and Vlambda of the target plasmid have obvious bands at 400bp bands, the carrier plasmids IgH, igK and Ig lambda have obvious bands at 5000bp bands, and the result is expected to indicate that the construction of the recombinant plasmid of the antibody heavy-light chain variable region gene library is successful.
3.5 screening of monoclonal antibodies
We screened monoclonal antibody sequences from a library of heavy and light chain individual total plasmids. (1) The method comprises the following steps Sequentially transfecting HEK239T cells by using the screened 3 sample heavy-light chain combinations IH + IK, IH + Ilambda, IIH + IIK, IIH + IIlambda, IIIH + IIIK and IIIH + IIIlambda, and screening out a plasmid combination (2) secreting a specific antibody: screening IIIH and IIIlambda according to OD values, averagely dividing transformed IIIH and IIIlambda into 8 parts, sequentially numbering extracted plasmids as 1H, 2H and 3H. (3) The method comprises the following steps Screening out 1H and 3 lambda from IIIH and IIIlambda 8 small portions, taking 20ng of plasmid for transformation, respectively selecting 30 portions of monoclonal colonies numbered as 1H in sequence1、1H2、1H3.......1H30And 3 λ1、 3λ2、3λ3.......3λ301H and 3 λ are also used1、3λ2、3λ3.......3λ303 λ and 1H1、1H2、1H3.......1H30HEK239T cells are combined and transfected to screen out a plasmid combination secreting specific antibodies. (4) The method comprises the following steps We selected 10 heavy chain monoclonal plasmids with higher OD values from them: 1H3、1H10、1H17、1H18、1H21、1H23、 1H24、1H27、1H28、1H9And 10 light chain monoclonal plasmids: 3 lambda2、3λ6、3λ7、3λ8、3λ10、3λ11、 3λ14、3λ15、3λ20、3λ29. Combining the heavy-light chain monoclonal plasmids two by two, sequentially transfecting HEK293T cells with the 100 plasmid combinations, screening out the monoclonal plasmid combination secreting specific antibodies, and detecting the antibody expression condition in cell supernatant by Elisa. Table 2 (FIG. 4 is a quantitative chart) shows Elisa results of the first 20 plasmid combinations with higher OD among 100 combinations.
TABLE 2 Elisa screening of monoclonal antibody heavy and light chain combinatorial plasmids
Figure BDA0003685410860000101
Screening to obtain heavy and light chain monoclonal sequences, selecting 5 heavy and light chain monoclonal sequences respectively, and performing one-to-one pairing in vitro transfection expression, wherein the heavy chain monoclonal sequences of the 5 heavy chains: 1H10,1H16,1H17,1H3,1H218And 5 light chain monoclonal sequences: 3 lambda5，3λ7,3λ8,3λ1,3λ10And finally obtaining 5 monoclonal antibodies with high affinity after ELISA screening, wherein the antibody combination is as follows: 1H10+3λ5、1H16+3λ7、1H17+3λ8、1H3+3λ1、1H18+3λ10(the results correspond to FIG. 4). The five plasmid combinations had OD values above 1.1, with very good affinity.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications. It is intended that the scope of the invention be defined by the claims and their equivalents.
SEQUENCE LISTING
<110> university of Nanchang
<120> high-affinity fully human monoclonal antibody against rabies virus and application thereof
<130> 1
<160> 20
<170> PatentIn version 3.5
<210> 1
<211> 356
<212> DNA
<213> 1H10 heavy chain variable region nucleotide sequence
<400> 1
gtacattccg aggtgcagct ggtgcagtct gggggaggct tggtccagcc tggggggtcc 60
ctgagactct cctgtgcagc ctctgtggag gcctttagta catattggat gaactgggtc 120
cgccaggctc cagggaaggg gctggagtgg gtggccaaca taaaccgaga tggaagtcag 180
aaatactatg tggactctgt ggagggccga ttcaccatct ccagagacaa cgccaagagc 240
tcactgtatc tgcaaatgaa cagcctgaga gccgatgaca cggctgtcta ttactgtatg 300
agtggctacg attccggcca ctggggccag ggatccctgg tcaccgtctc ctcagc 356
<210> 2
<211> 392
<212> DNA
<213> 3 lambda 5 light chain variable region nucleotide sequence
<400> 2
ttcaattccc aggctgtggt gactcaggag ccctcactga ctgtgtcccc aggagggaca 60
