Antibody or antigen binding fragment for resisting agkistrodon acutus venom and application of antibody or antigen binding fragment

Abstract

The invention discloses an anti-agkistrodon acutus venom antibody or antigen binding fragment and application thereof. The anti-agkistrodon acutus venom antibody or antigen binding fragment can be specifically bound with agkistrodon acutus venom and comprises three heavy chain complementary determining regions, and the amino acid sequences of the anti-agkistrodon acutus venom antibody or antigen binding fragment are shown as amino acid sequences coded by SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. The amino acid sequences of the three light chain complementarity determining regions are shown as amino acid sequences coded by SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16. The anti-agkistrodon acutus venom antibody or antigen binding fragment can be specifically bound with agkistrodon acutus venom without binding with agkistrodon acutus venom, viper venom, cobra venom and bungarus multicinctus venom, and can be used for detecting agkistrodon acutus venom. The monoclonal antibody disclosed by the invention has a neutralizing and protecting effect on agkistrodon acutus venom, and has important significance in assisting clinical treatment, reducing complications and improving the survival rate of patients.

Description

Technical Field

[0001] The present invention belongs to the field of immunology, and relates to an antibody or antigen-binding fragment against agkistrodon quinquefasciatus venom and an application thereof, and specifically relates to an antibody or antigen-binding fragment against agkistrodon quinquefasciatus venom, a test strip and an application thereof. Background Art

[0002] Deinagkistrodon acutus, commonly known as the five-step snake, is a medium-to-large tube-toothed venomous snake of the genus Deinagkistrodon in the family Viperidae. It has a large, obviously triangular head, an upturned snout, and cheek pits. The back of the head of the juvenile is light brown, the back of the body is pink-brown, and the tip of the tail is light yellow. It is widely distributed in China. The venom of the five-step snake is mainly composed of snake venom metalloproteinases (SVMPs), snake venom serine proteases (SVSPs), C-type lectins (CLECs), 5'-nucleotidase, nuclease, and snake venom phospholipase A2 (SVPLA2). The content of SVMPs, CLECs, PLA2, SVSP, and L-amino-acidoxidase (LAAOs) is relatively high in protein components.

[0003] China is one of the countries with the richest snake resources, with snakes distributed throughout the country. Cases of venomous snake bites such as pit vipers, five-step snakes and cobras are common. There are great differences in venomous snakes in various provinces and regions, and the level of snake bite treatment in different regions is uneven, and there is still a certain mortality and disability rate. Venomous snake bites are an acute injury, with rapid onset and rapid disease progression after the bite. Severe cases produce severe poisoning symptoms or even death, which is significantly related to the amount of venom excreted by the venomous snake and the length of time from the bite to the doctor. Therefore, rapid and accurate toxin detection is of great significance for assisting clinical treatment, reducing complications and improving patient survival. In the field of snake venom diagnosis, the biggest problem is how to distinguish one snake venom from other snake venoms. First, snake venom is not a single protein or polypeptide, but has multiple components; second, the same components in different types of snake venom will interfere with the diagnosis. Therefore, it is necessary to detect the unique components of five-step snake venom. The preparation of a monoclonal antibody that can specifically bind to five-step snake venom has become the key to solving the above problems.

[0004] Currently, in the detection of snake venom, the hybridoma cells corresponding to the monoclonal antibodies have problems such as low antibody production, unknown antibody sequences, and easy changes in cell stability. Summary of the invention

[0005] In the first aspect, in order to obtain antibodies or antigen-binding fragments against five-step snake venom that are different from the prior art, and to obtain independently developed neutralizing antibodies or five-step snake venom detection reagents that specifically bind to five-step snake venom, the present invention provides the following technical solutions:

[0006] An antibody against Agkistrodon acutus venom or an antigen-binding fragment binding to Agkistrodon acutus venom, characterized in that it can specifically bind to Agkistrodon acutus venom and comprises a heavy chain variable region and a light chain variable region:

[0007] c1) the three heavy chain complementary determining regions of the heavy chain variable region, HC-CDR1, HC-CDR2, and HC-CDR3, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively; and

[0008] c2) the three light chain complementary determining regions of the light chain variable region, LC-CDR1, LC-CDR2, and LC-CDR3, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively:

[0009] The antibody against Agkistrodon acutus venom is a monoclonal antibody;

[0010] The antigen-binding fragment that binds to Agkistrodon acutus venom is:

[0011] d1) single chain Fv; or

[0012] d2) disulfide-linked Fv; or

[0013] d3) Fab fragment; or

[0014] d4) F(ab')2 fragment; or

[0015] d5) Fab' fragment.

[0016] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region are as shown in the amino acid sequences encoded by SEQ ID NO:2 and SEQ ID NO:10, respectively, or have at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences encoded by SEQ ID NO:2 and SEQ ID NO:10, respectively.

[0017] In some embodiments, the amino acid sequence of the heavy chain constant region of the monoclonal antibody is shown in SEQ ID NO:22, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:24.

