KR20030048714A - Soluble Protein For Prevention and Treatment Of Rotavirus Infection, Eggs Containing The Same And Method For Producing Thereof - Google Patents

Abstract

PURPOSE: A process of preparing a water-soluble protein consisting of producing an adsorbed protein of rotavirus by gene recombination, immunizing laying hens using the same as an antigen and separating a water-soluble protein from an egg yolk of the immunized laying hens is provided. The water-soluble protein has a strong binding property to rotavirus and, therefore, an egg and a protein separated therefrom can be used as an agent for the detection, prevention and treatment of rotavirus which causes diarrhea in an infant or a baby. CONSTITUTION: A water-soluble protein is prepared by the steps of: (a)inserting DNA sequence coding an adsorbed protein of rotavirus into a vector containing one or more expression control sequences which are operatively linked to the DNA sequence and control the expression thereof; (b)transforming a host with the recombinant expression vector; (c)culturing a transformant in a medium and under conditions suitable for expression of the DNA sequence; (d)crushing the transformant and recovering a pure adsorbed protein; (e)immunizing laying hens with the recovered adsorbed protein; and (g)separating a water-soluble protein from an egg yolk of the immunized laying hens.

Description

Soluble Protein For Prevention and Treatment Of Rotavirus Infection, Eggs Containing The Same And Method For Producing Thereof}

The present invention relates to eggs comprising water soluble proteins for the prevention and treatment of rotavirus infection and to water soluble proteins isolated from egg yolk of such eggs. More specifically, the present invention is isolated from eggs and egg yolks, which are isolated from egg and egg yolk, which are released from laying hens immunized with the adsorbing protein of rotavirus as an antigen and useful for preventing and treating diarrhea in infants and young children. And water soluble proteins useful for preventing and treating diarrhea in infants.

Rotavirus is a pathogen that causes diarrhea in humans, monkeys, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, chickens, and turkeys. In particular, it is known as a main cause of diarrhea in infants and young children.

Rotavirus belongs to the Reoviridae family by classification, and has a capsid structure of two layers, inside and outside, without an envelope. It has a tetrahedron with a diameter of about 70 nm and has a core of about 50 nm in diameter at the inner capsid center. Inside the core is a genome consisting of 11 segments of linear double-stranded RNA. The genome of rotavirus encodes six structural proteins (VP1, VP2, VP3, VP4, VP6, VP7) and five nonstructural proteins (NSP1, NSP2, NSP3, NSP4, NSP5) (Bernstein DI and RL Ward, Rotavirus: textbook of pediatric infectious diseases, 4th ed.Philadelphia, WB Saunders. 1901-1902 (1998).

Of these 11 genomic segments, in particular the fourth and ninth genomic segments (in some strains corresponding to the seventh or eighth genomic segments) are important in the development of vaccines and diagnostic agents (Fields Virology, 3rd ed., vol 12, pp. 1625-1629, BN Fields et al., Lippincott-Raven Publishers, (1996).

On the other hand, rotaviruses isolated from monkeys and livestock usually proliferate relatively well in cultured cells, so that the culture or passage is easy. However, in the culture by human culture of rotavirus, even though viral antigens are produced, infectious viral particles occur in successive abortive infections which cannot be obtained by successive passages, thus making subcultures extremely difficult. Therefore, a human rotavirus prepared between a human rotavirus and a rotavirus such as a monkey or a cow and aimed at improving the proliferative capacity of the human rotavirus or pretreatment with trypsin A study was performed to inoculate rotavirus by rotational culture using a maintenance medium mixed with derived cell strain MA104 (ECACC No. 85102918) and AGMK (African Green Monkey Kidney) cells and mixed with trypsin. However, even in such a rotational culture, there is a disadvantage in that it is not possible to recover the amount of virus antigen necessary for the manufacture of vaccines and diagnostic agents.

