KR100471114B1 - Antibody For Prevention And Treatment Of Enterohemorrhagic E. coli Infection, Eggs Containing Thereof And Method For Producing Thereof - Google Patents

Abstract

The present invention relates to an egg comprising an antibody, which is scattered from an immunized laying hen using the hemorrhagic Escherichia coli adhering protein as an antigen and useful for preventing and treating enteritis, food poisoning and hemorrhagic E. coli. The present invention further relates to antibodies which are isolated from egg yolk of said eggs and which are useful for preventing and treating intestinal hemorrhagic Escherichia coli. The present invention also relates to a method of making said eggs and antibodies.

Description

Antibodies for preventing and treating intestinal hemorrhagic E. coli infections, eggs containing them and methods for their preparation {Antibody For Prevention And Treatment Of Enterohemorrhagic E. coli Infection, Eggs Containing Thereof And Method For Producing Thereof}

The present invention relates to an egg comprising an antibody useful for preventing and treating an infection of enteral hemorrhagic E. coli and an antibody isolated from the egg yolk of such an egg. More specifically, the present invention relates to eggs and egg yolks containing antibodies which are used to prevent and treat intestinal hemorrhagic E. coliosis, which are scattered from an immunized laying hen using antigen as an antigen, which acts as an important pathogenic factor for enterohemostatic E. coli infection. The present invention relates to antibodies that are isolated and useful for preventing and treating intestinal bleeding E. coli. In addition, the present invention relates to a method for producing the egg and the antibody.

Escherichia coli is characterized by Enterohemorrhagic E. coli (EHEC), Enterotoxigenic E. coli (ETEC), Entero pathogenic E. coli according to toxic traits, routine epidemiology, clinical traits and pathophysiological mechanisms. coli (EPEC) and enteroin vasive E. coli (EIEC).

Among them, hemorrhagic E. coli is a causative agent of hemorrhagic colitis (HC), which produces vero toxin and damages the intestinal epithelial cells after mounting in the intestine (Morrison DM, Am. J. Clin. Pathol 86, 108-112 ( 1985)). Intestinal hemorrhagic Escherichia coli was reported in 1982 when E. coli O157: H7 was discovered after a hamburger food poisoning case. E. coli O157: H7 is an E. coli that can cause hemolytic uremic syndrome (HUS), especially in children and the elderly, and infections have been reported at all ages.

On the other hand, antibodies using antisera or colostrum, monoclonal antibodies have been developed to prevent and treat infections caused by E. coli hemorrhagic E. coli, but it is expensive to obtain a large amount 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 period of 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)). Tokoro et al. Have proposed a method for treating intestinal infectious diseases with substances obtained from eggs produced by chickens immunized with the pathogen as an antigen, and antibodies obtained from egg albumin and egg yolk (US Patent No. 5080895). ). In addition, yolk antibody against pathogenic E. coli using a gene vaccine (International Patent Publication No. 01/48018), yolk antibody for the treatment of intestinal infectious diseases of piglets (European Patent Publication No. 0955061, Korean Patent No. 267747, Korean Patent 0267746), yolk antibodies to rotavirus (Korean Patent Publication No. 2001-16599), and the like have been disclosed. The methods described above use live viruses or bacteria, attenuated and inactivated viruses or pathogenic related genes of viruses, viruses and bacteria as antigens for inducing antibodies to laying hens.

However, when using a bacterial or virus strain itself as an antigen, there is a disadvantage that the antibody titer does not appear higher than when using a single antigen because it contains many heterologous proteins.

The present inventors have prepared the C-terminal amino acid residue of the adhesion protein, which acts as an important pathogenic factor for the infection of enterohemorrhagic Escherichia coli, by genetic recombination, and using it as an antigen to immunize the laying hens, and then the egg yolk of the eggs laid from the immunized hens. Antibodies useful for the prevention and treatment of intestinal hemorrhagic E. coliosis caused by infection of intestinal hemorrhagic E. coli were isolated.

Accordingly, the present invention provides eggs containing antibodies useful for preventing and treating intestinal hemorrhagic E. coliosis, which are scattered from an immunized laying hen using antigens as attachment antigens, which act as important pathogenic factors for enterohemostatic E. coli infection.