[0018] In some embodiments, the anti-Agaman snake venom antibody or the antigen-binding fragment that binds to Agaman snake venom has a leader peptide with an amino acid sequence as shown in SEQ ID NO: 18 and SEQ ID NO: 20, located at the N-terminus of its heavy chain variable region and the N-terminus of its light chain variable region, respectively.

[0019] In some embodiments, the anti-Agamoebae venom antibody or antigen-binding fragment that binds Agamoebae venom is a monospecific, bispecific, or multispecific antibody or antigen-binding fragment.

[0020] In the second aspect, the present invention further discloses a nucleic acid, which comprises a nucleic acid fragment encoding an antibody against A. acutus venom or an antigen-binding fragment binding to A. acutus venom as described in the first aspect; the nucleic acid fragment comprises a heavy chain variable region nucleic acid fragment and a light chain variable region nucleic acid fragment, encoding the heavy chain variable region and the light chain variable region, respectively. The heavy chain variable region nucleic acid fragment and the light chain variable region nucleic acid fragment may be connected or not connected. When connected, the nucleic acid fragment corresponds to one sequence encoding both the heavy chain variable region and the light chain variable region; when not connected, the nucleic acid fragment corresponds to two sequences encoding the heavy chain variable region and the light chain variable region, respectively.

[0021] In some embodiments, the nucleotide sequences are as shown in SEQ ID NO: 3, 5, 7, 11, 13, 15 to encode HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, LC-CDR3 of the anti-Agamandra venom antibody or the antigen-binding fragment that binds to Agamandra venom, respectively.

[0022] In some embodiments, it comprises the nucleotide sequences shown in SEQ ID NO: 1 (heavy chain variable region) and SEQ ID NO: 9 (light chain variable region).

[0023] In the third aspect, the present invention further discloses one or more expression vectors, wherein the target gene inserted therein comprises the nucleic acid described in the second aspect. The expression vectors herein simultaneously insert the nucleic acids encoding the heavy chain variable region and the light chain variable region; or comprise a heavy chain variable region plasmid and a light chain variable region plasmid into which the nucleic acids encoding the heavy chain variable region and the light chain variable region are inserted respectively.

[0024] In a fourth aspect, the present invention further discloses a host cell, which comprises one or more expression vectors described in the third aspect.

[0025] In a fifth aspect, the present invention further discloses a composition comprising the anti-Agamoeboides venom antibody or the antigen-binding fragment binding to Agamoeboides venom described in the first aspect.

[0026] In a sixth aspect, the present invention further discloses a test strip, the detection line of which is coated with the anti-Agaman snake venom antibody or the antigen-binding fragment binding to Agaman snake venom as described in the first aspect; the test strip can be used for the detection of Agaman snake venom.

[0027] In a seventh aspect, the present invention further discloses the use of the anti-Agamandra venom antibody or the antigen-binding fragment binding to Agamandra venom described in the first aspect in the preparation of a drug for treating Agamandra bites.

[0028] The present invention discloses an antibody or antigen-binding fragment that can specifically distinguish five-step snake venom without being interfered by other types of snake venom. The antibody or antigen-binding fragment that binds to five-step snake venom composed of 6 CDRs of amino acid sequence such as SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16 can specifically bind to five-step snake venom and not bind to agkistrodon venom, viper venom, cobra venom, and bungarus venom, and can be used for the detection of five-step snake venom. The test strip prepared by the monoclonal antibody of the present invention can quickly detect and determine whether the patient has been poisoned by five-step snake venom. The monoclonal antibody of the present invention has a neutralizing protective effect on five-step snake venom, which is of great significance for auxiliary clinical treatment, reducing complications and improving patient survival rate.

[0029] The present invention uses snake venom with complex components as antigen to immunize mice, screens and obtains antibodies or antigen-binding fragments with specific CDRs, and proves that they can specifically bind to the five-step snake venom, which has very important practical significance. In addition, the antibody of the present invention can be prepared using Chinese Hamster Ovary Cells (CHO) system, which can effectively solve the problems of low antibody yield of hybridoma cells, unknown antibody sequence, and easy change of cell stability.

[0030] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0032] Figure 1 This is the result of the titer determination of the anti-Agaman snake venom monoclonal antibody W14B5G5.

[0033] Figure 2 This is the electrophoresis result of the anti-Agamo snake venom monoclonal antibody W14B5G5. Among them, M represents Marker, R represents reducing, and NR represents non-reducing.

[0034] Figure 3 This is the result of the plasmid experiment.

[0035] Figure 4 This is the result of the sensitivity test of the five-step snake venom rapid test paper.

[0036] Figure 5 It is the result of the five-step snake venom rapid test paper detecting cross snake venom. DETAILED DESCRIPTION

[0037] In order to facilitate understanding by those skilled in the art, some terms appearing in this document are explained and illustrated.

[0038] As used herein, the singular forms "a", "an", and "the" include plural forms unless the context indicates otherwise. Thus, for example, "an agent" may be understood to include a plurality of agent components.

