KR20230111815A - Manufacturing method of single domain vhh antibody for preventing or treating early mortality syndrome - Google Patents

Abstract

The present invention relates to a method for producing a single domain VHH antibody, comprising the step of injecting flagellin together with an antigen into a camelid animal in the step of immunizing a camelid animal to prepare a single domain VHH antibody for the prevention or treatment of shrimp premature death syndrome. The method can selectively increase the production of antigen-specific IgG ₂ and IgG ₃ among various antibodies.

Description

Manufacturing method of single domain VHH antibody for preventing or treating shrimp premature death syndrome {MANUFACTURING METHOD OF SINGLE DOMAIN VHH ANTIBODY FOR PREVENTING OR TREATING EARLY MORTALITY SYNDROME}

The present invention relates to a method for producing a single domain VHH antibody for preventing or treating shrimp premature mortality syndrome, and in the step of immunizing camelids to produce a single domain VHH antibody, flagellin together with a causative agent of shrimp premature death syndrome or a virus antigen is injected into camelid animals, thereby producing a single domain VHH antibody for preventing or treating premature shrimp death syndrome, which can selectively increase the production of antigen-specific IgG ₂ and IgG ₃ among various antibodies. .

A nanobody is a variable region of a heavy chain antibody (VHH) in a single domain antibody (SDA), and refers to a single domain antibody isolated from a camelid animal, a so-called VHH antibody. Since VHH antibodies are single-domain molecules, they are expressed in cells without supramolecular assemblies, unlike antibodies consisting of four chains, namely two light chains and two heavy chains, and are more stable and potent than normal antibodies. In addition, since the VHH antibody is a very small molecule of about 15 kDa, it can bind to a hidden epitope that is inaccessible to conventional antibodies, and when applied for treatment, efficient penetration and rapid clearance are possible due to its small molecular weight. Moreover, it is emerging as a candidate for intracellular probing because it is more likely to bind to the same intracellular and extracellular folds.

VHH antibodies are prepared by obtaining B cells from camelids immunized with an antigen, and then selecting antibodies using a phage display method. At this time, when an antigen is injected into a camel to immunize camelids, various antibodies (IgG, IgA, IgD, IgM, IgE, etc.) are produced in the camel's B cells. In particular, camels produce three IgG isotypes composed of IgG ₁ , IgG ₂ , and IgG ₃ . Among them, IgG ₂ and IgG ₃ are antibodies having a structure without a light chain and a CH1 domain, from which VHH antibodies are produced. However, among various antibodies produced by immunized camels, production of IgG ₂ and IgG ₃ is remarkably low, making it difficult to produce VHH antibodies. Therefore, it is necessary to develop a method for efficiently producing VHH antibodies by selectively increasing the amount of IgG ₂ and IgG ₃ produced.

Flagellin is a globular protein constituting flagellar fibers in bacteria, and its molecular weight varies greatly depending on the species of bacteria (30,000 to 70,000), and it is characterized by the absence of cysteine and tryptophan in its amino acid composition. As it is known that flagellin can induce an immunomodulatory effect by binding to the receptor of Toll-like receptor-5 (TLR-5), a receptor that recognizes molecular patterns associated with pathogens, its potential as an adjuvant for vaccines has been proven, and this is also disclosed in patent literature. Specifically, Korean Patent Registration No. 0795839 relates to a vaccine adjuvant containing flagellin, a component of bacterial flagella, as an active ingredient. It is disclosed that the flagellin component protein stimulates the production of interleukin-8 in epithelial cells, promotes the maturation of dendritic cells, and increases antigen-specific immune responses. However, in the past, only the function of flagellin as an adjuvant for vaccines has been demonstrated, and when flagellin is injected with an antigen in a camelid animal, the function of selectively enhancing the production of IgG ₂ and IgG ₃ among various antibodies has not been mentioned.

On the other hand, as a defense mechanism against disease, antibodies are included in substances produced to protect not only humans but also various mammals and birds from infectious diseases to protect themselves or their offspring. Most mammals, similar to humans, produce IgG of about 150 kDa. Conventionally, a method of producing pathogenic bacteria-specific antibodies from serum obtained by immunizing mammals such as rabbits, cows, and goats has been widely used to prepare antibodies against various pathogenic bacteria. However, since the antibody collected through this method is a large molecule of about 150 kDa, it has disadvantages in that penetration is not efficient and rapid clearance is difficult to occur when applied for treatment. Therefore, there is a need to use smaller antibody-derived molecules to efficiently treat disease.