gtcactctca cctgtggctc cagtactgga gctgtcacca gtggtcttta tgcctactgg 120
ttccagcaga agcctggcca agtcccccgg acactgagta ctggagctgt catttataat 180
ataagcaaca aacagtcctg gacccctgcc cggttctcag gctccctcct tgggggcaaa 240
gctgccctga ccctttcggg tgcgcagcct gaggatgagg ctgactatta ctgcttgctc 300
tcctatagtc gtactcgggt gttcggcgga gggaccaagc tgaccgtcct aggtcagccc 360
aaggctgccc cctcggtcac tctgttccca cc 392
<210> 3
<211> 356
<212> DNA
<213> 1H16 heavy chain variable region nucleotide sequence
<400> 3
gtacattctg aagtgcagct ggtggagtct gggggaggct tggtccagcc tggggggtcc 60
ctgagactct cctgtgcagc ctctggattc acctttagta catattggat gaactgggtc 120
cgccaggctc cagggaaggg gctggagtgg gtggccaaca taaaccgaga tggaagtcag 180
aaatactatg tggactctgt ggagggccga ttcaccatct ccagagacaa cgccaagagc 240
tcactgtatc tgcaaatgaa cagcctgaga gccgatgaca cggctgtcta ttactgtatg 300
agtggctacg attccggcca ctggggccag ggatccctgg tcaccgtctc ctcagc 356
<210> 4
<211> 386
<212> DNA
<213> 3 lambda 7 light chain variable region nucleotide sequence
<400> 4
tccaattctc aggctgtggt gacccaggag ccctcactga ctgtgtcccc aggagggaca 60
gtcactctca cctgtggctc cagtactgga gctgtcacca gtggtcttta tgcctactgg 120
acagtcactt tccagcagaa gcctggccaa gtcccccgga cactgattta taatataagc 180
aacaaacagt cctggacccc tgcccggttc tcaggctccc tccttggggg caaagctgcc 240
ctgacccttt cgggtgcgca gcctgaggat gaggctgact attactgctt gctctcctat 300
agtcgtactc gggtgttcgg cggagggacc aagctgaccg tcctaggtca gcccaaggct 360
gccccctcgg tcactctgtt cccgcc 386
<210> 5
<211> 350
<212> DNA
<213> 1H17 heavy chain variable region nucleotide sequence
<400> 5
gtacattctg aggtgcagct gttggagtct gggggaggct tggtccagcc tggggggtcc 60
ctgagactct cctgtgcagc ctctggattc acctttagta catattggat gaactgggtc 120
cgccaggctc cagggaaggg gctggagtgg gtggccaacc gagatggaag tcagaaatac 180
tatgtggact ctgtggaggg ccgattcacc atctccagag acaacgccaa gagctcactg 240
tatctgcaaa tgaacagcct gagagccgat gacacggctg tctattactg tatgagtggc 300
tacgattccg gccactgggg ccagggatcc acggtcaccg tctcctcagc 350
<210> 6
<211> 371
<212> DNA
<213> 3 lambda 8 light chain variable region nucleotide sequence
<400> 6
tcttgggcca attttatgct gactcaggag ccctcactga ctgtgtcccc aggagggaca 60
gtcactctca cctgtggctc cagtactgga gctgtcacca gtggtcttta tgcctactgg 120
ttccagcaga agcctggcca agtcccccgg acactgattt ataatataag caacaaacag 180
tcctggaccc ctgcccggtt ctcaggctcc ctccttgggg gcaaagctgc cctgaccctt 240
tcgggtcctg aggatgaggc tgactattac tgcttgctct cctatagtcg tactcgggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtcagccca aggctgcccc ctcggtcact 360
ctgttcccgc c 371
<210> 7
<211> 383
<212> DNA
<213> 1H3 heavy chain variable region nucleotide sequence
<400> 7
gtacattctc aggtgcagct ggtggagtct gggggaggcg tggtccagcc tgggaggtcc 60
ctgagactct cctgtgcagc ctctggattc accttcagta gctatgctat gcactgggtc 120
cgccaggctc caggcaaggg gctggagtgg gtggcagtta tatcatatga tggaaataaa 180
tactacgcag actccgtgaa gggccgattc accatctcca gagacaattc caagaacacg 240
ctgtatctgc aaatgaacag cctgagagat gaggacacgg ctgtgtatta ctgtgcgaga 300
gctcctgatt gtagtggtgg tagctgctcc actgaatact tccagcactg gggccagggc 360
accctggtca ccgtctcctc agc 383
<210> 8
<211> 371
<212> DNA
<213> 3 lambda 1 light chain variable region nucleotide sequence
<400> 8
tcctgggccc agtctgtgct gacgcaggag ccctcactga ctgtgtcccc aggagggaca 60
gtcactctca cctgtggctc cagtactgga gctgtcacca gtggtcttta tgcctactgg 120
ttccagcaga agcctggcca agtcccccgg acactgattt ataatataag caacaaacag 180
tcctggaccc ctgcccggtt ctcaggctcc ctccttgggg gcaaagctgc cctgaccctt 240
tcgggtgcgc agcctgagga tgaggctgac tattactgct tgctctccta tagtcgtact 300
cgggtgttcg ggaccaagct gaccgtccta ggtcagccca aggctgcccc ctcggtcact 360
ctgttcccac c 371
<210> 9
<211> 368
<212> DNA
<213> 1H18 heavy chain variable region nucleotide sequence
<400> 9
gtacattccg aggtgcagct ggtgcagtct gggggaggct tggtccagcc tggggggtcc 60
ctgagactct cctgtgcagc ctctggattc acctttagta catattggat gaactgggtc 120
cgcctctccc agactccagg gaaggggctg gagtgggtgg ccaacataaa ccgagatgga 180
agtcagaaat actatgtgga ctctgtggag ggccgattca ccatctccag actctccgac 240