[0039] Herein, unless otherwise stated, the terms “comprises”, “includes” or “comprising” mean that the listed values, steps or components are included, but other values, steps or components are not excluded.

[0040] In this document, "subject" or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples.

[0041] As used herein, the term "antibody" refers to an immunoglobulin that can specifically recognize and bind to an antigen, and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies or antibody fragments.

[0042] Herein, the term "Fragment of variable region (Fv)" refers to a domain formed by connecting the heavy chain variable region and the light chain variable region of an antibody with a short peptide, which recognizes and specifically binds to an antigen epitope.

[0043] In this article, CDR region or "complementarity determining region" refers to the region in the variable region of an antibody that is highly variable in sequence and forms a loop that is structurally determined and / or contains antigen contact amino acid residues. CDR is primarily responsible for the binding of the antibody to the antigen epitope and determines the specificity of the antibody. In a given heavy chain or light chain variable region amino acid sequence, the specific amino acid sequence of each CDR is determined using any one of many known numbering rules or a combination thereof, including, for example, Kabat, Contact, AbM and Chothia. The CDR of an antibody of the present invention can be determined according to any rule or a combination thereof in the art.

[0044] Since the venom of pit viper, Agkistrodon acutus venom, etc. are all multi-component snake venoms with complex ingredients, although the monoclonal antibody W14B5G5 is obtained in the present invention, it is not clear which component in Agkistrodon acutus venom the monoclonal antibody specifically detects. However, experiments have shown that this monoclonal antibody can indeed specifically bind to Agkistrodon acutus venom from a variety of snake venoms. Therefore, in this article, antibodies or antigen-binding fragments with 6 CDRs of monoclonal antibody W14B5G5 are referred to as "antibodies against Agkistrodon acutus venom or antigen-binding fragments that bind to Agkistrodon acutus venom." Herein, "specifically binding to Agkistrodon acutus venom" does not mean that it specifically binds to various components in Agkistrodon acutus venom, but that it can specifically bind to components in Agkistrodon acutus venom, but it is not specifically analyzed what components they are specifically targeting.

[0045] In this article, the light chain variable region (VL) and the heavy chain variable region (VH) both include three complementary determining regions (CDR1, CDR2, CDR3) and four framework regions (FR1, FR2, FR3, FR4). The three CDRs of the light chain variable region (VL) are LC-CDR1, LC-CDR2, and LC-CDR3; the three CDRs of the heavy chain variable region (VH) are HC-CDR1, HC-CDR2, and HC-CDR3.

[0046] The anti-Agamo venom antibody or antigen-binding fragment thereof of the present invention comprises a substitution, insertion or deletion. The anti-Agamo venom antibody of the present invention comprises a modification of a light chain variable region, a heavy chain variable region, a light chain or a heavy chain, and the amino acid sequence thereof after modification is different from the amino acid sequence from which the antibody is derived. For example, an amino acid sequence derived from the same specified protein may be similar to the starting sequence, for example, having a certain percentage identity, for example, it may have a percentage identity of 90%, 92%, 96%, 98% with the starting sequence.

[0047] In the present invention, "identity" refers to the percentage of bases (or amino acids) in the two sequences being compared when the sequences are aligned between two peptides or between two nucleic acid molecules. Software programs known in the art can be used to determine the alignment and homology percentage or sequence identity, such as BLASTN and BLASTP.

[0048] "Antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, de-immunized antibodies, or Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain antibodies and epitope-binding fragments of any of the above.

[0049] Fab fragment (Antigen-binding fragment), also called antigen-binding fragment, refers to the antigen-binding region of an antibody, which contains a complete light chain and a heavy chain variable region and the CH1 domain of the constant region.

[0050] The F(ab')2 fragment contains two Fab fragments and a partial fragment of the hinge region, and the heavy chains are connected by disulfide bonds. The disulfide bonds between the heavy chains of the F(ab')2 fragment are broken to form two Fab' fragments.

[0051] The terms "treat" or "treat" or "alleviate" or "improve" are used interchangeably herein and refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or preventive benefit. As used herein, therapeutic benefit generally refers to eradication or reduction of the severity of the underlying condition being treated. In addition, therapeutic benefit is achieved by eradicating, reducing the severity, or reducing the incidence of one or more physiological symptoms associated with the underlying condition so that an improvement is observed in the animal (although the animal may still be afflicted with the underlying condition). For preventive benefit, the risk of disease in animals at risk of developing a particular disease can be reduced. As used herein, the term "therapeutic effect" generally includes therapeutic benefit and / or preventive benefit as described above. Preventive effects include delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping or reversing the progression of a disease or condition, or any combination thereof.

[0052] The term "subject" or "individual" or "animal" or "patient" used in this application refers to a human or non-human animal, including mammals or primates, for whom diagnosis, prognosis, alleviation, prevention and / or treatment of a disease or condition is desired. Mammalian subjects include humans, livestock animals, farm animals, and zoo or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, etc.

[0053] As used herein, the term "in vivo" generally refers to events that occur within an animal's body.