Various vaccines against human rotavirus infection have been developed since 1985. The mainstream is the development of a live vaccine by the so-called Zenerian Approach, which substitutes a non-human-like diluted virus with similar antigenicity (WO 92/01784; EPO 130906; Japanese Patent Laid-Open No. 06-328107; 'Modern Vaccinology', pp. 213-229, E. Kurstak, Plenum Medical Book Company (1994), USA; 'Vaccines', 2nd ed., Pp. 809-822, SA Plotolokin and EA Mortimer, WB Saundorscompany 1994, USA; 'The Jordan Report-Accelerated Development of Vaccines 1996', p. 46 and p. 68, published by the National Institute of Health, USA). However, due to the accumulation of clinical trial data on these live vaccines, the preventive effect is insufficient and the sale is prohibited due to side effects such as gastroenteritis.

In addition, antibodies using antisera, colostrum, and monoclonal antibodies have been developed to prevent and treat rotavirus infection, but it is expensive to obtain large amounts of serum antibodies and there are technical problems due to separation and storage. In the case of the serum antibody, it is not practical because there are expensive technologies and necessary expenses according to separation and storage of serum and immunoglobulin from blood. In the case of colostrum, it is not possible to produce a sufficient amount of colostrum antibody because it is secreted only for a limited time.

Recently, several researchers have begun to study antibody proteins using eggs from laying hens. Laying eggs, especially yolk of the egg, are known as abundant sources of specific antibodies (Gassmann, et al., FASEB J, 4, 2528-2538 (1990)). Korean Patent Publication No. 2001-16599 discloses a yolk antibody against rotavirus produced by rotating culture of rotavirus isolated from feces of an infant and then inactivating it to use as an antigen to induce an immune response to a laying hen. . In addition, European Patent Publication No. 955061 discloses a composition for oral administration for the prevention and treatment of porcine intestinal infections, including egg yolk antibodies produced by inducing an immune response to a laying hen using swine rotavirus as an antigen.

The invention uses live bacteria or attenuated and inactivated viruses as antigens for inducing rotavirus antibodies in laying hens. However, when the virus itself of the rotavirus is used as an antigen, since the virus contains many heterologous proteins, the antibody titer does not appear higher than that of the single antigen.

The present inventors prepared the adsorbed protein of rotavirus, which causes diarrhea in infants and young children, by genetic recombination, using the antigen as an antigen to immunize the laying hens, and separating the water-soluble protein from the egg yolk of the eggs laid from the immunized laying hens, Thus, it was confirmed that the water-soluble protein thus obtained may be useful for preventing and treating diarrhea in infants and toddlers by exhibiting a high binding capacity to rotavirus that causes diarrhea in infants and toddlers.

Thus, in one aspect, the present invention uses adsorbent proteins of rotavirus that cause diarrhea in infants and infants produced by genetic recombination as antigens to be spawned from immunized laying hens and useful for the prevention and treatment of infant and infant diarrhea. Provide eggs containing water soluble proteins.

In another aspect, the present invention provides a method for the prevention of infant and infant diarrhea, which is isolated from egg yolk from eggs laid from an immunized laying hen using antigen-adsorbed proteins of rotavirus that cause diarrhea in infants and infants produced by genetic recombination. It provides a water soluble protein useful for treatment.

In another aspect, the present invention provides a method of controlling the expression of (a) one or more expression control sequences operatively linked to a DNA sequence encoding an adsorbent protein of a rotavirus and controlling its expression. (b) transform the host with a recombinant expression vector formed therefrom, and (c) incubate the resulting transformants under medium and conditions appropriate for the expression of the DNA sequence. (d) crushing the cultured transformant and recovering substantially pure adhesion protein from the lysate, (e) immunizing the laying hen with the recovered attachment protein, and (f) spawning eggs from the immunized laying hen. The present invention provides a method of producing an egg containing a water-soluble protein useful for preventing and treating diarrhea in infants and toddlers.

In another aspect, the present invention provides a method of controlling the expression of (a) one or more expression control sequences operatively linked to a DNA sequence encoding an adsorbent protein of a rotavirus to regulate its expression. sequence), (b) transform the host with a recombinant expression vector formed therefrom, and (c) the resulting transformants are cultured under appropriate media and conditions to allow the DNA sequence to be expressed and (d) disrupting the cultured transformant and recovering substantially pure adhesion protein from the lysate, (e) immunizing the laying hens with the recovered attachment protein, (f) spawning eggs from the immunized laying hens, (g A method of preventing and treating diarrhea in infants and young children, characterized by the step of separating the water-soluble protein from the egg yolk of the laid eggs. Provided are methods for preparing soluble proteins.