In another aspect, the present invention provides antibodies useful for preventing and treating intestinal hemorrhagic E. coliosis, which are isolated from egg yolks from eggs laid from an immunized laying hen using antigens as attachment antigens, which act as important pathogenic factors for enteric hemorrhagic E. coli infections. .

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 hemorrhagic Escherichia coli attachment protein. inserted into a vector comprising a control sequence, (b) transforming the host with a recombinant expression vector formed therefrom, and (c) culturing the resulting transformant under medium and conditions suitable for causing the DNA sequence to be expressed. (D) crushing the cultured transformant and recovering substantially pure urease from the lysate, (e) immunizing the laying hens with the recovered attachment protein, and (f) spawning eggs from the immunized laying hens. It is characterized in that the present invention provides a method for producing an egg containing an antibody useful for preventing and treating enterohemorrhagic E. coli.

In another aspect, the present invention provides a method for controlling the expression of (a) one or more expression control sequences operatively linked to a DNA sequence encoding an adherent protein of E. coli. inserted into a vector comprising a control sequence, (b) transforming the host with a recombinant expression vector formed therefrom, and (c) culturing the resulting transformant under medium and conditions suitable for causing the DNA sequence to be expressed. (D) disrupting the cultured transformants and recovering substantially pure urease from the lysates, (e) immunizing the laying hens with the recovered attachment proteins, (f) spawning eggs from the immunized laying hens, (e) And separating the antibody from the egg yolk of the laid egg, thereby producing an antibody useful for preventing and treating enterohemorrhagic E. coli. Provide a method.

One aspect of the invention provides eggs containing antibodies useful for preventing and treating intestinal hemorrhagic E. coliosis, which are spawned from an immunized laying hen using the C-terminal amino acid residues of intimin that act as an important pathogenic factor for intestinal hemorrhagic E. coli infections as antigens. to provide.

The major pathogenic factors of intestinal hemorrhagic Escherichia coli are the production and adhesion of Shigella-like toxins (vero toxins). Intestinal hemorrhagic E. coli has a 60MDa plasmid that is involved in intestinal epithelial cell attachment and has an eae gene on the chromosome that shows attaching-effacing to adherent cells. The eae gene encodes 97 kDa intimin, a protein that regulates intestinal epithelial cell adhesion. Intestinal hemorrhagic E. coliosis typically begins with a short prognosis of mild non-hemorrhagic diarrhea, with hemorrhagic diarrhea within 24 hours, with severe abdominal pain and cramps.

As a pathogenic factor of enterohemorrhagic Escherichia coli according to the present invention, it is an adhesion protein of intestinal hemorrhagic E. coli O157: H7. More preferably, it is a 34 kDa protein (His) ₆ -IntC expressed from a plasmid in which the C-terminal amino acid residue of the intimin protein is fused with six histidines.

Pathogenic factors of enterohemorrhagic Escherichia coli according to the present invention can be prepared by genetic recombination. The preparation of pathogenic factors by genetic recombination can use known techniques. In general, the production of pathogenic factors by genetic recombination isolates genomic DNA from pathogens, amplifies the target gene by performing a polymerase chain reaction using the genomic DNA as a template, and converts the DNA sequence of the target gene into one. Or inserted into a vector comprising more than one expression control sequence to control expression of the DNA sequence operatively linked to the DNA sequence and to host the recombinant expression vector formed therefrom. Transfecting or transforming, culturing the resulting transfected or transformant under medium and conditions appropriate for the DNA sequence to be expressed, and isolating and purifying the recombinant protein produced from the cultured transformant. It is composed.

The genomic DNA of intestinal hemorrhagic E. coli was inoculated into the culture medium for intestinal hemorrhagic E. coli and cultured under suitable culture conditions to obtain the cells. Then, the cells were lysed by adding a lysis buffer solution to the protein from the supernatant obtained by centrifugation. It can be obtained by extracting pure DNA only by removing RNA and the like. The culture medium of intestinal hemorrhagic Escherichia coli is not particularly limited, but a penassay broth or tryptone, yeast extract consisting of beef extract, yeast extract, peptone, glucose, NaCl, K ₂ HPO ₄ and KH ₂ PO ₄ And NaCl (LB broth) may be used, and ampicillin and kanamycin may be used as needed. Preferably, the culture conditions are incubated for 10 hours to 30 hours in the temperature range 30 ℃ to 40 ℃. Centrifugation of the culture solution is carried out at 10,000 rpm to 15,000 rpm for 1 minute to 10 minutes.