[0054] As used herein, the term "in vitro" generally refers to an event that occurs outside an animal. For example, an in vitro cell function test or any animal in vitro assay. In vitro assays include cell-based assays in which dead or live cells are used. In vitro assays also include cell-free assays in which intact cells are not used.

[0055] As used herein, the term "administering" refers to delivering a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the present invention to a subject. Administration may be systemic or topical. Administration may be performed by an administration device, such as a syringe. Modes of administration include, but are not limited to, embedding, nasal inhalation, spraying, injection, and the like. Routes of administration include inhalation, intranasal, oral, intravenous, subcutaneous, or intramuscular administration, and the like.

[0056] The nucleotide sequences shown in this article are arranged from the 5' end to the 3' end; the amino acid sequences are arranged from the amino terminus (N terminus) to the carboxyl terminus (C terminus).

[0057] Herein, for the sake of brevity, "antibodies against Agkistrodon acutus venom or antigen-binding fragments that bind to Agkistrodon acutus venom" are referred to as "antibodies against Agkistrodon acutus venom or antigen-binding fragments".

[0058] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0059] Example 1. Obtaining mouse monoclonal antibodies.

[0060] 1.1 Immunization procedures:

[0061] (1) Agkistrodon acutus venom (from Shanghai Seron Biotechnology Co., Ltd.) was used as the immunogen (antigen). All Balb / c mice used for immunization (Shanghai Shengchang Biotechnology Co., Ltd.) were 6-8 weeks old, female, and healthy purebred mice weighing 18-20 g.

[0062] (2) First immunization: Mix 20 μg of antigen with 200 μl of complete Freund's adjuvant to make a 400 μl solution, and inject it into multiple subcutaneous points of Ba1b / c mice. Second to fourth immunization: Two weeks after the last immunization, mix 20 μg of antigen (200 μl) with 200 μl of incomplete Freund's adjuvant to make a 400 μl solution, and inject it into multiple subcutaneous points of Ba1b / c mice. Seven days after the fourth immunization, blood was collected from the tail vein of the mice, and the antibody titer of the mouse serum was measured by enzyme-linked immunosorbent assay (ELISA). When the antibody titer reached 1:1 million or more, cell fusion was prepared. Three days before cell fusion, booster immunization was performed, and 20 μg of antigen was used to immunize the mice during the booster immunization.

[0063] 1.2 Cell fusion:

[0064] (1) SP2 / 0 mouse myeloma cells were purchased from ATCC, USA. The culture medium was changed one day before fusion to keep the SP2 / 0 myeloma cells in good growth condition.

[0065] (2) Spleen cells: Take a mouse immunized with Agkistrodon acutus venom, bleed it, and kill it by breaking its neck. Then soak it in 75% alcohol for 3-4 minutes. Transfer the mouse to a clean bench. Take out the mouse spleen, put it in a cell culture dish, add 15ml of serum-free RPM1640 culture medium, and gently crush it with a glass rod until there are no tissue clumps and the cells are uniform. Then, wash the mouse spleen cells three times with serum-free RPM1640 culture medium, count them and set aside.

[0066] (3) Sp2 / 0 myeloma cells in the logarithmic growth phase were taken, washed three times with serum-free RPM1640 medium, and counted for later use.

[0067] (4) Mouse spleen cells and Sp2 / 0 myeloma cells were mixed at a ratio of 10:1, centrifuged at 1500 rpm for 7 min, and the supernatant was washed away to prepare for fusion.

[0068] (5) Slowly add 1 ml of PEG (1450) within 1 min and shake gently for 90 s; then add 5 ml of serum-free RPM1640 culture medium within 2.5 min, and finally add 5 ml of serum-free RPM1640 culture medium to terminate the reaction. After standing for 5 min, centrifuge at 1280 rpm for 8 min, discard the supernatant, add regular RPM1640 culture medium (containing 10% fetal bovine serum) to prepare a cell suspension.

[0069] (6) The above cell suspension was added at 2 x 10 4 The cells were seeded into 96-well plates at a density of 100 cells / well, 200 μl per well, and incubated in a 37°C, 5% CO2 cell culture incubator. After 5 days of culture, the culture medium was replaced with the conventional RPM1640 medium containing HAT (25X), and the cells were continued to be incubated in a 37°C, 5% CO2 cell culture incubator. HAT refers to hypoxanthine (H), aminopterin (A), and thymidine (T).

[0070] 1.3 Obtaining single cell clones by limiting dilution method

[0071] (1) Use the limiting dilution method for screening. Gently blow the hybridoma cells to be cloned in the culture well and count them; adjust the cells to 3-10 cells / ml. Take a new cell culture plate and add 100μl of diluted cells to each well. Continue incubation in a 37℃, 5% CO2 cell culture incubator; change the medium on the 7th day, and change the medium once every 2-3 days thereafter; on the 8th-9th day, cell monoclonal formation can be seen, and the supernatant in the well plate is taken for ELISA detection. The ELISA detection method is shown in 1.4.

[0072] (2) The confirmed cell clone is subcloned 2-3 times to ensure that the cell colony comes from the same hybridoma cell.