aacgccaaga gctcactgta tctgcaaatg aacagcctga gagccgatga cacggctgtc 300
tattactgta tgagtggcta cgattccggc cactggggcc agggatccac ggtcaccgtc 360
tcctcagc 368
<210> 10
<211> 368
<212> DNA
<213> 3 lambda 10 light chain variable region nucleotide sequence
<400> 10
tccaattccc agactgtggt gacccaggag ccctcactga ctgtgtcccc aggagggaca 60
gtcactctca cctgtggctc cagtactgga gctgtcacca gtggtcttta tgcccagcag 120
aagcctggcc aagtcccccg gacactgatt tataatataa gcaacaaaca gtcctggacc 180
cctgcccggt tctcaggctc cctccttggg ggcaaagctg ccctgaccct ttcgggtgcg 240
cagcctgagg atgaggctga ctattactgc ttgctctcct atagtcgtac tcgggtgttc 300
ggcggaggga ccaagctgac cgtcctaggt cagcccaagg ctgccccctc ggtcactctg 360
ttcccgcc 368
<210> 11
<211> 118
<212> PRT
<213> 1H10 heavy chain variable region amino acid sequence
<400> 11
Val His Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
1 5 10 15
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Val Glu Ala Phe
20 25 30
Ser Thr Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ala Asn Ile Asn Arg Asp Gly Ser Gln Lys Tyr Tyr Val
50 55 60
Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser
65 70 75 80
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Met Ser Gly Tyr Asp Ser Gly His Trp Gly Gln Gly Ser
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 12
<211> 130
<212> PRT
<213> 3 lambda 5 light chain variable region amino acid sequence
<400> 12
Phe Asn Ser Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser
1 5 10 15
Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
20 25 30
Thr Ser Gly Leu Tyr Ala Tyr Trp Phe Gln Gln Lys Pro Gly Gln Val
35 40 45
Pro Arg Thr Leu Ser Thr Gly Ala Val Ile Tyr Asn Ile Ser Asn Lys
50 55 60
Gln Ser Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys
65 70 75 80
Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Asp Tyr
85 90 95
Tyr Cys Leu Leu Ser Tyr Ser Arg Thr Arg Val Phe Gly Gly Gly Thr
100 105 110
Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
115 120 125
Phe Pro
130
<210> 13
<211> 118
<212> PRT
<213> 1H16 heavy chain variable region amino acid sequence
<400> 13
Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
1 5 10 15
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 25 30
Ser Thr Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ala Asn Ile Asn Arg Asp Gly Ser Gln Lys Tyr Tyr Val
50 55 60
Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser
65 70 75 80
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Met Ser Gly Tyr Asp Ser Gly His Trp Gly Gln Gly Ser
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 14
<211> 128
<212> PRT
<213> 3 lambda 7 light chain variable region amino acid sequence
<400> 14
Ser Asn Ser Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser
1 5 10 15
Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
20 25 30
Thr Ser Gly Leu Tyr Ala Tyr Trp Thr Val Thr Phe Gln Gln Lys Pro
35 40 45
Gly Gln Val Pro Arg Thr Leu Ile Tyr Asn Ile Ser Asn Lys Gln Ser
50 55 60
Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala
65 70 75 80
Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys
85 90 95
Leu Leu Ser Tyr Ser Arg Thr Arg Val Phe Gly Gly Gly Thr Lys Leu
100 105 110
Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
115 120 125
<210> 15
<211> 116
<212> PRT
<213> 1H17 heavy chain variable region amino acid sequence
<400> 15
Val His Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
1 5 10 15
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 25 30
Ser Thr Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ala Asn Arg Asp Gly Ser Gln Lys Tyr Tyr Val Asp Ser
50 55 60
Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Met Ser Gly Tyr Asp Ser Gly His Trp Gly Gln Gly Ser Thr Val
100 105 110
Thr Val Ser Ser
115
<210> 16
<211> 123
<212> PRT
<213> 3 lambda 8 light chain variable region amino acid sequence
<400> 16
Ser Trp Ala Asn Phe Met Leu Thr Gln Glu Pro Ser Leu Thr Val Ser
1 5 10 15
Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
20 25 30
Thr Ser Gly Leu Tyr Ala Tyr Trp Phe Gln Gln Lys Pro Gly Gln Val
35 40 45
Pro Arg Thr Leu Ile Tyr Asn Ile Ser Asn Lys Gln Ser Trp Thr Pro
50 55 60
Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu
65 70 75 80
Ser Gly Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Leu Ser Tyr Ser
85 90 95
Arg Thr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
115 120
<210> 17
<211> 127
<212> PRT
<213> 1H3 heavy chain variable region amino acid sequence
<400> 17
Val His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
1 5 10 15
Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 25 30
Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Asn Lys Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ala Pro Asp Cys Ser Gly Gly Ser Cys Ser Thr Glu
100 105 110
Tyr Phe Gln His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 18
<211> 123
<212> PRT
<213> 3 lambda 1 light chain variable region amino acid sequence
<400> 18
Ser Trp Ala Gln Ser Val Leu Thr Gln Glu Pro Ser Leu Thr Val Ser
1 5 10 15
Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
20 25 30
Thr Ser Gly Leu Tyr Ala Tyr Trp Phe Gln Gln Lys Pro Gly Gln Val
35 40 45
Pro Arg Thr Leu Ile Tyr Asn Ile Ser Asn Lys Gln Ser Trp Thr Pro
50 55 60
Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu
65 70 75 80
Ser Gly Ala Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Leu Ser
85 90 95
Tyr Ser Arg Thr Arg Val Phe Gly Thr Lys Leu Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
115 120
<210> 19
<211> 122
<212> PRT
<213> 1H18 heavy chain variable region amino acid sequence
<400> 19
Val His Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
1 5 10 15
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 25 30
Ser Thr Tyr Trp Met Asn Trp Val Arg Leu Ser Gln Thr Pro Gly Lys
35 40 45
Gly Leu Glu Trp Val Ala Asn Ile Asn Arg Asp Gly Ser Gln Lys Tyr
50 55 60
Tyr Val Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Leu Ser Asp
65 70 75 80
Asn Ala Lys Ser Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp
85 90 95
Asp Thr Ala Val Tyr Tyr Cys Met Ser Gly Tyr Asp Ser Gly His Trp
100 105 110
Gly Gln Gly Ser Thr Val Thr Val Ser Ser
115 120
<210> 20
<211> 122
<212> PRT
<213> 3 lambda 10 light chain variable region amino acid sequence
<400> 20
Ser Asn Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser
1 5 10 15
Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
20 25 30
Thr Ser Gly Leu Tyr Ala Gln Gln Lys Pro Gly Gln Val Pro Arg Thr
35 40 45
Leu Ile Tyr Asn Ile Ser Asn Lys Gln Ser Trp Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala
65 70 75 80
Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Leu Ser Tyr Ser Arg
85 90 95
Thr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
100 105 110
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
115 120

Claims (7)

1. A high affinity fully human monoclonal antibody against rabies virus having the following heavy and light chain variable regions:
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 11; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 12.
2. The monoclonal antibody of claim 1, wherein the antibody specifically binds to rabies virus G protein.
3. A nucleic acid molecule encoding the monoclonal antibody of claim 1 or 2, having the following heavy chain variable region and light chain variable region:
the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 1; the variable region of light chain has the nucleotide sequence as shown in SEQ ID No. 2.
4. An expression vector comprising the nucleic acid molecule of claim 3.
5. A host cell comprising the expression vector of claim 4.
6. Use of the monoclonal antibody or binding fragment thereof of claim 1 or 2 in the preparation of a medicament for the detection, treatment or prevention of rabies virus infection.
7. A composition comprising a therapeutically effective amount of the monoclonal antibody of claim 1, and a pharmaceutically acceptable carrier.
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