The gene encoding the adherent protein of enterohepatitis E. coli can be prepared by subjecting the isolated genomic DNA to a polymerase chain reaction. The primers used in the polymerase chain reaction can be synthesized by referring to the nucleotide sequences obtained from the gene bank.

The gene prepared by the above method is cloned into a suitable vector. One aspect of the present invention provides a recombinant plasmid pQE: (His) ₆ -IntC which can contain and express a nucleic acid sequence encoding the adhesion protein of enterohemorrhagic Escherichia coli which is a pathogenic factor according to the present invention. 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, pQE is used.

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.

The host cell of the invention may be a prokaryotic or eukaryotic cell. In addition, a host having a high DNA introduction efficiency and a high expression efficiency of the introduced DNA is usually used. 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 vector pQE: (His) ₆ -IntC of the present invention into a host cell, conventional transformation methods such as DEAE-dextran, calcium phosphate method and electroporation method can be used.

Separation of the recombinant protein from the transformant or transfectant and its culture can be carried out by conventional known methods. In order to remove unnecessary cell debris, cell lysates or cell cultures may be centrifuged, followed by precipitation, dialysis, various column chromatography, and the like. Ion exchange chromatography, gel-exclusion chromatography, gel-filtration chromatography, HPLC, reverse phase-HPLC, preparative SDS-PAGE, affinity columns and the like are examples of column chromatography. Among these, as the ion exchange resin generally used in ion exchange chromatography, cellulose, dextran, agarose and polyacrylamide and derivatives thereof can be used. In addition, gel-exclusion chromatography is widely used for protein separation on dextran, agarose and polyacrylamide, and can be separated by eluting the protein sample step by step or by concentration gradient. Gel filtration chromatography is a method of separating according to the size of the molecule is a very useful affinity chromatography that takes advantage of the natural affinity of the protein for specific molecules such as substrates or interferers. This is a method of separating proteins by covalently binding a molecule that binds to a protein to a non-insoluble support such as agarose and then using it as a support to selectively adsorb the appropriate protein. Preferably, the E. coli culture transformed according to the present invention is centrifuged to obtain the cells, and then lysed buffer solution is added to dissolve the cells and attached to pure E. coli bacteria using a column filled with Ni-NTA resin. Protein fractions are separated.

The method of immunizing a laying hen using the recombinant protein prepared according to the present 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, immunization is performed three times at about 2 week intervals, and the next inoculation is at about 8 week intervals. The eggs are collected from the immunized laying hens, the antibodies are separated therefrom, the amount of antibody derived is measured and finally when the increase reaches a sufficient state, the eggs are finally recovered.

Measurement of specific antibody titers transferred to eggs of the laying hens can be measured according to conventional methods in the art. Preferably, an enzyme immunosorbent method or a microtiter method is used. More preferably, the antigenic protein attached to the microplate is reacted with the egg yolk of the immunized egg according to the present invention, followed by the reaction of the secondary antibody diluted with alkaline phosphate, and the phosphate sublate tablet as a substrate solution. In addition, Enzyme Linked Immunosorbent Assay (ELISA) is used, which is a method for quantifying the protein induced by measuring enzymatic color reaction at a wavelength of about 405 nm.

The present invention also provides antibodies useful for preventing and treating intestinal hemorrhagic E. coliosis, which are isolated from egg yolks from eggs laid from immunized laying hens using antigens as adhesion antigens, which act as important pathogenic factors for enteric hemorrhagic E. coli infections.

Isolation of the antibody from the yolk of the immunized egg can be obtained by removing the yolk lipid component and solids in the yolk. Preferably, the antibody 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, thaw the freezing body to centrifugation, The formed supernatant can be separated by filtration. More preferably, egg yolk is separated from the recovered eggs, diluted to 5 to 15 times by adding distilled water, and then the pH of the diluted solution is adjusted to about 5.0, and then frozen at -10 ° C to -30 ° C for at least about 24 hours, and frozen. The sieve is thawed at room temperature and centrifuged for 20 to 40 minutes at 5,000 to 15,000 × g, 10 ° C. to 20 ° C., and the supernatant formed is separated by filtration with filter paper.