1 is a construct of a recombinant vector for expressing the adsorbed protein Vp8 of rotavirus that causes diarrhea in infants and toddlers.

2 is a photograph of isolated, purified SDS-PAGE of the expressed protein (lane 1: protein marker, lane 2: MBP-Vp8).

Figure 3 shows the Western blot results using the recombinant protein Vp8 of rotavirus and its antiserum.

Figure 4 is the result of measuring the binding ability of the water-soluble protein isolated from egg yolk released from the egg vaccinated from the immunized laying hens with rotavirus by indirect fluorescent antibody method (a: positive control, b: negative control, c water-soluble) Protein Vp8).

In the present invention, an adsorbent protein is used as a pathogenic factor of rotavirus that causes diarrhea in infants and toddlers, and representatively, Vp8 is exemplified. Here, the term "infant" refers to a child at the age of 2 weeks to 2 years of age and the term "infant" refers to a child at the age of 2 to 6 years.

The fourth genome segment of the 11 genome segments of rotavirus encodes the structural protein Vp4 exposed as spikes on the surface of the viral particles. Vp4 functions as a hemagglutinin, neutralizing antigen, protease-promoted infectivity, pathogenicity, membrane fusion, adhesion to cells, and is cut into Vp5 and Vp8 by proteases. In particular, Vp8 is known to have five of eight Vp4 neutralizing antibody determinants.

Pathogenic factors of rotavirus that cause diarrhea in infants and toddlers according to the present invention are produced by genetic recombination. The preparation of pathogenic factors by genetic recombination can use known techniques. In general, the preparation of pathogenic factors by genetic recombination isolates genomic RNA or DNA from the pathogen, and performs a polymerase chain reaction therefrom to prepare cDNA, amplify the target gene using the generated cDNA as a template, and the target Inserting a DNA sequence of a gene into a vector comprising one or more expression control sequences to control expression of the DNA sequence operatively linked to the DNA sequence and therefrom The host is transfected or transformed with the formed recombinant expression vector, the resulting transfected or transformant is incubated in a medium and conditions appropriate for the DNA sequence to be expressed, and the recombinant protein produced from the cultured transformant is Separating and purifying.

The genomic RNA of rotavirus can be isolated by infecting the rotavirus in cultured cells, culturing in a culture medium containing the nutrients necessary for the growth of the cultured cells, dissolving the recovered culture solution and using a virus separation column. . As cultured cells, Vero cells (CV-1), which are African green monkey kidney cells, are used. Suitable media are known in the art and generally include carbon sources, nitrogen sources, essential amino acids, vitamins and minerals, as well as other components, such as growth factors or serum that may be required by certain host cells. Preferably, it is incubated for one week in a preservation medium in which 2% fetal bovine serum is added to DMEM (Dulbeco's minimum essen tial medium). When the incubation is completed, the culture solution is recovered, the RNA extraction buffer is added to dissolve the cells, and passed through the virus separation kit column to completely remove the genomic DNA and protein to separate only pure rotavirus RNA.

Rotavirus cDNA can be prepared by reverse transcription-polymerase chain reaction of the RNA of the rotavirus. The primers used for the reverse transcriptase-polymerase chain reaction are synthesized with reference to the nucleotide sequence of human rotavirus Vp8 (M96825) obtained from the Gene Bank.

The Vp8 gene of rotavirus can be produced by amplifying by polymerase chain reaction using the rotavirus cDNA as a template. The produced Vp8 gene is cloned into a suitable vector. The term "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. The vector may be a plasmid, phage particles, or simply a potential genomic insert. For example, pUC8, pBR322, pET / Rb, pGEX, pET28a and the like can be used, and preferably pMAL-c2x (NEB) is used. An expression vector exemplified in the present invention is recombinant plasmid pMAL: Vp8.