[0073] 1.4 Identification of positive clones by ELISA

[0074] (1) Antigen coating: Coat the ELISA plate with 0.2 μg / ml of Agkistrodon acutus venom solution, Agkistrodon acutus venom solution, Viper venom solution, Cobra venom solution, and Bungarus cobra venom solution. Wash the ELISA plate after coating.

[0075] (2) Blocking: Block the ELISA plate with 2% bovine serum albumin solution and wash the ELISA plate after blocking.

[0076] (3) Primary antibody incubation: Take the supernatant of the single cell clone well and add it to the ELISA plate. Incubate in a 37°C incubator for 90 min. After the incubation, wash the ELISA plate.

[0077] (4) Secondary antibody incubation: Add goat anti-mouse secondary antibody HRP (Southernbiotech, USA) to the ELISA plate and incubate in a 37°C incubator for 90 min. After the incubation, wash the ELISA plate.

[0078] (5) Color development: Add the single-component color development solution 3,3',5,5'-tetramethylbenzidine (TMB) to the ELISA plate and incubate in a 37°C incubator for 5 min.

[0079] (6) Termination: Add 50 μl of 2 mol / L sulfuric acid to the ELISA plate.

[0080] (7) OD value detection: Detect the ELISA plate at a wavelength of 450 nm.

[0081] (8) Criteria for positive wells: For the supernatant from the same well, when the antigen to be detected is Agkistrodon acutus venom, the OD value 检测孔 / OD value 阴性对照 >2.1; When testing other snake venoms, OD value 检测孔 / OD value 阴性对照 <2.1. The single cell clone corresponding to the supernatant is the desired positive hybridoma cell.

[0082] 1.5 Filter Results

[0083] (1) After screening, a monoclonal antibody against Agkistrodon acutus venom was obtained. The monoclonal antibody against Agkistrodon acutus venom was named W14B5G5. Figure 1 As shown, monoclonal antibody W14B5G5 specifically binds to Agkistrodon acutus venom and has no binding to other snake venoms.

[0084] Example 2 Ascites Preparation and Monoclonal Antibody Purification

[0085] 2.1 Ascites preparation

[0086] (1) 0.3-0.5 ml of pristane or liquid paraffin is injected intraperitoneally into Balb / c mice. After 7-15 days, the selected hybridoma cell lines are expanded and cultured and then inoculated into the peritoneal cavity of Balb / c mice.

[0087] (2) Seven days after the injection of hybridoma cells, the abdomen of the mice began to swell and was observed daily. When the mice had difficulty eating, walking, or had dark fur, they were killed and ascites was extracted once.

[0088] (3) Mice were killed by dislocating the cervical vertebrae and disinfected by soaking in 75% alcohol for 5 min;

[0089] (4) Use surgical scissors to make a small incision in the mouse abdomen, peel off the surrounding skin to expose the abdominal cavity, then make a small incision in the abdominal cavity, insert a rubber-tipped dropper into the abdominal cavity and aspirate the effusion into a clean 15 ml centrifuge tube;

[0090] (5) Centrifuge at 3000 rpm for 10 min. The colorless and transparent middle layer is ascites.

[0091] 2.2 Antibody purification

[0092] (1) The ascites was diluted with PBS pH 7.4, centrifuged, and the supernatant was purified by protein G affinity chromatography.

[0093] (2) Equilibration: Equilibrate the purification column with 0.4 M PB buffer (pH 7.0);

[0094] (3) Loading: Slowly pass the diluted ascites supernatant through the column to ensure that the antibody is better bound to the protein G column;

[0095] (4) Washing: Wash the column with equilibration buffer;

[0096] (5) Elution: The antibody bound to the column was eluted with 0.1 M glycine buffer (pH 2.7), and 1 M Tris-HCl (pH 8.0) was added to neutralize the glycine to keep the pH at a neutral value suitable for antibody storage.

[0097] (6) The purified monoclonal antibody was subjected to SDS-PAGE electrophoresis. The results were as follows: Figure 2 As shown, the heavy and light chains of the antibody can be clearly seen.

[0098] Example 3 Recombinant plasmid construction and amplification

[0099] 3.1 Nucleotide Sequencing

[0100] (1) The monoclonal hybridoma cells were sent to Shanghai Bio-Innovation Biotechnology Co., Ltd. for sequencing to obtain the nucleotide sequences of the antibody heavy chain and light chain. Table 1 shows the sequences of the important regions of the monoclonal antibody.

[0101] Table 1. Nucleotide and amino acid sequences of important regions of monoclonal antibody W14B5G5

[0102]

[0103]

[0104]

[0105] The nucleotide sequence of the heavy chain constant region of W14B5G5 is shown below (SEQ ID NO: 21):

[0106] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA

[0107] The amino acid sequence of the heavy chain constant region of W14B5G5 (SEQ ID NO: 22) is:

[0108] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0109] The nucleotide sequence of the light chain constant region of W14B5G5 is shown below (SEQ ID NO: 23):

[0110] CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAAC AGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

[0111] The amino acid sequence of the light chain constant region of W14B5G5 (SEQ ID NO: 24) is:

[0112] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0113] 3.2 Expression vector construction

[0114] (1) Appropriate upstream and downstream primers were constructed for the heavy chain and light chain of W14B5G5, respectively, and PCR amplification was performed on the target sequence (heavy chain or light chain of W14B5G5) to obtain PCR products.