Recently, as interest in improving dietary habits for preventing adult diseases has rapidly increased worldwide, the demand for aquatic products has continuously increased, and thus the production of fish aquaculture is rapidly increasing. Therefore, aquaculture has become a major economic means in many countries, and disease outbreaks in farmed fish have a great impact on economic development. Among farmed fish, Vibrio bacteria and white spot virus are known as the main causes of disease in shrimp. Vibrio genus is a gram-negative bacillus, which has one at one end and two or more cirrus depending on the species and moves actively. The causes of shrimp disease include Vibrio parahaemolyticus , Vibrio harveyi , and Vibrio anguillarum , which are called enteritis vibrio. When shrimp are infected with these bacteria, they die within 24 to 72 hours. In addition, the white spot syndrome virus (WSSV) has an attachment similar to a tail, and has a rod-shaped capsid and an envelope. When a shrimp is infected with the white spot virus, white spots appear on the shrimp's carapace, appendage and cuticle, the hepatopancreas turns red, and 3 to 5 days after infection, it dies. Therefore, there is a need for mass production of antibodies for preventing and treating premature mortality caused by various bacteria and viruses as described above.

Accordingly, as a result of intensive research efforts to overcome the problems of the prior art, the present inventors have confirmed that VHH can be efficiently produced by selectively increasing the production of IgG ₂ and IgG ₃ when flagellin is used to prepare VHH antibodies for preventing or treating premature shrimp death syndrome, thereby completing the present invention.

KR 10-0795839 B

Therefore, the main object of the present invention is the antigen-specific IgG ₂ and IgG ₃ It is to provide a method for producing a single domain VHH antibody for preventing or treating shrimp premature death syndrome, which can efficiently produce VHH antibodies by selectively increasing production, antibodies produced through the production method, and uses thereof.

According to one aspect of the present invention, the method for producing a single domain VHH antibody for preventing or treating shrimp premature death syndrome provides a method for producing a single domain VHH antibody comprising the step of injecting and immunizing a camelid animal with a mixture of a causative bacteria or virus antigen of shrimp premature death syndrome and flagellin.

Nanobodies refer to single domain antibodies, so-called VHH antibodies, isolated from camelids. Since the VHH antibody is a very small molecule of about 15 kDa, technologies for antibody therapeutics based thereon are being developed. In general, VHH antibodies are prepared by obtaining and selecting B cells from camelid animals immunized with an antigen. At this time, various antibodies (IgG, IgA, IgD, IgM, IgE, etc.) as well as IgG ₂ and IgG ₃ are produced in the B cells of immunized camelid animals. Among them, IgG ₂ and IgG ₃ have a structure without a light chain and a CH1 domain, from which VHH antibodies are produced. However, the IgG ₂ and IgG ₃ produced It is difficult to produce VHH antibodies because the amount produced is remarkably low. Accordingly, the inventors of the present invention have confirmed that, when flagellin is injected together with an antigen when immunizing a camel, the production of IgG ₂ and IgG ₃ is selectively increased, thereby efficiently producing a VHH antibody for preventing or treating premature shrimp death syndrome. The present invention was completed.

In the method for producing a single domain VHH antibody of the present invention, the causative agent of the shrimp premature death syndrome is one or more bacteria selected from the group consisting of Vibrio parahaemolyticus, Vibrio harveyi and Vibrio anguillarum, and the virus is a white spot syndrome virus (WSSV).

In the method for producing a single domain VHH antibody of the present invention, the flagellin is derived from a bacterium selected from the group consisting of Salmonella typhimurium , Vibrio vulnficus and Listeria monocytogenes .

In the method for producing a single domain VHH antibody of the present invention, the mixing ratio of the outer membrane protein or antigen and flagellin may be 5 to 20:1, preferably 10:1. For example, when the content of outer membrane protein or antigen is 1 mg, flagellin may be 0.05 to 0.2 mg. As the ratio of flagellin increases in the mixing ratio, the antigen-specific IgG ₂ and IgG ₃ It is difficult to efficiently produce a single domain VHH antibody because it is not selectively produced.

According to an experimental example of the present invention, as a result of confirming the production of IgG ₂ and IgG ₃ according to the mixing of the outer membrane protein or antigen and flagellin, it was confirmed that when alpaca was immunized by mixing the outer membrane protein or antigen with flagellin (Example), the production of antigen-specific IgG ₂ and IgG ₃ was selectively increased compared to the case where alpaca was immunized by injecting only the outer membrane protein or antigen (Comparative Example) (see Experimental Example 1). These results suggest that, in immunization with Alcapa to prepare a single domain VHH antibody, when flagellin is used, the production of IgG ₂ and IgG ₃ for VHH antibody production can be selectively increased, so that VHH antibody can be produced efficiently.

According to another aspect of the present invention, the present invention provides a single domain VHH antibody for preventing or treating shrimp premature death syndrome represented by the amino acid sequence of SEQ ID NO: 1 prepared by a manufacturing method comprising injecting and immunizing a mixture of a causative bacteria or virus antigen and flagellin of shrimp premature death syndrome into a camelid animal.

According to another aspect of the present invention, the present invention provides a single domain VHH antibody for preventing or treating shrimp premature death syndrome represented by the amino acid sequence of SEQ ID NO: 2 prepared by a manufacturing method comprising the step of injecting and immunizing a mixture of a causative bacteria or virus antigen and flagellin of shrimp premature death syndrome into a camelid animal.

The single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 1 of the present invention has specific binding ability to one or more bacteria selected from the group consisting of Vibrio parahaemolyticus , Vibrio harveyi and Vibrio anguillarum, in particular, Vibrio parahaemolyticus, and has an amino acid sequence of SEQ ID NO: 2 of the present invention. Domain VHH antibodies have specific binding ability to white spot syndrome virus (WSSV).

The term 'single domain VHH antibody' used herein while referring to Vibrio parahaemolyticus, Vibrio harvey and Vibrio anguillarum is a specific antibody to Vibrio parahaemolyticus, Vibrio harvey and Vibrio anguillarum, and the term 'single domain VHH antibody' used while referring to white spot virus is a specific antibody to white spot virus, the CDRs being part of a single domain polypeptide, heavy chain antibodies, light This includes antibodies naturally devoid of chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.

The single domain antibody may be derived from species such as mouse, human, camel, llama, goat, rabbit or cow, but the single domain antibody in the present invention is derived from camelids such as camels, dromedaries, llamas, alpacas and wild llamas. The single domain antibody used in the present invention is a naturally occurring single domain antibody known as a heavy chain antibody without a light chain. More specifically, this variable region derived from a heavy chain antibody naturally devoid of a light chain is called a VHH or nanobody to distinguish it from the VH of a conventional 4-chain immunoglobulin.

The single domain VHH antibody of the present invention may include variants of the amino acid sequences described in the attached sequence listing within the range of specifically recognizing Vibrio parahaemolyticus, Vibrio harvey and Vibrio anguillarum, or white spot virus. For example, changes may be made to the amino acid sequence of an antibody to improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the antibody.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule represented by SEQ ID NO: 3 encoding a single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 1.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule represented by SEQ ID NO: 4 encoding a single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 2.

As used herein, the term "nucleic acid molecule" has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, include not only natural nucleotides, but also sugar or base site modified analogs (analogues).

Considering the mutations having the aforementioned biological equivalent activity, the nucleic acid molecule encoding the single domain VHH antibody of the present invention is construed to include a sequence showing substantial identity with the sequences listed in the Sequence Listing. The above substantial identity means a sequence showing at least 61% homology, preferably 70% homology, more preferably 80% homology, and most preferably 90% homology, when the sequence of the present invention and any other sequence are aligned so as to correspond as much as possible and the aligned sequences are analyzed using an algorithm commonly used in the art.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule represented by SEQ ID NO: 3.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule represented by SEQ ID NO: 4.

As used herein, the term “vector” is a means for expressing a gene of interest in a host cell and includes a plasmid vector, a phagemid vector, a cosmid vector, a bacteriophage vector, an adenovirus vector, a retrovirus vector, and a viral vector such as an adeno-associated virus vector.

According to a preferred embodiment of the present invention, in the vector of the present invention, a nucleic acid molecule encoding a single domain VHH antibody is operatively linked to a promoter.

As used herein, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, whereby the control sequence controls the transcription and/or translation of the other nucleic acid sequence.

According to another aspect of the present invention, the present invention provides a host cell transformed with a recombinant vector containing the nucleic acid molecule represented by SEQ ID NO: 3.

According to another aspect of the present invention, the present invention provides a host cell transformed with a recombinant vector containing the nucleic acid molecule represented by SEQ ID NO: 4.

Any host cell capable of stably cloning and expressing the vector of the present invention can be used as any host cell known in the art, for example, Escherichia coli, Bacillus strain, Streptomyces , Pseudomonas , Proteus mirabilis or Staphylococcus cus ), but is not limited thereto.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating premature death syndrome of shrimp comprising at least one of the single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 1 and the single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

Since the single domain VHH antibody according to the present invention binds specifically to antigens for strains such as Vibrio parahaemolyticus, Vibrio harvey and Vibrio anguillarum, or white spot virus, the single domain VHH antibody alone or in combination with a conventional pharmaceutically acceptable carrier can be used as a pharmaceutical composition for preventing or treating shrimp mortality syndrome caused by the antigens.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, alginic acid, gelatin, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It is not. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.

As a method of administering the pharmaceutical composition of the present invention to shrimp, any method capable of administering to shrimp can be used, preferably, a single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 1 and a single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 2 After mixing at least one of the feed additive raw materials and excipients, the mixture may be compressed into pellets to prepare a feed and fed by oral administration, but is not limited thereto.

According to another aspect of the present invention, the present invention provides a feed additive composition for shrimp premature death syndrome comprising at least one of the single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 1 and the single domain VHH antibody represented by the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

The feed additive of the present invention may be used in its original form or additionally known carriers such as cereals and their by-products acceptable for livestock, stabilizers, etc. may be added. If necessary, organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, sodium phosphate, potassium phosphate, acidic pyrophosphate, polyphosphates (polyphosphates), polyphenols, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice Extracts, natural antioxidants such as chitosan, tannic acid, and phytic acid, antibiotics, antibacterial agents, and other additives may be added, and the shape may be in a suitable state such as powder, granule, pellet, or suspension.

As described above, the method for preparing a single domain VHH antibody for preventing or treating premature shrimp death syndrome according to the present invention can selectively increase the production of antigen-specific IgG ₂ and IgG ₃ among various antibodies produced in camelid animals. Accordingly, since IgG ₂ and IgG ₃ selectively increased than various antibodies can be used for preparing single domain VHH antibodies, single domain VHH antibodies can be produced more efficiently.

1 and 2 are results confirming the amount of IgG ₂ and IgG ₃ produced according to Test Example 1 (FIG. 1; Vibrio parahaemolyticus outer membrane protein; FIG. 2; white spot virus).
3 and 4 are results of screening specific antibodies by panning according to Test Example 3 (FIG. 3; control, FIG. 4; Example).
5 and 6 are results confirming the specificity of the single domain VHH antibody according to Experimental Example 6 (FIG. 5a: Reactivity to Vibrio parahaemolyticus, FIG. 5b: Reactivity to Vibrio harvey, FIG. 6 Reactivity to VP28 antigen of white spot virus).
7 to 9 are results confirming the inhibitory effect on shrimp premature death syndrome according to Experimental Example 7 (Fig. 7: inhibitory effect on Vibrio parahaemolyticus, Figs. 8 and 9: inhibitory effect on VP28 antigen of white spot virus).

Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.

Example: Preparation of a mixture for immunization of camelid animals (1)

A mixture for immunizing camelid animals was prepared with the ingredients and contents in Table 1, and the specific experimental method is as follows.

1) Bacterial antigen production

In order to use the outer membrane protein (hereinafter referred to as OMP) present in the outer membrane of Vibrio parahaemolyticus as an antigen, OMP was extracted from Vibrio parahaemolyticus. Vibrio parahaemolyticus was cultured for 24 hours at 37 ° C. in a dedicated medium (1.5 L). After confirming colony formation, the culture medium is centrifuged (8,000 g, 4° C., 10 min) to recover cells. The recovered cells were resuspended in 35 ml lysis buffer (20 mM Tris-HCl, 1 mM PMSF, pH 8) and sonicated. After sonication, centrifugation (10,000 g, 10 min, 4 ℃) to obtain a supernatant. The supernatant was centrifuged (20,000 g, 4° C., 1 h) using an ultra-high-speed centrifuge to obtain a pellet. The pellet was resuspended in 10 ml of 20 mM Tris-HCl (pH 8) containing 2% sarkosyl, left at room temperature for 1 hour, and centrifuged (20,000 g, 4C, 1 h) in an ultra-high-speed centrifuge to recover the OMP antigen. The OMP antigen was washed twice with 30 ml PBS and used as an alpaca immunization antigen.

2) production of viral antigens

White spot syndrome virus (WSSV) was cultured using host cells. When cell lesions were confirmed about 2 days after infection with the virus, the freezing and thawing process was repeated three times to isolate the virus, and the supernatant containing the virus was recovered through centrifugation. In order to inactivate the recovered virus, the virus culture medium was inactivated by heat treatment at 60 ° C. for 30 minutes, and additionally ultrasonicated to produce viral antigens.

3) Production of flagellin

Flagellin was isolated from a standard strain of Salmonella enteritidis . Specifically, the strain was inoculated and cultured at 1% per 1.5 L of LB medium. It was cultured at 37°C for 24 hours, and strains were recovered by centrifugation (6,000 g, 30 min) at 4°C. The recovered strain was suspended in 300 ml PBS and washed twice, and the strain was resuspended in PBS, the pH was adjusted to 2.0 with hydrochloric acid, and stirred for 30 minutes. After stirring, the supernatant was collected by centrifugation (14,000 g) at 4°C for 15 minutes, titrated to pH 7.4 with 1 M sodium hydroxide, added to the supernatant to make 65% ammonium sulfate, and left at 4°C overnight. The supernatant was centrifuged again (20,000 g, 15 min). After centrifugation, the precipitated pellet was resuspended in 40 ml of PBS, put in a dialysis tube, and dialyzed on PBS for 48 hours to produce flagellin.

4) Preparation of antigen for alpaca immunization

Mixtures for alpaca immunization were prepared by mixing 1 mg each of the OMP antigen prepared in 1) and the virus antigen prepared in 2) above with the same amount of adjuvant (Titer Max®) as the antigen solution and then ultrasonically homogenized. The mixture was mixed with flagellin to complete antigen preparation for alpaca immunization.

antigen antigen content Flagellin content Vibrio parahaemolyticus outer membrane proteins and white spot virus 1 mg per each antigen 0.2mg

Comparative Example: Preparation of a mixture for immunization of camelid animals (1)

A mixture for immunizing camelid animals was prepared to include the ingredients and contents in Table 2. Except for not including flagellin, the Example and the manufacturing method are the same.

antigen antigen content Flagellin content Vibrio parahaemolyticus outer membrane proteins and white spot virus 1 mg per each antigen 0.0mg

Experimental Example 1: Induction of immune response in camels, IgG from separated blood ₂ and IgG ₃ Check the creation of

The mixture prepared in Example was dispersed in 2 to 4 places on camels and administered subcutaneously. Thereafter, the same amount of the mixture was administered in the same manner 5 times at intervals of 2 weeks. Two weeks after the final administration, blood was collected in transfusion packs containing anticoagulants, and plasma was separated by centrifugation (6,000 g, 30 min). Protein A and Protein G columns (GE Healthcare) were used to separate IgG ₁ , IgG ₂ and IgG ₃ from plasma.

In order to identify antigen-specific IgG ₁ , IgG ₂ and IgG ₃ , 100 ng of each of the viral and bacterial antigens diluted in PBS was coated on a 96-well plate overnight at 4° C., and an ELISA-based antigen-antibody reaction was confirmed. The antigen-coated plate was washed three times with PBS-T (0.05% Tween 20), and then blocked for 1 hour at room temperature with PBS supplemented with 3% BSA. After BSA blocking, the plate was washed three times with PBS-T, and each antibody was added at 10 nM each to induce an antigen-antibody reaction at room temperature for 1 hour. The plate was washed with PBS-T, and then treated with anti-alpaca IgG (rabbit polycolnal antibody, 1:5,000). The plate was washed and reacted with anti-rabbit IgG-HRP (goat antibody, 1:10,000) at room temperature for 1 hour. Plates washed with PBS-T were added with TMB substrate and then stop solution was added to each well. The antigen-antibody reaction was confirmed by measuring the absorbance at 450 nm using a plate reader, and the results are shown in FIGS. 1 and 2 .

As a result, as can be seen in FIGS. 1 and 2, when the mixture of antigen and flagellin was injected to immunize alpaca, the production of antigen-specific IgG ₂ and IgG ₃ was selectively increased compared to when only the antigen was injected.

Experimental Example 2: Synthesis of cDNA using leukocytes isolated from immunized camels and preparation of a single domain antibody library

After alpaca immunization, 100 ml of blood was collected and peripheral blood mononuclear cells (PBMC) were isolated using Ficoll solution. Total RNA was extracted from the isolated PBMC using RNAiso Plus (TAKARA BIO). cDNA was synthesized from the recovered total RNA using SuperScript III (Invitrogen).

The VHH gene was amplified using polymerase chain reaction (PCR). The forward primer sequence of IgG ₂ and IgG ₃ is 5'-AGKTGCAGCTCGTGGAGTCNGGNGG-3' (SEQ ID NO: 5), the reverse primer sequence of IgG ₂ is 5'-GGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO: 6), and the reverse primer of IgG ₃ is 5'-TTGTGGTTTTGGTGTCTTGGG-3' (SEQ ID NO: 7).

The polymerase chain reaction was carried out using the above primers using the specifically synthesized cDNA as a template, and the PCR reaction was 95 ° C for 2 min, 96 ° C for 30 sec, 58 ° C for 30 sec, 72 ° C for 1 min, 72 ° C. The alpaca VHH gene was amplified under conditions of 5 min and 30 cycles.

The VHH gene amplified by PCR reaction was forward primer (SfiⅠ Restriction enzyme site addition, 5´-TGCTCCTCGCGGCCCAGCCGGCCATGGCTCAGGTGCAGCTCGTGGAGTCTGG-3´) (SEQ ID NO: 8) and reverse primer (SpeⅠ Restriction enzyme site added, 5′-ATGATGATGTGCACTAGTTTGTGGTTTTGGTGTCTTGGG-3′ (SEQ ID NO: 9) (IgG₃), 5'-ATGATGATGTGCACTAGTGGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO: 10) (IgG₂) was used to perform PCR (20 cycles of 2 min at 95 ° C, 30 sec at 96 ° C, 30 sec at 58 ° C, 1 min at 72 ° C and 5 min at 72 ° C) to add a restriction enzyme site. A VHH gene fragment to which a restriction enzyme site was added was inserted into a phagemid vector (pKSTV03). The phagemid vector into which the VHH gene is inserted is a host capable of infecting phage.E. coli TG-1 cells (Lucigen) were transformed. the transformedE. coli An appropriate amount of TG-1 was inoculated into 500 ml of 2YT-AG (2YT medium, 100 μg/ml ampicillin 2% glucose), cultured (37°C, 1.5 h), infected with M13KO7 helper phage (Invitrogen, m.o.i = 10), and cultured for 16 hours. In order to collect the phage floating in the upper layer by centrifuging the culture medium, the phage were precipitated and recovered using a PEG (polyethylene glycol)/NaCl precipitation method.

Experimental Example 3: Selection of specific antibodies by panning

A panning experiment was conducted using the phages recovered above to increase the number of phages specifically binding to the antigen. Specifically, 4 μg of antigen was added to 2 ml of PBS, then placed in an immunotube (Nunc) and left overnight at 4 ° C. The next day, washed three times with PBS-T and blocked for 2 hours at room temperature using 3% skim milk diluted in PBS. Then, it was washed 3 times with PBS-T. In order to select phages that bind to the antigen, 3 ml of the phage-containing solution was added and allowed to react for 2 hours. Then, the solution in the test tube was discarded and washed 10 times with PBS-T. To elute and collect phages bound to the antigen, 1 ml of 0.1 M glycine/HCl (pH 2) was added and allowed to stand at room temperature for 10 minutes to elute the phages. Thereafter, 50 μl of 2M Tris-base was added to neutralize the mixture. By repeating the panning process, phages specifically binding to the antigen were obtained and increased.

As shown in FIG. 4, when selecting antigen-specific antibodies using a panning technique after obtaining the VHH gene through immunization with alpaca antigen + flagellin, sufficient positive phage can be obtained in two rounds of panning compared to the control group (FIG. 3, immunization with alpaca antigen alone).

Experimental Example 4: Selection and confirmation of positive clones

200 μl of 2YT-AG medium was added to each well of a 96-well plate, and a single colony of E. coli TG-1 containing the VHH gene was inoculated into each well using a sterilized toothpick. After incubation (37 ° C, 4 hours), M13KO7 helper phage (moi = 5) was added and infected. After infection with helper phages, E. coli was centrifuged to remove uninfected helper phages, and precipitated E. coli was suspended in 250 μl of 2YT-AK. Then, it was incubated for 16 hours at 37°C. The culture solution was centrifuged to recover the phage solution floating in the upper layer, and ELISA was performed.

To identify phage clones binding to the antigen, 100 ng of the antigen was coated on a 96-well plate overnight at 4°C. The antigen-coated plate was washed three times with PBS-T. Thereafter, the plate was blocked for 1 hour at room temperature with 3% BSA. After blocking, the plate was washed three times with PBS-T, and then phage solution was added and reacted at room temperature for 1 hour. The plate was washed three times with PBS-T, phage-specific antibody was diluted 1:2000 using Blocking, and reacted at room temperature for 1 hour. The plate was washed three times with PBS-T, the HRP-attached antibody was diluted 1:5000 and reacted at room temperature for 1 hour. After washing five times, a TMB substrate was added and a stop solution was added to each well. The absorbance was evaluated by measuring the absorbance at 450 nm using a plate reader.

Experimental Example 5: Identification of single domain VHH antibodies for each antigen

Among the 26 outer membrane protein (V1) and 12 white spot virus (WSSV) phage clones present in the outer membrane of Vibrio parahaemolyticus selected in Experimental Example 4, one V1 and one WSSV phage clone was selected, and the VHH gene sequence of the positive phage clone was analyzed by BI Prism 3100 Genetic Analyzer (Applied Biosystems0) to analyze the amino acid sequence. The amino acid sequence of the VHH gene of the V1-positive phage clone is shown in SEQ ID NO: 1, and the amino acid sequence of the VHH gene of the WSSV-positive phage clone is shown in SEQ ID NO: 2.

Experimental Example 6: Confirmation of specificity of single domain VHH antibody

The reactivity of each of the selected VHH antibodies against Vibrio parahaemolyticus and white spot virus was confirmed.

Specifically, first, after diluting a specific antigen (V1 or VP28) using a coating buffer, dispensing to 100 ng/well, and overnight at 4°C. The next day, after washing each well three times with washing buffer (PBS/T (0.05% Tween-20)), 200 μl/well of Blocking buffer (0.5% skim milk in PBS) was dispensed into each well, followed by reaction at room temperature for 1 hr. After washing each well three times with washing buffer (PBS/T (0.05% Tween-20)), the VHH antibody to be measured was diluted with dilution buffer (0.5% skim milk in PBS/T (0.05% Tween-20)), treated with 100 μl per well, and reacted at room temperature for 1 hr. Then, after washing three times, Biotin anti-HA was diluted 1:5,000 in dilution buffer, and 100 μl/well was dispensed, followed by reaction at room temperature for 1 hr. After completion of the reaction, after washing three times, Streptavidin HRP was diluted 1:10,000 in dilution buffer, dispensed at 100 μl/well, and reacted at room temperature for 30 min. Finally, after washing 3 times, 100 μl/well of TMB solution was dispensed for color development. After completion of the reaction, 100 μl/well of stop solution (1.6N H2SO4) is dispensed to stop color development, and absorbance is measured at 450 nm using a microplate reader. After drawing this result using the Graphpad prism program, the EC ₅₀ , which is the average value of the maximum infection value and the minimum infection value, determines the value at which the VHH antibody binds to a specific antigen (V1 or VP28) by 50% to determine the degree of binding of the VHH antibody. The results are shown in FIGS. 5 and 6.

As can be seen in FIG. 5, the specific binding EC ₅₀ values (effective concentration of drug that causes 50% of the maximum response) of the V1 VHH antibody according to the present invention to Vibrio parahaemolyticus (V1) (FIG. 5a) and Vibrio harvey (Vb) (FIG. 5b) were 0.975 ng/ml and 1.720 ng/ml, respectively, as well as Vibrio parahaemolyticus. Reactivity to bay was also excellent.

In addition, as can be seen in FIG. 6, the specific binding EC ₅₀ value (effective concentration of drug that causes 50% of the maximum response) of the WSSV VHH antibody according to the present invention to the VP28 antigen of the white spot virus was 2.055 μg/ml, showing excellent reactivity to the white spot virus.

Experimental Example 7: Confirmation of inhibitory effect on shrimp premature mortality syndrome

The inhibitory effect of the VHH antibody prepared according to the present invention against bacteria or viruses causing premature shrimp death syndrome was confirmed.

IC ₅₀ , which is the inhibitory effect of bacteria or viruses causing premature shrimp death syndrome, is measured as follows. Specifically, V1, the cause of death syndrome, was smeared on each TSB agar plate, and then incubated at 28 ° C for 24 hours. After culturing, take a single colony, inoculate it into TSB broth medium, and incubate at 28℃ for 24 hours by Shaking incubation. Prepare all strains with a bacterial count of about 1 X 10 ⁹ cfu/mL. Dilute 10,000 times with diluent (medium) to prepare about 1 X 10 ⁵ cfu/mL of all strains. 100 μl/ml of the prepared bacterial solution is dispensed per well (the final number of inoculated bacteria is 5 X 10 ⁴ cfu/mL). Put a VHH antibody sample into each well so that it enters the final 200 μl, and then measure the bacterial growth rate. After incubation at 28℃, 150 rpm for 24 hours, the values are measured with an ELISA Reader (OD 600nm). After drawing a graph using the Graphpad prism program by referring to the OD 600nm immediately after bacterial inoculation and after 24 hours of incubation for the growth rate, determine the IC ₅₀ (IC ₅₀ , inhibitory concentration of drug that causes 50% of the maxium inhibition) value at a concentration that inhibits bacterial growth by 50%. The results are shown in Table 3 and FIG. 7 below.

VHH
density
(μg/ml) 0.40132 0.60198 0.90297 1.35446 2.03169 3.04754 4.57131 6.85697 OD600
(bacterial growth rate) 0.7475 0.7665 0.7625 0.75 0.7525 0.7045 0.594 0.535

As can be seen in Table 3 and Figure 7, the concentration that inhibits the growth of Vibrio parahaemolyticus (V1) by about half (IC ₅₀ , inhibitory concentration of drug that causes 50% of the maxium inhibition) is the V1 VHH antibody concentration of 2.5 μg / ml. The growth of Vibrio parahaemolyticus (V1) was inhibited by 50%.

In addition, the ability of the VP28 VHH antibody to inhibit WSSV virus infection was determined by adding VP28 VHH diluted by concentration to animal cells infected with VP28 expressing pseudo-virus, the main cell membrane protein of WSSV, and the degree of infection of animal cells by the VP28 expressing pseudo-virus was determined by the number of GFP fluorescent cells, and the concentration that inhibited pseudo-virus infection by half was IC ₅₀ (IC ₅₀ , inhibitory concentration of drug that causes 50% of The maxium inhibition is called the concentration. The resulting values are graphed using the Graphpad prism program, and then the IC ₅₀ value is determined at a concentration that inhibits viral infection by 50%.

As can be seen in Figures 8 and 9, the degree of infection of WSSV (White Spot Syndrome Virus) was analyzed by the VP28 expressing pseudo-virus system, and the concentration that inhibits infection by about half (IC ₅₀ , inhibitory concentration of drug that causes 50% of the maxium inhibition) inhibited VP28 expressing pseudo-virus infection by 50% at a WSSV VHH antibody concentration of about 10.3 μg / ml.