Host cells transformed or transfected with the expression vectors described above constitute another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. As used herein, the term "transfection" means that the expression vector is accepted by the host cell whether or not any coding sequence is actually expressed. "Host cells" can be prokaryotic or eukaryotic. In addition, a host having a high DNA introduction efficiency and a high expression efficiency of the introduced DNA is usually used. For example, known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast can be used, preferably E. coli.

As a method for transforming the recombinant plasmid pMAL: Vp8 of the present invention into the host cell, conventional transformation methods such as DEAE-dextran, calcium phosphate method, electroporation method, and the like can be used.

Isolation of the Vp8 protein from the transformant or transfectant and its culture can be carried out by conventional known methods. In order to remove unnecessary cell debris, the cell lysate or cell culture is centrifuged, followed by precipitation, dialysis, various column chromatography, and the like. For example, ion exchange chromatography, gel-penetration chromatography, HPLC, reverse phase-HPLC, SDS-PAGE for preparation, affinity columns, and the like can be used for column chromatography. Preferably, the supernatant is recovered by ultrasonic crushing E. coli transformed according to the present invention by centrifugation at 5,000 to 9,000Xg, 10 to 40 minutes at 4 ° C and the recovered supernatant is purified using a amylose column to obtain a pure Vp8 protein fraction. To separate.

Immunization to laying hens using the Vp8 protein prepared according to the invention as an antigen can be carried out according to conventional techniques in the art. It does not specifically limit as a scattering system used for this invention, For example, it is preferable to use a white leg horn system, a Rhode island type, a high-line brown type etc. with a high scattering rate. Routes for administering the antigenic protein to the laying hens include intravaginal, intramuscular, intraocular or subcutaneous injection and the like. Antigen proteins of the invention can be used to increase their immunity using an adjuvant such as Freund's complete adjuvant or incomplete adjuvant.

Additional antigen boosters can be performed until sufficient antibodies are obtained. Preferably, three to four immunizations are performed at about 2 week intervals, and the next inoculation is at about 8 week intervals. Eggs are collected from the immunized laying hens, the desired protein is isolated therefrom, the amount of protein derived is measured, and when the increase reaches a steady state, the eggs are finally recovered.

The water-soluble protein according to the present invention is to separate the egg yolk from the recovered eggs, dilute it with distilled water, adjust the pH of the dilution to 4.0 to 6.0 and freeze, centrifugation by thawing the freezing body, formed supernatant Preferably, egg yolk is separated from the recovered eggs, diluted 5 to 15 times with distilled water, and then the pH of the dilution is adjusted to about 5.0, followed by 24 to -10 ° C to -30 ° C. After freezing for more than the hour, the frozen body is thawed at room temperature, centrifuged at 5,000 to 15,000 xg, 10 to 20 ℃ for 20 to 40 minutes, and the formed supernatant is separated by filtration with filter paper.

Determination of the amount of protein derived can be determined according to a conventional method in the art, and preferably, by enzyme immunosorbent or microtiter method. More preferably, the recombinant protein Vp8 attached to the microplate is reacted with a water-soluble protein isolated from egg yolk of an immunized egg according to the present invention, followed by a reaction of a secondary antibody diluted with alkaline phosphate, followed by phosphate as a substrate solution. Enzyme Linked Immunosorbent Assay (ELISA) is used to quantify the amount of protein induced by measuring the color reaction by the action of the enzyme at about 405 nm wavelength by adding the substrate.

On the other hand, the protein isolated from the egg yolk of the egg produced according to the present invention has the property of strongly binding to rotavirus. The indirect fluorescent antibody method was observed under a fluorescence microscope by fixing a rotavirus-infected cell on the fixed surface of the microscope slide, reacting it with a water-soluble protein according to the present invention, and then adding a secondary antibody combined with a fluorescent dye (Indirect). Fluorescent Assay (IFA). In this case, if there is no fluorescence in the cytoplasm of the infected or uninfected control cells, the negative or the titer of the reaction is the same in the infected or non-infected cells, or if the fluorescence is in the cell nucleus, the result is compared with the undetermined and control cells. Positive cases are determined in the cytoplasm of infected cells. As a result of analyzing the water-soluble protein according to the present invention by indirect fluorescent antibody method, fluorescence was observed in the cytoplasm of the infected cells as compared with the control cells. Therefore, it was confirmed that the water-soluble protein according to the present invention strongly binds to rotavirus.