  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

Genomic DNA Isolation of Intestinal Hemorrhagic E. Coli O157: H7

Enteric hemorrhagic E. coli O157: H7 strain EDL932 was prepared by penassay broth; 1.5 g beef extract, yeast extract 1.5 g, peptone 5 g, glucose 1 g, NaCl 3.5 g, K ₂ HPO ₄ 3.68 g, KH ₂ PO ₄ 1.32 g 1 l ^-1 ). When the culture was completed, the cells were recovered and centrifuged once. The cells were resuspended with 0.15 M PBS (phosphate-buffered saline [pH 7.4]) and then the same amount of lysis buffer (50 mM Tris-HCl, 50 mM EDTA, 0.3% SDS, 20 μg / ml proteinase K [pH 8.0]) was added. The solution was added and lysed at 50 ° C. for 2 hours. After centrifugation, the supernatant was collected, extracted twice with phenol-chloroform-isoamyl alcohol (25: 24: 1, v / v), chloroform extraction was performed once, followed by ethanol precipitation. The separated genomic DNA was dissolved in TE buffer (10 mM Tris, 1 mM EDTA [pH 8.0]), quantified and used as template DNA for PCR while storing at 4 ° C.

Example 2

Eae Gene Cloning of E. Coli O157: H7

5 ml of intestinal hemorrhagic E. coli O157: H7 was taken and centrifuged at 13,000 rpm for 2 minutes, suspended in 120 µl of Buffer I (50 mM glucose, 25 mM Tris Goat [pH8.0], 10 mM EDTA [pH8.0]) and Buffer II. 240 μl (1% SDS, 0.2N sodium hydroxide) was added and mixed well. After reacting for 5 minutes at 0 ° C., 180 μl of acetate was added to 3M potassium acetonitrile and vortex mixed for 10 seconds. The supernatant was centrifuged to obtain plasmid DNA by chloroform / isoamyl alcohol DNA extraction.

The eae gene was PCR amplified using genomic DNA of E. coli O157: H7 isolated above as a template. DNA sequence of the eae gene was obtained from the GenBank (EMBL, Nucleotide Sequence Database, Cambridge, UK.), from which the primers involved in polymerase chain reaction (PCR) were synthesized. The PCR mixture was subjected to PCR with a total composition of 50 μl, including 0.2 μM forward primer, 0.2 μM reverse primer, 1 μl of chromosomal DNA, 200 μM dNTP, and 2 units of Taq polymerase.

PCR conditions were performed 30 times (cycle) after the initial denaturation step (95 ℃, 3 minutes), 95 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 2 minutes step by step, and finally The reaction was carried out at 72 ° C. for 10 minutes. After completion of the reaction, amplification of the target sequence was confirmed by agarose gel electrophoresis of 1.2%. As a result, the portion of 2800bp (69 ~ 2873) gene amplified by polymerase chain reaction was located from 2027 to 2873.

Example 3

(His) ₆ -IntC Fusion

The PCR product obtained in Example 2 was cloned into plasmid pCR2.1 (Invitrogen Co.), and then transformed into E. coli TOP10F '(Invitrogen Co.,) and plasmids were isolated. The isolated plasmid was digested with restriction enzymes BamH I and Kpn I (Promega Co.) to obtain a DNA fragment of 870 bp. An expression vector pQE vector prepared by cleaving with the same restriction enzyme as the DNA fragment was ligated (FIG. 1). The expression vector pQE was transformed into E. coli M15 [pREP4] (Qiagen, Inc.) to select a positive clone.

Example 4

Expression and Isolation of Recombinant Protein (His) ₆ -IntC

The positive clones obtained in Example 3 were taken and inoculated into 10 ml of LB broth (Tryptone 10g / L, Yeast Extract 10g / L, NaCl 10g / L) and incubated overnight at 37 ° C. Fresh 100 ml LB broth was inoculated with overnight incubated bacteria and shaken to obtain an absorbance (A ₆₀₀ ) of 0.6. 0.5 mM of isopropyl-β-D-thio-galactopyranoside (IPTG) was added thereto, followed by further incubation for 4 hours. