JPH06100591A - Peptide, antibody derived therefrom and anti-newcastle disease agent containing the same antibody - Google Patents

Abstract

PURPOSE:To obtain an effective and safe preventing agent for infection and onset of Newcastle disease (fowl plague) or a therapeutic agent therefor so as to inhibit the infection with the Newcastle virus and propagation of the infection. CONSTITUTION:The objective peptide comprises an amino acid sequence from the glycine residue at the 6-position to the arginine residue at the 11-position in the amino acid sequence expressed by sequence No. 1 in a sequence table and is composed of 6-15 amino acid residues. Furthermore, the objective antibody is derived from the peptide and the objective anti-Newcastle disease agent contains the antibody as an active ingredient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to peptides, antibodies derived therefrom, and anti-Newcastle disease drugs containing the antibodies as active ingredients.

[0002]

Newcastle disease, often also referred to as "poultry plague", is one of the most serious diseases affecting poultry. The causative pathogen is Newcastle disease virus (NDV), which belongs to the paramyxovirus. Viral infections often cause respiratory disorders with severe nervous system abnormalities leading to paralysis and death, which in turn spread to hens housed in ten thousand units per poultry and have a serious economic impact on poultry raising operations. . For this viral disease, vaccines such as Newcastle disease inactivating preventive solution (Sato strain, Ishii strain) and live virus preventive solution (B1 strain) have been prepared and put into practical use.

However, accidents caused by insufficient inactivation with the inactivation preventive solution or additives such as preservatives and adjuvants, and reversion of the pathogenicity of the live vaccine virus strain with the live virus preventive solution have occurred. Accidents due to stray microorganisms during the manufacturing process often occur. Therefore, it is required to provide more effective and safe means as a measure for preventing infection, disease onset, or treatment of Newcastle disease due to virus infection.

[0004]

NDV has a characteristic of causing cell fusion in infected cells to form megakaryocytes, and this fusion phenomenon is caused by F protein, which is a glycoprotein present on the outer membrane of the virus. Is triggered. The F protein is produced as a precursor (F ₀ ) having a molecular weight of about 68 kd, and is then cleaved by limited degradation by a trypsin-like endoprotease, resulting in two disulfides of F ₁ (56 kd) and F ₂ (12 kd). It is known to express a biological activity only after forming a binding polypeptide (Nagai Y.
Et al., Virology Volume 72, 494-
508 (1976)). The amino acid sequence of the limited degradation recognition site is well conserved among viruses that cause cell fusion, and it has been shown that NDV has extremely high homology in both virulent and attenuated strains (R. L.
Glickman et al., Journal of Virology, Vol. 62,
Pp. 354-356 (1988), N. et al. S. Milla
r et al., Journal of General Birology (J
individual of General Virolog
y) 69, 613-620 (1988)).

The amino terminus of the F ₁ polypeptide is a highly hydrophobic site, through which the virus fuses with the cell membrane of target cells. It has been shown that a synthetic peptide corresponding to the amino terminus or an analog thereof suppresses membrane fusion of Sendai virus and measles virus belonging to paramyxovirus (SCH Stuart-Har).
ris and J. Oxford, "Problems of Antiviral Therapy" (Problem
ms of Antivirus Therapy),
13-34, Academic Press (Academi
c Press) 1983).

[0006] In addition, a rabbit antiserum against the F protein blocks the membrane fusion between the virus and the target cells and suppresses the spread of infection to the parainfluenza virus Simian virus 5 (SV5) belonging to the same paramyxovirus. It has been shown in in Vitro for (D.C.Mer
z et al., Journal of Experimental Media (Journal of Experimenta).
151 Medicine, 151, pp. 275-288,
(1980)). However, the antigen recognition site on the F protein involved in virus neutralization and the mechanism of infection defense have not been clarified, and the efficacy of an antibody recognizing the F protein has not been confirmed for NDV.

[0007] In the present invention, focusing on the polypeptides corresponding to the limited degradation recognition site of F protein of NDV, the cleavage of the protein is inhibited by producing an antibody that recognizes them, resulting in viral infection and infection. It is an object of the present invention to provide an effective and safe NDV infection preventive agent, disease preventive agent, or therapeutic agent for preventing the spread of E. coli.

[0008]

[Means for Solving the Problems] The inventors of the present invention synthesized a certain peptide portion presumed to interact with trypsin on the F protein of NDV, and the antibody derived from the peptide was synthesized by the F protein. The present invention has been completed based on the finding that the cleavage of NDV can be inhibited to suppress the growth of NDV.

That is, the present invention includes the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing, including the amino acid sequence from the 6th glycine residue to the 11th arginine residue, and containing 6 to 15 amino acid residues. A peptide consisting of
The present invention relates to an antibody derived from the peptide and an anti-Newcastle disease drug containing the antibody as an active ingredient.

Cysteine may be added to the C-terminal of the amino acid sequence of the peptide of the present invention for ligation with a carrier protein described later. Specific examples of the peptides of the present invention include the peptides shown in SEQ ID NO: 2, 3 or 4 in the sequence listing.

The peptide of the present invention can be obtained by a conventional liquid phase method, solid phase method or the like, but more conveniently, it can be obtained by using a commercially available peptide synthesizer by the solid phase method.

In the solid-phase method, the amino acid corresponding to the amino acid residue is condensed from the C-terminal side of the peptide on a polymeric support to form a peptide bond.
As the condensation method, for example, a dicyclohexylcarbodiimide (DCC) method, an acid anhydride method, an active ester method or the like can be used. As a protecting group for α-amino group, t-butoxycarbonyl (hereinafter Boc) group or 9
-Fluorenylmethyloxycarbonyl (hereinafter referred to as Fmo
The group c) can be used. For example, when synthesizing using an automatic peptide synthesizer, it is preferable to appropriately select in consideration of the properties of amino acids and synthesize according to the manual instruction of the apparatus.

When synthesizing by the liquid phase method, as in the solid phase method,
It can be performed according to a usual peptide synthesis method. Known methods include, for example, the method described in Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd., 1975, and the like.

Next, elimination from the protective group and the support is carried out by α
When the Boc group is used as the amino protecting group, hydrogen fluoride containing a scavenger, trifluoromethanesulfonic acid or the like is used. As the scavenger, thioanisole, ethanedithiol, dimethyl sulfide and the like are used. In addition, Fmoc was added to the α-amino protecting group.
When the group is used, a mixed solution containing phenol and trifluoroacetic acid can be used.

The peptide can be purified by using commonly used ion exchange resins, partition chromatography, gel chromatography, reverse phase liquid chromatography, etc., alone or in combination. The amino acid composition of the purified peptide can be easily measured by an amino acid analyzer.

The peptides of the present invention are mainly used for inducing antibodies.

The antibody of the present invention is derived from the above peptide and thus specifically reacts with the peptide. In addition, the antibody specifically inhibits the cleavage of the F protein existing on the outer membrane of Newcastle disease virus by a trypsin-like protease. The antibody of the present invention includes a monoclonal antibody or a polyclonal antibody.

The antibody of the present invention can be produced by the following method. That is, it is produced by obtaining an immunoglobulin fraction from the serum of an immunized animal and then purifying the immunoglobulin fraction by affinity chromatography. Rabbits as animals to be immunized,
Goat, mouse, rat, etc. can be used. For immunization, an immunogen obtained by linking the peptide of the present invention with a carrier protein is mixed with Freund's adjuvant, and 0.05-50 mg, 7 times at a time, subcutaneously, intramuscularly or intraperitoneally in an animal.
It is carried out by administering at intervals of -30 days for 2 to 15 weeks. Blood is collected from the immunized animal 3 to 10 days after the final immunization, and the serum portion is separated and obtained by a commonly-used method. As a method for obtaining an immunoglobulin fraction from serum, a known ammonium sulfate fractionation method is used. In the purification by affinity chromatography, the peptide of the present invention that is an antigen is used, and only the antibody that binds to the peptide is separated and obtained.

The monoclonal antibody can be produced by the so-called cell fusion method.

The antibody of the present invention inhibits the production of new infectious virus from Newcastle disease virus-infected cells, as will be shown in the test examples described below.
It is effective as a disease preventive or therapeutic drug. A Newcastle disease infection preventive agent, disease preventive agent or therapeutic agent containing the antibody of the present invention as an active ingredient is a carrier or a diluent, for example, as an injection after a sterilization operation using a membrane filter, a nasal spray, etc. Be administered to.

The present invention will be described in more detail below with reference to examples, but these do not limit the present invention.

[0022]

【Example】

 Example 1 Preparation of peptide (1) Synthesis of peptide represented by SEQ ID NO: 2 in Sequence Listing.

The Boc-Cys-PAM (PAM: 4-
(Oxymethyl) phenylacetamidomethyl) resin 71
Using 6 mg (Cys content 0.7 mM / g) as a raw material, an automatic peptide synthesizer (Applied Biosystems 43
0A) was used for the solid phase synthesis. Regarding the synthetic procedure, the properties of amino acids were appropriately considered in accordance with the synthetic manual instruction of this device. According to a conventional method for the synthesis of this peptide, 1.4 g of a protected peptide resin was obtained by reacting 7 L-amino acids each in an amount of 2 mM from the C-terminal Cys according to the above-mentioned amino acid sequence.
The protective group for the side chain functional group of the amino acid used in this reaction includes 2,4,6-trimethylbenzenesulfonyl group for the guanidino group of Arg and p- for the mercapto group of Cys.
A methoxybenzyl group was used.

After drying, 1.4 g of this protected peptide resin was stirred with a mixed solution of 0.7 ml of ethanedithiol and 1.4 ml of thioanisole under ice cooling for 10 minutes, 14 ml of trifluoroacetic acid was added and stirred for 10 minutes. . 1.4 ml of trifluoromethanesulfonic acid was added to this mixed solution, which was stirred for 10 minutes, and further stirred at room temperature for 50 minutes. Then, 50 ml of anhydrous ether was added to precipitate the crude peptide, and the crude peptide and the desorbed resin were filtered with a glass filter. 5 for crude peptide on glass filter
After trifluoroacetic acid (ml) was added and dissolved, anhydrous ether was added to this to obtain a crude peptide precipitate. This product was separated, washed with anhydrous ether several times, and dried under reduced pressure. 200 mg of the peptide thus obtained was purified by high performance liquid chromatography (HPLC) under the following conditions. The column is reverse-phase ODS (trade name: ULT
RON S-C ₁₈ ) was used to separate and purify with acetonitrile containing 0.1% trifluoroacetic acid by a linear gradient method (acetonitrile 100% flow rate 10 ml / min for 60 minutes). The retention time of the peptide under this condition is 2
It was 4.2 minutes. The required elution fraction was concentrated and freeze-dried to obtain 90 mg of the desired peptide.

Regarding the amino acid analysis of the peptide, after hydrolyzing with 6N hydrochloric acid at 110 ° C. for 20 hours, it was measured with an amino acid analyzer L8500 (manufactured by Hitachi Ltd.). Amino acid analysis results

(2) Synthesis of peptide represented by SEQ ID NO: 4 in the Sequence Listing By synthesizing the peptide by the same method as in the above (1) and further purifying (purification is carried out under the condition of HPLC, the retention time of the peptide is 28. After 4 minutes, 90 mg of the desired peptide was obtained. Amino acid analysis results

Example 2 Preparation of Anti-NDV Peptide Antibody (1) Ligation of Peptide to Carrier Protein When ligating to carrier protein, keyhole limpet hemocyanin (hereinafter KLH, Calbiochem), 5
Peptide 20m synthesized by the method of Example 1 (1) in 1 ml of sodium phosphate buffer (0.2 M, PH 8.0) containing 0 mM DTT (dithiothreitol).
g was dissolved. KLH 9 mg in 20 mM sodium borate buffer-0.15 M sodium salt solution (PH 9.0)
0.5 M phosphate buffer (PH 7.0) 10
The solution was returned to near neutral with 0 μl, and a solution prepared by dissolving 0.8 mg of succinyl 4- (p-maleimidophenyl) butyrate (hereinafter SMPB, Pierce) as a coupling agent in dioxane was gradually added to the solution. It was The obtained mixed liquid was stirred for 1 hour and then purified by column chromatography. Sephadex G-25 (Pharmacia) was used as a column, and a physiological saline phosphate buffer solution (hereinafter, PBS, 20) was used.
Separated and purified with mM phosphoric acid, 150 mM NaCl, pH 7.2).

The carrier protein solution was dropped and mixed while stirring the peptide solution thus pretreated. The mixture was gently stirred at room temperature for 3 hours and dialyzed against PBS overnight.

The thus-synthesized KLH carrier-linked peptide was subjected to polyacrylamide gel electrophoresis, and it was confirmed that an immunogen was certainly produced due to an increase in molecular weight. La each sample containing 10 mM DTT
In addition to emmli's sample buffer (62.5 mM Tris-HCl buffer pH 6.8, 2% sodium dodecyl sulfate, 0.01% promophenyl blue and 10% glycerol) [Laemmli, Nature, 2].
27, p. 680 (1970)], and electrophoresis was performed using 8% separating gel. The protein in the separation gel was confirmed by staining with Coomassie Brilliant Blue.

(2) Inoculation of test animal KLH-connecting peptide 50 obtained in Example 2 (1) above
0 μg / 160 μl was diluted with PBS and emulsified with Complete Freund's adjuvant (ICN Immunobiologicals) at a ratio of 1: 1. Then 250 Japanese white rabbits (1.5 kg, male, Shimizu Experimental Materials Co., Ltd.)
μg / 1 ml emulsion was inoculated by subcutaneous injection. Subsequently, inoculation was performed twice with a complete Freund's adjuvant at 1-week intervals, and booster injection was performed with a KLH-ligated peptide containing 1: 1 mixture of incomplete Freund's adjuvant (Nacalai Tesque, Inc.) 4 times at 1-week intervals. Animals were bled 5 days after the final immunization. Serum was purified by a known ammonium sulfate fractionation method (50% saturated ammonium sulfate) to obtain an immunoglobulin fraction in serum, which was used as a partially purified product. Thus, an immunoglobulin fraction containing anti-peptide antibody 28.3
mg / ml was obtained. The peptides obtained in Example 1 (2) were treated in the same manner as in Examples 2 (1) and (2), and similar results were obtained. Thereafter, antibodies against them were respectively labeled with Fcl-2 and Fcl-2. This is almost the same as Fcl-1.

(3) Immunological assay (enzyme-linked immunosorbent assay (ELISA) using anti-peptide serum against peptide-coated plate)

In this test, it was determined that the antiserum produced in the animal by the peptide of the present invention specifically binds to the peptide. As a result of the test, it was confirmed that the peptide of the present invention has immunogenicity and induces the antibody in the test animal.

Falcon 3912 Microtest III ^™ Flexible Assay Plate (Becton Dickinson Labware) was used in the assay. Peptide 50μ on this plate at a concentration of 100μg / ml in PBS
1 was coated and the plate was incubated for 30 minutes at 37 ° C. After that, the plate is inverted and all the holes are emptied, and RIA buffer (PBS / 0.1% sodium azide, 0.5% bovine serum albumin) /0.05% Twe is used.
Washed 3 times with en20. The blotting plate was dried by tapping on a paper towel. After sucking the plate, 100 μl of RIA buffer was added to each well and incubated at 37 ° C. for 30 minutes. The plate was then washed and blotted as in the previous case.

Then, the immunoglobulin fraction containing the anti-peptide antibody obtained by the method of Example 2 (2) was assayed on the prepared coated plate. First, the immunization fraction is 1: 5
Antibody responses to peptide antigens were assayed at 0 followed by serial 2-fold dilutions in RIA buffer.

After incubating at 37 ° C. for 30 minutes, the plate was washed and blotted as described above. Then 1: 4000 diluted goat anti-rabbit I in RIA buffer
50 μl of gG alkaline phosphatase (Tago) was added to each well and incubated at 37 ° C. for 30 minutes. The plate was then washed and blotted as in the previous case. DEA buffer (10% diethanolamine buffer (pH 9.8) /0.02% sodium azide, 0.
50 μl of 4 mM p-nitrophenyl phosphate (Sigma) in 0.1% magnesium chloride hexahydrate) was added to the wells. After 15 minutes, the enzymatic reaction was stopped with 25 μl of 1M NaOH. The plates were then analyzed spectrophotometrically at 405 nm using a microplate reader (Biorad, Model 2550 EIA Reader). As a result, since the absorbance decreased in a dilution-dependent manner, it was observed that the antisera of Fcl-1 and Fcl-2 against both peptides bind to the peptides.

(4) Purification of anti-peptide serum When assaying the virus action, affinity column chromatography may be used for purification in order to exclude other virus inhibitors. Here, a purification method using FMP-Activated Avid Gel F (Bioprobe International) is described.

40 mg K in 15 ml sodium carbonate-sodium bicarbonate buffer (50 mM, pH 9.4)
Dissolve LH. 1.5g of dry gel (FMP-Activated Avid Gel F) for this KLH solution
Was gradually added with stirring, and the liquid was gently stirred at 4 ° C. for 10 hours. Then 30 ml of sodium carbonate
With sodium bicarbonate buffer (50 mM, pH 9.4),
Subsequently, 30 ml of phosphate buffer (50 mM, pH 7.
The gel was washed in 2). KLH thus obtained
The binding gel was 15 ml of phosphate buffer (50 mM, pH
It was suspended in 7.2). 20 mg of SMPB (succinyl 4) dissolved in 0.3 ml of dioxane was added to this KLH (keyhole limpet hemocyanin) binding gel solution.
-(P-maleimidophenyl) butyrate),
The mixture was gently stirred at room temperature for 1 hour. Then 30
KLH with ml of phosphate buffer (50 mM, pH 7.2)
-The SMPB-bound gel was washed. The obtained KLH-SM
PB binding gel is 20m to inactivate unreacted hydroxyl groups
Suspended in 50 ml Tris-HCl buffer (0.1 M, pH 8.5) containing M 2-mercaptoethanol,
The mixture was gently stirred at 4 ° C for 6 hours. Then KL in the following order
The H-SMPB binding gel was washed. 40 ml sodium acetate buffer (10 mM, pH 4.5), 40 ml sodium acetate buffer saline (10 mM, pH 4.
5, 0.5 M NaCl), 40 ml Tris-HCl buffer (10 mM, pH 8.5), 40 ml Tris-HCl buffer saline (10 mM, pH 8.5, 0.5 MN)
aCl) and finally 40 ml of borate buffer saline (20 mM, pH 8.5, 0.15 M NaCl).

KLH-SMPB thus obtained
About 15 ml of the anti-peptide antibody-containing partially purified product obtained in Example 2 (2) above was mixed with the binding gel and gently stirred at 4 ° C. for 10 hours. After that, the gel and the anti-peptide antibody-containing liquid were filtered and separated with a glass filter. Gel on a glass filter, 10 ml of 20 mM borate buffer saline (0.15 M NaCl) followed by 10 ml of 20 m
M borate buffer saline (0.4 M NaCl) was added to wash the gel. The filtrate and washings were collected and stored at 4 ° C until the next operation.

Then, the antigen peptide is bound to FMP-activated avid gel F.

20 ml of phosphate buffer (0.5 M, pH 8.0) containing 0.05 M dithiothreitol
16 mg of the antigenic peptide was dissolved therein. To this peptide solution, 2 g of dry gel (FMP-Activated Avid Gel F) was gradually added while stirring the solution, and after the addition, the mixture was stirred at room temperature for 15 minutes. After that, the peptide-gel mixture was gently stirred at room temperature for 8 hours, and then 50 ml.
The gel was washed with phosphate buffer (50 mM, pH 8.0). The peptide-bonded gel thus obtained was treated as described above to inactivate unreacted hydroxyl groups in the same manner as in the case of KLH-SMPB.
Then, the peptide-bonded gel was washed in the same manner as described above, and finally suspended in 20 ml of 20 mM borate buffer saline (pH 8.5, 0.15 M NaCl).

Finally, the binding between the peptide-containing gel and the anti-peptide antibody-containing liquid recovered by the above-mentioned operation and the elution of the anti-peptide antibody will be described. Mix the prepared peptide-bonded gel with the anti-peptide antibody-containing solution, and mix at 10
Stir gently for a period of time. This binds the anti-peptide antibody to the peptide binding gel. This anti-peptide antibody-bonded gel was placed in an appropriate glass column (1.2 cmφ × 18 cm) and 50 ml of 20 mM borate buffer saline (pH
8.5, 0.15M NaCl), then 50m
l of 20 mM borate buffer saline (pH 8.5,
0.4 M NaCl) and finally 20 mM borate buffer saline (pH 8.0, 0.4 M NaCl). For the final washing, the eluate was measured with a spectrophotometer, and washing was performed at a measurement wavelength of 280 nm until the absorbance reached 0.01 to 0.02.

The anti-peptide antibody-binding affinity gel column thus prepared was supplied with 0.1 M glycine-hydrochloric acid buffer (pH 2.4) to elute the anti-peptide antibody. The eluate was collected by 0.5 ml, and the antibody component was collected in the range of fraction numbers 13 to 18 in the fractions 1 to 30. The IgG content in the affinity-purified anti-peptide antibody thus obtained was 0.3 mg / ml.

Example 3 Properties of Purified Anti-Peptide Antibody The reaction specificity of the purified anti-NDV peptide antibody of the present invention will be described by taking Fcl-1 as an example.

As a result of analysis by the known Western blotting method, it was confirmed that the purified Fcl-1 antibody specifically reacts with the synthetic peptide used as the antigen. Further, it was revealed that this antibody specifically reacts with the F protein among the constituent components of purified NDV (Miyadera strain: distributed by Professor Nagai of Nagoya University), and mainly recognizes the F ₂ protein portion.

Further, the inhibitory action of the present antibody on the cleavage of F protein was examined according to the following procedure. BHK-21 cells grown in a monolayer on a 24-well plate (immature hamster kidney-derived cell line, ATCC clone No. CCL-10)
Were infected with NDV diluted with Eagle's MEM medium containing 5% calf serum (manufactured by Microbial Diseases Research Association) and incubated at 37 ° C.
It was cultured in. After 4 hours, 35S-labeled methionine (100 μCi / ml) and purified antibody (0.03 mg / m 2
1) was added and the culture was further continued. After 30 hours, the infected cells were collected, lysed with a detergent, immunoprecipitated with a mouse monoclonal antibody against NDV F protein (distributed by Professor Kita of Hokkaido University), and the F protein was cleaved by electrophoresis. The condition was analyzed. When the purified antibody was not added, almost all of the F ₀ protein was cleaved, whereas when the purified antibody was added, the F ₀ protein was obviously inhibited by the cleavage. Further, these F ₀ proteins were mainly distributed on the cell surface. From the above, it was shown that an antibody against the limited degradation recognition site of F protein of NDV has the property of inhibiting the cleavage of F ₀ into F ₁ and F ₂ on the surface of infected cells.

Example 4 Assay for Action on Virus (Neutralization Test): Inhibitory Effect on Production of Infectious Virus from NDV-Infected Cells Incubation was carried out by adding Fcl-1 to the culture solution of NDV-infected cells and culturing the cells. It was assayed how much the production of new infectious virus from the cells was inhibited. NDV (70 plaque forming units / 200 μl) diluted with Eagle MEM medium containing 5% calf serum was adsorbed (at 37 ° C., 1 ° C.) to BHK-21 cells grown in a monolayer state on a 24-well plate.
After removing the unadsorbed virus, washed and serially diluted Fcl-1 (final concentration 0.1, 0.01, 0300).
1 ml of the same medium containing 1 and 0 mg / ml) was added, and
Culture was performed at 37 ° C. in a% carbon dioxide gas. After 20 hours, the infectious titer of NDV in the culture supernatant was calculated by the plaque method. The results are shown in Fig. 1.

The antibody against the cleavage site of the F protein of NDV showed an inhibitory effect of about 60% on the amount of virus produced in the absence of the antibody at a concentration of 0.1 mg / ml.
Therefore, it was revealed that the present antibody exhibits an infection-inhibiting effect by suppressing the cleavage of the virus F protein on the cell surface against the step of newly producing infectious virus from infected cells.

[0048]

INDUSTRIAL APPLICABILITY The antibody of the present invention has a
It inhibits the F protein of DV, F ₀ , from cleaving into F ₁ and F ₂ , and consequently the production of new infectious viral particles from virus-infected cells. Therefore, the present antibody can be used not only as an agent for preventing infection or disease onset of this virus in NDV-infected colonies, mainly in poultry, but also by suppressing the production of new virus against those already infected. , Available as a remedy.
At the same time, as described above, the antigen recognition site of this antibody is N
It is well conserved among clones with different DV, and it is considered that the frequency of antigenic changes is extremely low. Therefore, even if an NDV mutant appears, this antibody continues to act effectively to protect against infection. Can be demonstrated.

[0049]

[Brief description of drawings]

FIG. 1 shows the inhibitory effect of anti-peptide antibody on infectious NDV production.

[0050]

[Sequence list]

SEQ ID NO: 1 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence

SEQ ID NO: 2 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence

SEQ ID NO: 3 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence

[0053]

[Sequence list]

SEQ ID NO: 4 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] June 8, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Detailed explanation of the invention

[Correction method] Change

[Correction content]

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to peptides, antibodies derived therefrom, and anti-Newcastle disease drugs containing the antibodies as active ingredients.

[0002]

Newcastle disease, often also referred to as "poultry plague", is one of the most serious diseases affecting poultry. The causative pathogen is Newcastle disease virus (NDV), which belongs to the paramyxovirus. Viral infections often cause respiratory disorders with severe nervous system abnormalities leading to paralysis and death, which in turn spread to hens housed in ten thousand units per poultry and have a serious economic impact on poultry raising operations. . For this viral disease, vaccines such as Newcastle disease inactivating preventive solution (Sato strain, Ishii strain) and live virus preventive solution (B1 strain) have been prepared and put into practical use.

However, accidents caused by insufficient inactivation with the inactivation preventive solution or additives such as preservatives and adjuvants, and reversion of the pathogenicity of the live vaccine virus strain with the live virus preventive solution have occurred. Accidents due to stray microorganisms during the manufacturing process often occur. Therefore, it is required to provide more effective and safe means as a measure for preventing infection, disease onset, or treatment of Newcastle disease due to virus infection.

[0004]

NDV has a characteristic of causing cell fusion in infected cells to form megakaryocytes, and this fusion phenomenon is caused by F protein, which is a glycoprotein present on the outer membrane of the virus. Is triggered. The F protein is produced as a precursor (F ₀ ) having a molecular weight of about 68 kd, and is then cleaved by limited degradation by a trypsin-like endoprotease, resulting in two disulfides of F ₁ (56 kd) and F ₂ (12 kd). It is known to express a biological activity only after forming a binding polypeptide (Nagai Y.
Et al., Virology Volume 72, 494-
508 (1976)). The amino acid sequence of the limited degradation recognition site is well conserved among viruses that cause cell fusion, and it has been shown that NDV has extremely high homology in both virulent and attenuated strains (R. L.
Glickman et al., Journal of Virology, Vol. 62,
Pp. 354-356 (1988), N. et al. S. Milla
r et al., Journal of General Birology (J
individual of General Viro1og
y) 69, 613-620 (1988)).

The amino terminus of the F ₁ polypeptide is a highly hydrophobic site, through which the virus fuses with the cell membrane of target cells. It has been shown that a synthetic peptide corresponding to the amino terminus or an analog thereof suppresses membrane fusion of Sendai virus and measles virus belonging to paramyxovirus (SCH Stuart-Har).
ris and J. Oxford, "Problems of Antiviral Therapy" (Problem
ms of Antivirus Therapy),
13-34, Academic Press (Academi
c Press) 1983).

[0006] In addition, a rabbit antiserum against the F protein blocks the membrane fusion between the virus and the target cells and suppresses the spread of infection to the parainfluenza virus Simian virus 5 (SV5) belonging to the same paramyxovirus. In contrast, it has been shown in vitro (DC Mer.
z et al., Journal of Experimental Media (Journal of Experimenta).
151 Medicine, 151, pp. 275-288,
(1980)). However, the antigen recognition site on the F protein involved in virus neutralization and the mechanism of infection defense have not been clarified, and the efficacy of an antibody recognizing the F protein has not been confirmed for NDV.

[0007] In the present invention, focusing on the polypeptides corresponding to the limited degradation recognition site of F protein of NDV, the cleavage of the protein is inhibited by producing an antibody that recognizes them, resulting in viral infection and infection. It is an object of the present invention to provide an effective and safe NDV infection preventive agent, disease preventive agent, or therapeutic agent for preventing the spread of the disease.

[0008]

[Means for Solving the Problems] The inventors of the present invention synthesized a certain peptide portion presumed to interact with trypsin on the F protein of NDV, and the antibody derived from the peptide was synthesized by the F protein. The present invention has been completed based on the finding that the cleavage of NDV can be inhibited to suppress the growth of NDV.

That is, the present invention includes the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing, including the amino acid sequence from the 6th glycine residue to the 11th arginine residue, and containing 6 to 15 amino acid residues. A peptide consisting of
The present invention relates to an antibody derived from the peptide and an anti-Newcastle disease drug containing the antibody as an active ingredient.

Cysteine may be added to the C-terminal of the amino acid sequence of the peptide of the present invention for ligation with a carrier protein described later. Specific examples of the peptides of the present invention include the peptides shown in SEQ ID NO: 2, 3 or 4 in the sequence listing.

The peptide of the present invention can be obtained by a conventional liquid phase method, solid phase method or the like, but more conveniently, it can be obtained by using a commercially available peptide synthesizer by the solid phase method.

In the solid-phase method, the amino acid corresponding to the amino acid residue is condensed from the C-terminal side of the peptide on a polymeric support to form a peptide bond.
As the condensation method, for example, a dicyclohexylcarbodiimide (DCC) method, an acid anhydride method, an active ester method or the like can be used. As a protecting group for α-amino group, t-butoxycarbonyl (hereinafter Boc) group or 9
-Fluorenylmethyloxycarbonyl (hereinafter referred to as Fmo
The group c) can be used. For example, when synthesizing using an automatic peptide synthesizer, it is preferable to appropriately select in consideration of the properties of amino acids and synthesize according to the manual instruction of the apparatus.

When synthesizing by the liquid phase method, as in the solid phase method,
It can be performed according to a usual peptide synthesis method. Known methods include, for example, the method described in Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd., 1975, and the like.

Next, elimination from the protective group and the support is carried out by α
When the Boc group is used as the amino protecting group, hydrogen fluoride containing a scavenger, trifluoromethanesulfonic acid or the like is used. As the scavenger, thioanisole, ethanedithiol, dimethyl sulfide and the like are used. In addition, Fmoc was added to the α-amino protecting group.
When the group is used, a mixed solution containing phenol and trifluoroacetic acid can be used.

The peptide can be purified by using commonly used ion exchange resins, partition chromatography, gel chromatography, reverse phase liquid chromatography, etc., alone or in combination. The amino acid composition of the purified peptide can be easily measured by an amino acid analyzer.

The peptides of the present invention are mainly used for inducing antibodies.

The antibody of the present invention is derived from the above peptide and thus specifically reacts with the peptide. In addition, the antibody specifically inhibits the cleavage of the F protein existing on the outer membrane of Newcastle disease virus by a trypsin-like protease. The antibody of the present invention includes a monoclonal antibody or a polyclonal antibody.

The antibody of the present invention can be produced by the following method. That is, it is produced by obtaining an immunoglobulin fraction from the serum of an immunized animal and then purifying the immunoglobulin fraction by affinity chromatography. Rabbits as animals to be immunized,
Goat, mouse, rat, etc. can be used. For immunization, an immunogen obtained by linking the peptide of the present invention with a carrier protein is mixed with Freund's adjuvant, and 0.05-50 mg, 7 times at a time, subcutaneously, intramuscularly or intraperitoneally in an animal.
It is carried out by administering at intervals of -30 days for 2 to 15 weeks. Blood is collected from the immunized animal 3 to 10 days after the final immunization, and the serum portion is separated and obtained by a commonly-used method. As a method for obtaining an immunoglobulin fraction from serum, a known ammonium sulfate fractionation method is used. In the purification by affinity chromatography, the peptide of the present invention that is an antigen is used, and only the antibody that binds to the peptide is separated and obtained.

The monoclonal antibody can be produced by the so-called cell fusion method.

The antibody of the present invention inhibits the production of new infectious virus from Newcastle disease virus-infected cells, as will be shown in the test examples described below.
It is effective as a disease preventive or therapeutic drug. A Newcastle disease infection preventive agent, disease preventive agent or therapeutic agent containing the antibody of the present invention as an active ingredient is a carrier or a diluent, for example, as an injection after a sterilization operation using a membrane filter, a nasal spray, etc. Be administered to.

The present invention will be described in more detail below with reference to examples, but these do not limit the present invention.

[0022]

EXAMPLES Example 1 Preparation of peptide (1) Synthesis of peptide shown in SEQ ID NO: 2 in Sequence Listing.

The Boc-Cys-PAM (PAM: 4-
(Oxymethyl) phenylacetamidomethyl) resin 71
Using 6 mg (Cys content 0.7 mM / g) as a raw material, an automatic peptide synthesizer (Applied Biosystems 43
0A) was used for the solid phase synthesis. Regarding the synthetic procedure, the properties of amino acids were appropriately considered in accordance with the synthetic manual instruction of this device. According to a conventional method for the synthesis of this peptide, 1.4 g of a protected peptide resin was obtained by reacting 7 L-amino acids each in an amount of 2 mM from the C-terminal Cys according to the above-mentioned amino acid sequence.
The protective group for the side chain functional group of the amino acid used in this reaction includes 2,4,6-trimethylbenzenesulfonyl group for the guanidino group of Arg and p- for the mercapto group of Cys.
A methoxybenzyl group was used.

After drying, 1.4 g of this protected peptide resin was stirred with a mixed solution of 0.7 ml of ethanedithiol and 1.4 ml of thioanisole under ice cooling for 10 minutes, 14 ml of trifluoroacetic acid was added and stirred for 10 minutes. . 1.4 ml of trifluoromethanesulfonic acid was added to this mixed solution, which was stirred for 10 minutes, and further stirred at room temperature for 50 minutes. Then, 50 ml of anhydrous ether was added to precipitate the crude peptide, and the crude peptide and the desorbed resin were filtered with a glass filter. 5 for crude peptide on glass filter
After trifluoroacetic acid (ml) was added and dissolved, anhydrous ether was added to this to obtain a crude peptide precipitate. This product was separated, washed with anhydrous ether several times, and dried under reduced pressure. 200 mg of the peptide thus obtained was purified by high performance liquid chromatography (HPLC) under the following conditions. The column is reverse-phase ODS (trade name: ULT
RON S-C ₁₈ ) was used to separate and purify with acetonitrile containing 0.1% trifluoroacetic acid by a linear gradient method (acetonitrile 100% flow rate 10 ml / min for 60 minutes). The retention time of the peptide under this condition is 2
It was 4.2 minutes. The required elution fraction was concentrated and freeze-dried to obtain 90 mg of the desired peptide.

Regarding the amino acid analysis of the peptide, after hydrolyzing with 6N hydrochloric acid at 110 ° C. for 20 hours, it was measured with an amino acid analyzer L8500 (manufactured by Hitachi Ltd.). Amino acid analysis results

(2) Synthesis of peptide represented by SEQ ID NO: 4 in the Sequence Listing By synthesizing the peptide by the same method as in the above (1) and further purifying (purification is carried out under the condition of HPLC, the retention time of the peptide is 28. After 4 minutes, 90 mg of the desired peptide was obtained. Amino acid analysis results

Example 2 Preparation of Anti-NDV Peptide Antibody (1) Ligation of Peptide to Carrier Protein When ligating to carrier protein, keyhole limpet hemocyanin (hereinafter KLH, Calbiochem), 5
20 m of the peptide synthesized by the method of Example 1 (1) in 1 ml of sodium phosphate buffer (0.2 M, pH 8.0) containing 0 mM DTT (dithiothreitol).
g was dissolved. KLH 9 mg in 20 mM sodium borate buffer-0.15 M sodium salt solution (pH 9.0)
0.5 M phosphate buffer (pH 7.0) 10
The solution was returned to near neutrality with 0 μl, and a solution prepared by dissolving 0.8 mg of succinyl 4- (p-maleimidophenyl) butyrate (hereinafter, SMPB, Bias Co.) as a coupling agent in dioxane was gradually added to the solution. It was The obtained mixed liquid was stirred for 1 hour and then purified by column chromatography. Sephadex G-25 (Pharmacia) was used as a column, and a physiological saline phosphate buffer solution (hereinafter, PBS, 20) was used.
Separated and purified with mM phosphoric acid, 150 mM NaCl, pH 7.2).

The carrier protein solution was dropped and mixed while stirring the peptide solution thus pretreated. The mixture was gently stirred at room temperature for 3 hours and dialyzed against PBS overnight.

The thus-synthesized KLH carrier-linked peptide was subjected to polyacrylamide gel electrophoresis, and it was confirmed that an immunogen was certainly produced due to an increase in molecular weight. La each sample containing 10 mM DTT
In addition to emmli's sample buffer (62.5 mM Tris-HCl buffer pH 6.8, 2% sodium dodecyl sulfate, 0.01% bromophenyl blue and 10% glycerol) [Laemmli, Nature, 2].
27, p. 680 (1970)], and electrophoresis was performed using 8% separating gel. The protein in the separation gel was confirmed by staining with Coomassie Brilliant Blue.

(2) Inoculation of test animal KLH-connecting peptide 50 obtained in Example 2 (1) above
0 μg / 160 μl was diluted with PBS and emulsified with Complete Freund's adjuvant (ICN Immunobiologicals) at a ratio of 1: 1. Then 250 Japanese white rabbits (1.5 kg, male, Shimizu Experimental Materials Co., Ltd.)
μg / 1 ml emulsion was inoculated by subcutaneous injection. Subsequently, inoculation was performed twice with a complete Freund's adjuvant at 1-week intervals, and booster injection was performed with a KLH-ligated peptide containing 1: 1 mixture of incomplete Freund's adjuvant (Nacalai Tesque, Inc.) 4 times at 1-week intervals. Animals were bled 5 days after the final immunization. Serum was purified by a known ammonium sulfate fractionation method (50% saturated ammonium sulfate) to obtain an immunoglobulin fraction in serum, which was used as a partially purified product. Thus, an immunoglobulin fraction containing anti-peptide antibody 28.3
mg / ml was obtained. The peptides obtained in Example 1 (2) were also treated in the same manner as in Examples 2 (1) and (2), and similar results were obtained. Thereafter, the antibodies against them were treated with Fc1-2 and Fc1-2, respectively. It is abbreviated as Yohi Fc1-1.

(3) Immunological assay (enzyme-linked immunosorbent assay (ELISA) using anti-peptide serum against peptide-coated plate)

In this test, it was determined that the antiserum produced in the animal by the peptide of the present invention specifically binds to the peptide. As a result of the test, it was confirmed that the peptide of the present invention has immunogenicity and induces the antibody in the test animal.

Falcon 3912 Microtest III ^™ Flexible Assay Plate (Becton Dickinson Labware) was used in the assay. Peptide 50μ on this plate at a concentration of 100μg / ml in PBS
1 was coated and the plate was incubated for 30 minutes at 37 ° C. After that, the plate is inverted and all the holes are emptied, and RIA buffer (PBS / 0.1% sodium azide, 0.5% bovine serum albumin) /0.05% Twe is used.
Washed 3 times with en20. The blotting plate was dried by tapping on a paper towel. After sucking the plate, 100 μl of RIA buffer was added to each well and incubated at 37 ° C. for 30 minutes. The plate was then washed and blotted as in the previous case.

Then, the immunoglobulin fraction containing the anti-peptide antibody obtained by the method of Example 2 (2) was assayed on the prepared coated plate. First, the immunization fraction is 1: 5
Antibody responses to peptide antigens were assayed at 0 followed by serial 2-fold dilutions in RIA buffer.

After incubating at 37 ° C. for 30 minutes, the plate was washed and blotted as described above. Then 1: 4000 diluted goat anti-rabbit I in RIA buffer
50 μl of gG alkaline phosphatase (Tago) was added to each well and incubated at 37 ° C. for 30 minutes. The plate was then washed and blotted as in the previous case. DEA buffer (10% diethanolamine buffer (pH 9.8) /0.02% sodium azide, 0.
50 μl of 4 mM p-nitrophenyl phosphate (Sigma) in 0.1% magnesium chloride hexahydrate) was added to the wells. After 15 minutes, the enzymatic reaction was stopped with 25 μl of 1M NaOH. The plates were then analyzed spectrophotometrically at 405 nm using a microplate reader (Biorad, Model 2550 EIA Reader). As a result, since the absorbance decreased in a dilution-dependent manner, it was observed that the antisera of Fcl-1 and Fcl-2 against both peptides bind to the peptides.

(4) Purification of anti-peptide serum When assaying the virus action, affinity column chromatography may be used for purification in order to exclude other virus inhibitors. Here, a purification method using FMP-Activated Avid Gel F (Bioprobe International) is described.

40 mg K in 15 ml sodium carbonate-sodium bicarbonate buffer (50 mM, pH 9.4)
Dissolve LH. For this KLH solution, 1.5 g of dry gel (FMP-Activated Avid Gel F)
Was gradually added with stirring, and the liquid was gently stirred at 4 ° C. for 10 hours. Then 30 ml of sodium carbonate
With sodium bicarbonate buffer (50 mM, pH 9.4),
Subsequently, 30 ml of phosphate buffer (50 mM, pH 7.
The gel was washed in 2). KLH thus obtained
The binding gel was 15 ml of phosphate buffer (50 mM, pH
It was suspended in 7.2). 20 mg of SMPB (succinyl 4) dissolved in 0.3 ml of dioxane was added to this KLH (keyhole limpet hemocyanin) binding gel solution.
-(P-maleimidophenyl) butyrate),
The mixture was gently stirred at room temperature for 1 hour. Then 30
KLH with ml of phosphate buffer (50 mM, pH 7.2)
-The SMPB-bound gel was washed. The obtained KLH-SM
PB binding gel is 20m to inactivate unreacted hydroxyl groups
Suspended in 50 ml Tris-HCl buffer (0.1 M, pH 8.5) containing M 2-mercaptoethanol,
The mixture was gently stirred at 4 ° C for 6 hours. Then KL in the following order
The H-SMPB binding gel was washed. 40 ml sodium acetate buffer (10 mM, pH 4.5), 40 ml sodium acetate buffer saline (10 mM, pH 4.
5, 0.5 M NaCl), 40 ml Tris-HCl buffer (10 mM, pH 8.5), 40 ml Tris-HCl buffer saline (10 mM, pH 8.5, 0.5 MN)
aCl) and finally 40 ml of borate buffer saline (20 mM, pH 8.5, 0.15 M NaCl).

KLH-SMPB thus obtained
About 15 ml of the anti-peptide antibody-containing partially purified product obtained in Example 2 (2) above was mixed with the binding gel and gently stirred at 4 ° C. for 10 hours. After that, the gel and the anti-peptide antibody-containing liquid were filtered and separated with a glass filter. Gel on a glass filter, 10 ml of 20 mM borate buffer saline (0.15 M NaCl) followed by 10 ml of 20 m
M borate buffer saline (0.4 M NaCl) was added to wash the gel. The filtrate and washings were collected and stored at 4 ° C until the next operation.

Then, the antigen peptide is bound to FMP-activated avid gel F.

20 ml of phosphate buffer (0.5 M, PH 8.0) containing 0.05 M dithiothreitol
16 mg of the antigenic peptide was dissolved therein. To this peptide solution, 2 g of dry gel (FMP-Activated Rapid Gel F) was gradually added while stirring the solution, and after the addition, the mixture was stirred at room temperature for 15 minutes. After that, the peptide-gel mixture was gently stirred at room temperature for 8 hours and then 50 ml.
The gel was washed with phosphate buffer (50 mM, pH 8.0). The peptide-bonded gel thus obtained was treated as described above to inactivate unreacted hydroxyl groups in the same manner as in the case of KLH-SMPB.
Then, the peptide-bonded gel was washed in the same manner as described above, and finally suspended in 20 ml of 20 mM borate buffer saline (pH 8.5, 0.15 M NaCl).

Finally, the binding between the peptide-containing gel and the anti-peptide antibody-containing liquid recovered by the above-mentioned operation and the elution of the anti-peptide antibody will be described. Mix the prepared peptide-bonded gel with the anti-peptide antibody-containing solution, and mix at 10
Stir gently for a period of time. This binds the anti-peptide antibody to the peptide binding gel. This anti-peptide antibody-bonded gel was placed in an appropriate glass column (1.2 cmφ × 18 cm) and 50 ml of 20 mM borate buffer saline (pH
8.5, 0.15M NaCl), then 50m
l of 20 mM borate buffer saline (pH 8.5,
0.4 M NaCl) and finally 20 mM borate buffer saline (pH 8.0, 0.4 M NaCl). For the final washing, the eluate was measured with a spectrophotometer, and washing was performed at a measurement wavelength of 280 nm until the absorbance reached 0.01 to 0.02.

The anti-peptide antibody-binding affinity gel column thus prepared was supplied with 0.1 M glycine hydrochloride buffer (pH 2.4) to elute the anti-peptide antibody. The eluate was collected by 0.5 ml, and the antibody component was collected in the range of fraction numbers 13 to 18 in the fractions 1 to 30. The IgG content in the affinity-purified anti-peptide antibody thus obtained was 0.3 mg / ml.

Example 3 Properties of Purified Anti-Peptide Antibody The reaction specificity of the purified anti-NDV peptide antibody of the present invention will be described by taking Fcl-1 as an example.

As a result of analysis by the known Western blotting method, it was confirmed that the purified Fcl-1 antibody specifically reacts with the synthetic peptide used as the antigen. Further, it was revealed that this antibody specifically reacts with the F protein among the constituent components of purified NDV (Miyadera strain: distributed by Professor Nagai of Nagoya University), and mainly recognizes the F ₂ protein portion.

Further, the inhibitory action of the present antibody on the cleavage of F protein was examined according to the following procedure. BHK-21 cells grown in a monolayer on a 24-well plate (immature hamster kidney-derived cell line, ATCC clone No. CCL-10)
The cells were infected with NDV diluted in Eagle MEM medium containing 5% calf serum (manufactured by Microbial Diseases Research Association) and incubated at 37 ° C.
It was cultured in. After 4 hours, 35S-labeled methionine (100 μCi / ml) and purified antibody (0.03 mg / m 2
1) was added and the culture was further continued. After 30 hours, the infected cells were collected, lysed with a detergent, immunoprecipitated with a mouse monoclonal antibody against NDV F protein (distributed by Professor Kita of Hokkaido University), and the F protein was cleaved by electrophoresis. The condition was analyzed. When the purified antibody was not added, almost all of the F ₀ protein was cleaved, whereas when the purified antibody was added, the F ₀ protein was obviously inhibited by the cleavage. Further, these F ₀ proteins were mainly distributed on the cell surface. From the above, it was shown that the antibody against the limited degradation recognition site of F protein of NDV has the property of inhibiting the cleavage of F ₀ into F ₁ and F ₂ on the surface of infected cells.

Example 4 Assay for Action on Virus (Neutralization Test): Inhibitory Effect on Production of Infectious Virus from NDV-Infected Cells Incubation was carried out by adding Fcl-1 to the culture solution of NDV-infected cells and culturing the cells. It was assayed how much the production of new infectious virus from the cells was inhibited. NDV (70 plaque forming units / 200 μl) diluted in Eagle MEM medium containing 5% calf serum was adsorbed (37 ° C., 1 ° C.) to BHK-21 cells grown in a monolayer state on a 24-well plate.
After that, the unadsorbed virus was removed, washed, and serially diluted Fcl-1 (final concentration 0.1, 0.01, 0.00
1 ml of the same medium containing 1 and 0 mg / ml) was added, and
Culture was performed at 37 ° C. in a% carbon dioxide gas. After 20 hours, the infectious titer of NDV in the culture supernatant was calculated by the plaque method. The results are shown in Fig. 1.

The antibody against the cleavage site of the F protein of NDV showed an inhibitory effect of about 60% on the amount of virus produced in the absence of the antibody at a concentration of 0.1 mg / ml.
Therefore, it was revealed that the present antibody exhibits an infection-inhibiting effect by suppressing the cleavage of the virus F protein on the cell surface against the step of newly producing infectious virus from infected cells.

[0048]

INDUSTRIAL APPLICABILITY The antibody of the present invention has a
It inhibits the F protein of DV, F ₀ , from cleaving into F ₁ and F ₂ , and consequently the production of new infectious viral particles from virus-infected cells. Therefore, the present antibody can be used not only as an agent for preventing infection or disease onset of this virus in NDV-infected colonies, mainly in poultry, but also by suppressing the production of new virus against those already infected. , Available as a remedy.
At the same time, as described above, the antigen recognition site of this antibody is N
It is well conserved among clones with different DV, and it is considered that the frequency of antigenic changes is extremely low. Therefore, even if an NDV mutant appears, this antibody continues to act effectively to protect against infection. Can be demonstrated.

[0049]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

[0050] SEQ ID NO: length of the two sequences: 7 Type of sequence: amino acid Topology: linear sequence type: peptide

[0051] SEQ ID NO: the length of the 3 sequences: 8 SEQ type: amino acid Topology: linear sequence type: peptide

[0052] SEQ ID NO: 4 sequence Length: 9 Type of sequence: amino acid Topology: linear sequence type: peptide

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 shows the inhibitory effect of anti-peptide antibody on infectious NDV production.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // C07K 99:00

Claims (7)

[Claims] 1. A peptide comprising the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing, including the amino acid sequence from the 6th glycine residue to the 11th arginine residue, and comprising 6 to 15 amino acid residues. 2. The peptide according to claim 1, wherein cysteine is added to the C-terminal of the amino acid sequence. 3. The peptide according to claim 2, which is represented by SEQ ID NO: 2, 3 or 4 in the sequence listing. 4. A peptide comprising an amino acid sequence from the 6th glycine residue to the 11th arginine residue in the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing and comprising 6 to 15 amino acid residues Induced antibody. 5. The antibody according to claim 4, wherein the antibody inhibits cleavage of an F protein existing on the outer membrane of Newcastle disease virus by a trypsin-like protease. 6. An immunoglobulin fraction is obtained from the serum of an animal immunized with an immunogen in which the antibody is derived from the peptide and a carrier protein, and the immunoglobulin fraction is subjected to affinity chromatography using the peptide. The antibody according to claim 4 or 5, which is obtained by purifying the antibody. 7. A peptide comprising an amino acid sequence from the 6th glycine residue to the 11th arginine residue in the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing and comprising 6 to 15 amino acid residues An anti-Newcastle disease drug containing an induced antibody as an active ingredient.