Preferred embodiments are presented to aid in understanding the invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1

Cloning of the Vp8 Gene of Rotavirus

(1-1) Isolation of Genomic RNA from Rotavirus

After preserving the rotavirus strain Wa of the cell monolayer (CV-1), and then adding 2% Fetal Bovine Serum to DMEM (Dulbecco's minimum essential medium), a maintenance medium The virus was incubated for 1 week at. 150 μl of the virus culture supernatant was dissolved in guanidium thiocyanate sol. And then RNA was attached to a virus virus isolation kit column. Wash the column twice with wash buffer and 50 μl RNA was isolated with DEPC treated nuclease free water.

(1-2) Vp8 Gene Cloning of Rotavirus

Rotavirus Vp8 is part of the spike protein and is involved in neutralizing antibody formation. The primers involved in reverse transcriptase-polymerase chain reaction were synthesized by referring to the nucleotide sequence of human rotavirus Wa strain Vp8 (M96825) obtained from the Gene Bank (EMBL, Nucleotide Sequence Database, Cambridge, UK.). EcoRI and HindIII restriction enzyme recognition sites were inserted at the end of the primer. 15 μl viral RNA Into 2pmol primer and denatured at 92 ℃ for 10 minutes, 5unit RNase inhibitor (inhibitor), reverse transcription buffer, 200μM dNTP, 10unit MMLV reverse transcriptase was added 30μ final CDNA was synthesized by reacting at 40 ° C. for 1 hour. After inactivating reverse transcriptase at 98 ° C. for 5 minutes, polymerase chain reaction was performed.

PCR was carried out using the composition of the rotavirus cDNA synthesized above as a template and the reaction volume of 30 μl, with the composition of 20 pmol forward primer, 20 pmol forward primer, 250 μM dNTP, and 2.5 unit Taq DNA polymerase. . When the target gene was amplified, the reaction was performed 30 times at 92 ° C for 1 minute, 56 ° C for 1 minute, and 72 ° C for 1 minute. Amplified DNA was cloned using T4 ligase at the restriction sites of EcoR I and Hind III of the pMAL-c2x (NEB) expression vector (FIG. 1).

Forward primer: 5'-EcoRⅠ-GCTTCGCTCATTTATAGACAG-3 '

Reverse primer: 5'-HindIII-TTATGCTCTCTTATACTGTATCG-3 '

Example 2

Expression and Purification of Recombinant Protein Vp8

(2-1) Expression of Recombinant Protein

The positive clone obtained in Example 1 was taken and inoculated in LB medium containing 10 ml of ampicillin, followed by shaking culture at 37 ° C. overnight. Inoculated with E. coli cultured overnight in 1000mL of prepared LB medium shaken (A ₆₀₀ ) cultured to 1.0 and incubated for 16 hours at 37, 25 or 20 ℃ by adding 0.5 mM IPTG to induce expression. E. coli was obtained by centrifugation (8,000 rpm, 20 minutes, 4 ° C.), and then stored at −70 ° C. and used as a sample for the next experiment.

(2-2) Purification of Recombinant Protein Vp8

The transformed E. coli pellets of Example 2-1 were resuspended in column buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA). The suspension was subjected to sonication followed by centrifugation (9,000 × g, 30 minutes, 4 ° C.) to take the supernatant, appropriately diluted with column buffer and loaded onto the equilibrated amylose column. Washed with column buffer (12 times column volume), protein fractions were obtained with elution buffer (column buffer with 10 mM) and quantified with BCA assay kit (price). In addition, the protein was qualitatively examined by SDS-PAGE (FIG. 2) and Western blot to confirm the recombinant protein Vp8.

Example 3

Immunogenicity Test of Recombinant Protein Vp8

Infected with Rotavirus Wa strain (ATCC VR2018) on Vero cells (CV-1) with cell monolayers, and then added with 2% Fetal Bovine Serum (Dulbecco's minimum essential medium) The virus was incubated for one week. Infected cells were harvested, resuspended in Western blot sample buffer, left for 5 minutes at 95 ° C and subjected to SDS-PAGE. The protein separated by SDS-PAGE was transferred to a nitrocellulose membrane, and the antiserum of the Vp8 recombinant protein expressed in Escherichia coli DH5 was subjected to western blot using a antibody to confirm that the protein was about 30 kDa (Fig. 3).

Example 4

Immune Induction of Laying Hens Using Recombinant Protein Vp8

(4-1) Public animals

Animals used for induction of water-soluble proteins for the recombinant protein Vp8 obtained in the above example used 15-week-old egg-laying eggs of 36-week-old ISA-brown.

(4-2) Immune induction of laying hens

The prepared recombinant protein Vp8 (1 mg / ml) and Freund's complete adjuvant (Sigma Chemical Co.) were each mixed in equal amounts to emulsify. 1 ml of the emulsion was inoculated firstly by intramuscular injection into four quarters of 0.25 ml each of the laying hens. Booster injection was performed three times at two weeks interval after the first inoculation, and the next inoculation was performed every eight weeks. From the second inoculation, emulsification with Freund's incomplete adjuvant (Gibco) was performed in the same manner as the first inoculation. Eggs were collected daily and stored at 8 ° C. to be used for the experiment.

(4-3) Isolation of Water Soluble Proteins

Egg yolk was separated from the collected eggs and diluted 10-fold with distilled water (pH 5.0). The diluted solution was adjusted to pH 5.0 and frozen at -20 ° C for one day. After thawing at room temperature, only the supernatant was collected by centrifugation at 10,000 × g at 15 ° C. for 30 minutes. The supernatant was filtered through filter paper (Whatman No. 1) to separate the water soluble protein. And the separated protein was used as a sample of various experiments while storing at -20 ℃, and some were stored by freeze-drying.

(4-4) ELISA (enzyme-linked immunosorbent assay) analysis

Recombinant protein Vp8 was diluted with bicarbonate buffer (pH 9.6) to 5-10 μg / ml each and coated on a microplate (Microtest III flexible Assay plate, Falcon 3991) at 4 ° C. for 1 day. The coated plate was washed three times with washing solution (0.02M phosphate buffer, 0.13M NaCl, pH 7.2, 0.05% Tween20) and then left at room temperature for 2 hours with 5% skim milk solution (pH 7.3, Difco). . A water-soluble protein isolated from egg yolk of immunized eggs was diluted 3 times from 2,000 times to 1,458,000 times with a diluted solution of 5% skim milk solution and PBST (PBS containing 0.05% Tween-20) in an equal amount and then reacted at 37 ° C for 2 hours. I was. The secondary antibody was reacted for 2 hours at 37 ° C. by diluting alkaline phosphate-conjugated Affini Pure rabbit anti-chicken IgY (IgG, Jackson, USA) to 5,000-fold. As a substrate solution, the enzyme was dissolved in phosphate substrate tablets (ρ-nitrophenyl phosphate, Sigma-104) in 10% diethanolamine (dieethanolamine containing 0.5 mM MgCl ₂ pH 9.8) for 20 minutes. Reacted. The plate was washed six times during each procedure. The reaction inhibitor was 5M sodium hydroxide and measured the optical density (OD) at 405nm using a microplate reader (Molecular Devices; E Max), as shown in Table 1, it was confirmed that the egg-derived protein is induced.

Water Soluble Protein Induced by Recombinant Protein Vp8 Average O.D after inoculation (50000 fold dilution) Week 0 2 weeks 4 Weeks 12 Weeks 20 Weeks <0.01 0.23 0.71 0.98 1.15

Example 5

Investigation of the binding capacity of water soluble proteins to rotavirus

Rotavirus-infected cells were harvested and centrifuged three times with 0.01 M phosphate buffer (pH 7.2) to perform the indirect fluorescent antibody method. The washed infected cells were adjusted to a final cell number of 1 × 10 ⁶ cells / ml, incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator, and then fixed on antigen slides with 4 ° C. anhydrous acetone for 10 minutes. 20 μl of the antiserum of the Vp8 recombinant protein diluted 16-fold in 0.01 M phosphate buffer was added and reacted at 37 ° C. for 30 minutes. After the addition of μl it was observed under a fluorescence microscope.