[0115] (2) Gel recovery: Use a gel recovery kit to recover the PCR product.

[0116] (3) Double restriction digestion of the PCR product to expose restriction sites. Double restriction digestion of the pcDNA3.1 vector (Shanghai Bio-Tech Co., Ltd.) was performed to obtain a linearized vector.

[0117] (4) Recombinant plasmid ligation: The recombinant plasmids were ligated according to the reaction system in Table 2. The recombinant plasmids include a heavy chain recombinant plasmid and a light chain recombinant plasmid.

[0118] Table 2. Reaction system

[0119]

[0120] Note: The recombinant enzyme is from Shanghai Bio-Innovation Biotechnology Co., Ltd.

[0121] (5) The constructed plasmid was placed in a culture medium for 2-3 minutes to allow the temperature to drop, and then the transformation and bacterial solution coating experiments were performed at 37°C overnight.

[0122] 3.3 Colony screening experiment

[0123] (1) Pick a single colony from the overnight plate.

[0124] (2) Colony PCR was performed using BI-CMV-F (SEQ ID NO: 25): CGCAAATGGGCGGTAGGCGTG; BI-SEQ-R (SEQ ID NO: 26): AGCGTAAAAGGAGCAACATAGT.

[0125] (3) After the colony PCR results are correct, select the corresponding colony, add it to the culture medium and culture it overnight.

[0126] 3.4 Plasmid extraction (using the plasmid extraction kit of Tiangen Biochemical Technology Co., Ltd.)

[0127] (1) Take 1-5 mL of bacterial culture and centrifuge at 12,000 rpm for 1 min. Remove the supernatant as much as possible (if the bacterial culture is large, the bacterial precipitate can be collected into a centrifuge tube through multiple centrifugation).

[0128] (2) Add 250 μL of Solution I (25 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50 mM glucose, 100 μg / ml RNase A) to the centrifuge tube containing the bacterial cell pellet, and use a vortex oscillator to thoroughly suspend the bacterial cell pellet.

[0129] (3) Add 250 μL of solution II (250 mM NaOH, 1% SDS (sodium dodecyl sulfate)) to the centrifuge tube and gently invert the tube 6-8 times to fully lyse the cells.

[0130] (4) Add 350 μL of Solution III (3 M potassium acetate, 5 M acetic acid) to the centrifuge tube and gently invert it 6-8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 min and carefully transfer the supernatant to another clean centrifuge tube with a pipette, trying not to suck out the precipitate.

[0131] (5) Take the supernatant from the previous step, add 0.6 times the volume of anhydrous ethanol, and mix thoroughly (if the volume is too much, mix it twice and then load it onto the column).

[0132] (6) Add the mixed solution obtained in the previous step to the adsorption column (the adsorption column is inserted into the collection tube), place it at room temperature for 2 minutes, centrifuge it at 12000 rpm for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0133] (7) Add 750 μL of rinse solution to the adsorption column (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube.

[0134] (8) Add 700 μL of rinse solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube.

[0135] (9) Centrifuge at 12,000 rpm for 2 min. Leave the adsorption column open at room temperature or in a 50°C incubator for several minutes to remove the remaining rinse solution in the adsorption column.

[0136] (10) Place the adsorption column in a clean centrifuge tube, and drop 50-200 μL of elution solution (10 mM Tris-HCl, pH = 7.5, 80% ethanol) preheated in a 65°C water bath onto the center of the adsorption membrane. Leave the tube at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min.

[0137] (11) In order to increase the recovery efficiency of the plasmid, the obtained eluate (10 mM Tris-HCl, pH = 7.5) can be added back to the adsorption column, left at room temperature for 2 min, and centrifuged at 12000 rpm for 1 min. Figure 3 As shown, it is the expected size.

[0138] Example 4: Recombinant expression of monoclonal antibodies in cells

[0139] 4.1 Preparation of cells before transfection

[0140] (1) Take out the required CHO (Thermo Fisher Scientific) cell tube from liquid nitrogen and thaw it in a 37°C water bath for 1-2 min to thaw the cells quickly. Do not immerse the cell tube completely in water.

[0141] (2) Transfer the thawed cell suspension to 30 ml of preheated ExpiCHO TM The expression medium was inoculated into a 125 ml polycarbonate, disposable, sterile, breathable conical flask and cultured in a shaker at 37°C, 8% CO2 and relative humidity ≥ 80%. After 3-5 days, the CHO cells were cultured to an appropriate cell density and then counted.

[0142] 4.2 Cell transfection

[0143] (1) Take 150×10 6 CHO cell suspension was centrifuged and the supernatant was discarded. The cells were resuspended in 5 ml of transfection reagent (GenePμlser Electroporation Buffer, #1652676).