<110> ADbiotech Co., Ltd. <120> MANUFACTURING METHOD OF SINGLE DOMAIN VHH ANTIBODY FOR PREVENTING OR TREATING EARLY MORTALITY SYNDROME <130> P20-0450 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 122 <212> PRT <213> artificial sequence <220> <223> V1_L5_IgG3_long hinge type <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Val Ile Ser Phe Ser 20 25 30 Phe Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Arg Ile Thr Thr Arg Gly Thr Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Val Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met His Ser Leu Lys Pro Glu Asp Ser Ala Val Tyr Tyr Cys Ser 85 90 95 Leu Gly Ser Pro Pro Thr Asp Trp Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln 115 120 <210> 2 <211> 123 <212> PRT <213> artificial sequence <220> <223> WSSV_S95_IgG2_short hinge type <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Val Phe Ser Phe Ser 20 25 30 Phe Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Arg Ile Thr Thr Arg Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Lys Thr Leu Trp Gly Ala Asn Phe Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser Ala His His Ser Glu Asp Pro 115 120 <210> 3 <211> 366 <212> DNA <213> artificial sequence <220> <223> V1_L5_IgG3 <400> 3 caggtgcagc tcgtggagtc tgggggaggc ttggcgcagc ctggggggtc tctgagactc 60 tcctgtgcag tatctggaag agtcatcagc ttcagtttca tgggctggta ccgccaggct 120 ccagggaagc agcgcgagtt cgtcgcacgg attactactc gtggtacaac aagctatgca 180 gactccgtga agggccgttt caccgtctcc agagacaaca ccaagaacac ggtgtatctg 240 caaatgcaca gcctgaaacc tgaggattcg gccgtctatt actgtagcct tggatcgccc 300 ccaaccgact ggggccaggg gacccaggtc accgtctcct cagaacccaa gacaccaaaa 360 ccacaa 366 <210> 4 <211> 369 <212> DNA <213> artificial sequence <220> <223> WSSV_S95_IgG2 <400> 4 caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggaag agtcttcagc ttcagtttca tgggctggta ccgccaggct 120 ccagggaagc agcgcgagtt cgtcgcacgg attactactc gtggtactac gaactatgca 180 gactccgtga agggccgatt caccatctcc agaaacaacg ccaagaacac ggtgtatcta 240 caaatgaaca gcctgaaacc tgacgacacg gccgtctatt actgcaacgt gaaaaccctc 300 tggggcgcta acttctgggg ccagggaacc caggtcaccg tctcctcggc gcaccacagc 360 gaagacccc 369 <210> 5 <211> 25 <212> DNA <213> artificial sequence <220> <223> IgG2, IgG3 Forward primer <400> 5 agktgcagct cgtggagtcn ggngg 25 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> IgG2 reverse primer <400> 6 ggggtcttcg ctgtggtgcg 20 <210> 7 <211> 21 <212> DNA <213> artificial sequence <220> <223> IgG3 reverse primer <400> 7 ttgtggtttt ggtgtcttgg g 21 <210> 8 <211> 52 <212> DNA <213> artificial sequence <220> <223> VHH forward primer <400> 8 tgctcctcgc ggcccagccg gccatggctc aggtgcagct cgtggagtct gg 52 <210> 9 <211> 39 <212> DNA <213> artificial sequence <220> <223> VHH reverse primer (IgG3) <400> 9 atgatgatgt gcactagttt gtggttttgg tgtcttggg 39 <210> 10 <211> 38 <212> DNA <213> artificial sequence <220> <223> VHH reverse primer (IgG2) <400> 10 atgatgatgt gcactagtgg ggtcttcgct gtggtgcg 38

Claims (14)

A method for producing a single domain VHH antibody for preventing or treating shrimp premature death syndrome,
A method for producing a single domain VHH antibody comprising the step of injecting a mixture of an outer membrane protein or viral antigen and flagellin of a causative bacterium of shrimp premature death syndrome into a camelid animal to immunize it.
According to claim 1,
The causative agent of the shrimp premature death syndrome is one or more bacteria selected from the group consisting of Vibrio parahaemolyticus , Vibrio harveyi and Vibrio anguillarum , and the virus is a white spot virus (white spot syndrome virus, WSSV).
According to claim 1,
The flagellin is derived from a bacterium selected from the group consisting of Salmonella typhimurium , Vibrio vulnficus and Listeria monocytogenes .
According to claim 1,
The method of producing a single domain VHH antibody, wherein the mixing ratio of the outer membrane protein or antigen and flagellin is 5 to 20: 1.
A single domain VHH antibody for preventing or treating shrimp premature death syndrome represented by the amino acid sequence of SEQ ID NO: 1 prepared by the method of claim 1.
A nucleic acid molecule represented by SEQ ID NO: 3 encoding the single domain VHH antibody according to claim 5.
A recombinant vector comprising the nucleic acid molecule according to claim 6 .
A host cell transformed with the recombinant vector of claim 7.
A single domain VHH antibody for preventing or treating shrimp premature mortality syndrome represented by the amino acid sequence of SEQ ID NO: 2 prepared by the method of claim 1.
A nucleic acid molecule represented by SEQ ID NO: 4 encoding the single domain VHH antibody according to claim 9.
A recombinant vector comprising the nucleic acid molecule according to claim 10 .
A host cell transformed with the recombinant vector of claim 11.
A pharmaceutical composition for preventing or treating premature shrimp mortality syndrome comprising at least one of the single domain VHH antibodies according to claims 5 and 9 as an active ingredient.
A feed additive composition for the prevention or treatment of premature shrimp mortality syndrome comprising at least one of the single domain VHH antibodies according to claims 5 and 9 as an active ingredient.