As a result, it was confirmed that the water-soluble protein induced by Vp8 expressed as a recombinant protein in E. coli strongly binds to rotavirus (FIG. 4, a: positive control, b: negative E. coli, c water-soluble protein Vp8).

As mentioned above, eggs released from laying hens immunized with proteins involved in the adsorption of rotaviruses as antigens of the present invention and water-soluble proteins isolated from egg yolks of these eggs have strong binding capacity with rotaviruses. Therefore, the eggs recovered from the laying hen according to the present invention and the protein isolated from the eggs can be used as an agent for detecting, preventing and treating rotavirus that causes infant and infant diarrhea.

Claims (14)

Eggs containing water-soluble proteins that are spawned from laying hens immunized with adsorbent proteins of rotavirus as antigens and are useful for preventing and treating diarrhea in infants and young children. The egg according to claim 1, wherein the adsorbent protein of Rotavirus is Vp8. The egg of claim 1, wherein the adsorbed protein of the rotavirus is expressed from E. coli transformed with the recombinant plasmid pMAL: Vp8. A water-soluble protein isolated from egg yolks from eggs laid from laying hens immunized with adsorbent proteins of rotavirus as antigen and useful for preventing and treating diarrhea in infants and young children. The water-soluble protein according to claim 4, wherein the adsorbent protein of Rotavirus is Vp8. The water-soluble protein according to claim 4, wherein the adsorbent protein is expressed from E. coli transformed with the recombinant plasmid pMAL: Vp8. 7. The egg yolk according to any one of claims 4 to 6, wherein the egg yolk is diluted with distilled water, the pH of the diluted egg yolk solution is adjusted to 4.0 to 6.0 and frozen, and the frozen body is thawed to centrifugation. Water soluble protein isolated by filtration of the supernatant. (a) inserting a DNA sequence encoding an adsorbed protein of a rotavirus into a vector comprising one or more expression control sequences operatively linked to and controlling its expression (B) transforming the host with a recombinant expression vector formed therefrom, (c) culturing the resulting transformant under medium and conditions appropriate for the DNA sequence to be expressed, and (d) culturing the transformed Crushing the sieve and recovering substantially pure adsorbed protein from the lysate, (e) immunizing the laying hens with the recovered adsorbent protein, and (f) laying eggs from the immunized laying hens. A method of making an egg comprising a water soluble protein useful for preventing and treating diarrhea in infants. The method of claim 8, wherein said DNA sequence is gene Vp8. The method of claim 8, wherein the recombinant expression vector is recombinant plasmid pMAL: Vp8. (a) inserting a DNA sequence encoding an adsorbed protein of a rotavirus into a vector comprising one or more expression control sequences operatively linked to and controlling its expression (B) transforming the host with a recombinant expression vector formed therefrom, (c) culturing the resulting transformant under medium and conditions appropriate for the DNA sequence to be expressed, and (d) culturing the transformed Sieving and recovering substantially pure adsorbed protein from the lysate, (e) immunizing the laying hens with the recovered adsorbent protein, (f) spawning eggs from the immunized laying hens, and (g) water soluble from the egg yolk of the laid eggs. And separating the protein, thereby producing a water-soluble protein useful for preventing and treating diarrhea in infants and toddlers. . The method of claim 11, wherein said DNA sequence is gene Vp8. The method of claim 11, wherein the recombinant expression vector is recombinant plasmid pMAL: Vp8. The method of claim 11, wherein the separating the water-soluble protein from the egg yolk is carried out by diluting the egg yolk with distilled water, adjusting the pH of the diluted egg yolk solution to 4.0 to 6.0, freezing, and thawing the freezing body. And separating the formed supernatant by filtration.