[0144] (2) After mixing, add plasmid to a final concentration of 600 ng / μl. Repeatedly pipette the suspension to mix the CHO cells and plasmid DNA evenly.

[0145] (3) Add the mixed solution to the shock tube and place the shock tube into the electroporator to prepare for the shock.

[0146] (4) Set the electrotransfection parameters: voltage 160 V; pulse length 15 ms; electroporation tube width 2 mm; pulse number 1.

[0147] (5) Press the “start” key to execute the electroporation procedure.

[0148] (6) After the electroporation is completed, the cells in the electroporated tube are divided into shake flasks containing culture medium prepared in advance and incubated for 40 minutes.

[0149] (7) After the incubation, the cells were placed in a shaker at 37° C., 120 rpm, 8% CO 2 , and a relative humidity of ≥80% for culture. Monoclonal antibodies in the cell supernatant can be detected after 48 hours of culture.

[0150] 4.3 Antibody purification - Protein A affinity purification column purification

[0151] (1) Balanced chromatography column: 1xPBS, flow rate 1ml / min, 20ml

[0152] (2) Sample loading: flow rate 1 ml / min

[0153] (3) Washing: 1xPBS, flow rate 1ml / min, 20ml

[0154] (4) Elution: Sodium acetate buffer (PH 3.4), 1 ml / min, collect in separate tubes, about 1000 μl per tube. Collect 10 tubes in total, and read the absorbance at 280 nm using a NanoDrop instrument.

[0155] (5) Dialysis: Pipette high-concentration protein into a dialysis bag and place it in a beaker containing 1× PBS for dialysis. Perform dialysis three times.

[0156] Example 5: Preparation of a rapid test strip for the detection of Agkistrodon acutus venom and evaluation of its detection performance

[0157] 5.1 Probe preparation

[0158] (1) Add 5 mL of 100 mM 2-(N-morpholinyl)ethanesulfonic acid buffer to 0.125 mL of latex microsphere solution and mix well. Add a certain amount of N-hydroxysuccinimide (NHS) solution and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution. Oscillate at room temperature for 30 min.

[0159] (2) A certain amount of horse anti-Agamo snake venom polyclonal antibody (Shanghai Seron Biotechnology Co., Ltd.) was added to the above solution and incubated at room temperature for 2-3 hours. After the incubation, 1% BSA solution was added for blocking and the incubation was continued for 2-3 hours.

[0160] (3) The solution was centrifuged at 15,000 rpm for 10-30 minutes, the supernatant was discarded, and the precipitate was reconstituted with the preservation solution.

[0161] (4) The above solution is evenly soaked into the polyester fiber membrane and transferred to a 45°C oven for drying.

[0162] 5.2 Nitrocellulose membrane coating

[0163] (1) The nitrocellulose membrane was coated using an XYZ three-dimensional membrane stripper purchased from Shanghai Jinbiao Biotechnology Co., Ltd. The test line (T line) was coated with the anti-Agkistrodon quinquefasciatus venom recombinant monoclonal antibody W14B5G5, and the control line (C line) was coated with goat anti-horse secondary antibody purchased from Shanghai Solebao Company.

[0164] 5.3 Test strip assembly

[0165] Assemble the sample pad (glass fiber), nitrocellulose membrane (including T line and C line), polyester fiber membrane (including probe), and absorbent paper, and use a strip cutter to cut the large plate into 4 mm wide test papers. Put the cut test paper into the fixed position of the lower card shell of the outer packaging card shell, and then put the upper card cover of the card shell on, overlap and press. The sample pad, polyester membrane, nitrocellulose membrane, absorbent paper, and strip cutter were all purchased from Shanghai Jinbiao Biological Co., Ltd.

[0166] 5.4 Use of the Five-step Snake Venom Rapid Test Strips and Result Determination

[0167] (1) Take 3-4 drops of the sample and drip them into the sample well. Observe the results after 15 minutes. If both the T line and the C line are colored, it is a positive result. If only the C line is colored, it is a negative result. If only the T line is colored or no band is colored, it is invalid.

[0168] 5.5 Test strip detection performance evaluation

[0169] (1) Sensitivity test

[0170] Use physiological saline (0.9% NaCl, pH 7.2) to prepare 10 ng / ml, 50 ng / ml, and 100 ng / mL of Agkistrodon acutus venom solution as samples, add 100 μl of sample to each well, and observe the test results after 15 minutes. Figure 4 As shown: the test result of 10ng / mL is weakly positive; the test results of 50ng / ml and 100ng / mL are moderately positive and strongly positive; the minimum detection amount of the test paper is 10ng / ml.

[0171] (2) Crossover experiment

[0172] Prepare 500 ng / mL standard solutions of pit viper venom, viper venom, cobra venom, and bungarus venom (all from Shanghai Seron Biotechnology Co., Ltd.) in physiological saline (0.9% NaCl, pH 7.2) as samples, add 100 μl of sample to each well, and observe the test results after 15 minutes. Figure 5 As shown, the results of the tests for the other four snake venoms were negative.

[0173] Example 6: Evaluation of the protective effect of A. quinquefasciatus-specific antibodies on animals

[0174] 6.1 Preparation of diluent: weigh 8.5g sodium chloride, 4.5g boric acid, and 0.5g sodium tetraborate, add water for injection to make up to 1000ml, filter, and sterilize by wet heat at 121℃ for 30 minutes.

[0175] 6.2 Dilution of snake venom

[0176] Dilution of Agkistrodon acutus venom: Accurately aspirate the liquid glycerol Agkistrodon acutus venom stock solution and dilute it with diluent so that its 2LD50 volume (100μg) does not exceed 0.8ml. When mixing with anti-Acuto venom monoclonal antibody, add diluent to 2ml.

[0177] 6.3 Mixing:

[0178] (1) Experimental Group 2: 1 ml of 2.21 mg / ml anti-Agamus acutus monoclonal antibody W14B5G5 stock solution was added with twice the median lethal dose (2LD50) of Agamus acutus venom, and the diluent was added to 2 ml.

[0179] (2) Negative control group 2: Take 1 ml of the diluent, add 2LD50 test amount of Agkistrodon acutus venom, and add the diluent to 2 ml.

[0180] (3) Place the two mixed solutions in a 37°C water bath for 45 minutes and then immediately inject into mice.

[0181] 6.4 Injection: Inject 4 mice each in the experimental group 2 and the negative control group 2, with 0.4 ml injected intraperitoneally into each mouse.

[0182] 6.5 Observation: The experimental mice were observed once a day, and the incidence and death were recorded for 48-72 hours. The experimental results showed that in the negative control group 2, all 4 mice died within 24 hours; in the experimental group 2, only 1 mouse survived after 72 hours, with a mortality rate of 75%. Compared with the results of the negative control group, it was shown that the monoclonal antibody W14B5G5 that specifically binds to the venom of the five-step snake has a certain neutralizing protective effect on mice.

[0183] Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. Changes and improvements to the present invention will be possible without exceeding the concept and scope specified in the claims. In summary, the contents of the embodiments of this specification should not be understood as limiting the present invention.

Claims

1. An antibody against Agkistrodon acutus venom or an antigen-binding fragment thereof, characterized in that: Able to specifically bind to Agkistrodon acutus venom, including heavy chain variable region and light chain variable region: c1) the three heavy chain complementary determining regions of the heavy chain variable region, HC-CDR1, HC-CDR2, and HC-CDR3, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively; and SEQ ID NO: c2) the three light chain complementary determining regions of the light chain variable region, LC-CDR1, LC-CDR2, and LC-CDR3, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively: The antibody against Agkistrodon acutus venom is a monoclonal antibody; The antigen-binding fragment that binds to Agkistrodon acutus venom is: d1) single chain Fv; or d2) disulfide-linked Fv; or d3) Fab fragment; or d4) F(ab')2 fragment; or d5) Fab' fragment.

2. The anti-Agamoeboid venom antibody or the antigen-binding fragment binding to Agamoeboid venom according to claim 1, characterized in that: The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in the amino acid sequences encoded by SEQ ID NO:2 and SEQ ID NO:10, respectively, or have at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences encoded by SEQ ID NO:2 and SEQ ID NO:10, respectively.

3. The anti-Agamoebae venom antibody or the antigen-binding fragment binding to Agamoebae venom according to claim 1, characterized in that: The amino acid sequence of the heavy chain constant region of the monoclonal antibody is shown in SEQ ID NO:22, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

24.

4. The anti-Agamoebae venom antibody or the antigen-binding fragment binding to Agamoebae venom according to claim 1, characterized in that: It is a monospecific, bispecific or multispecific antibody or antigen-binding fragment.

5. A nucleic acid, characterized in that A nucleic acid fragment comprising an antibody against Agkistrodon acutus venom or an antigen-binding fragment binding to Agkistrodon acutus venom according to any one of claims 1 to 4; the nucleic acid fragment comprises a heavy chain variable region nucleic acid fragment and a light chain variable region nucleic acid fragment, encoding the heavy chain variable region and the light chain variable region, respectively.

6. An expression vector, characterized in that The inserted target gene comprises the nucleic acid according to claim 5.

7. A host cell, characterized in that Comprising one or more expression vectors according to claim 6.

8. A composition, characterized in that The invention comprises the anti-Agamo snake venom antibody or the antigen-binding fragment binding to Agamo snake venom according to any one of claims 1 to 4.

9. A test strip, characterized in that: The detection line is coated with the anti-Agamo snake venom antibody or the antigen-binding fragment binding to Agamo snake venom according to any one of claims 1 to 4; the test strip can be used for the detection of Agamo snake venom.

10. Use of the anti-Agamoebae venom antibody or the antigen-binding fragment binding to Agamoebae venom according to any one of claims 1 to 4 in the preparation of a drug for treating Agamoebae bites.

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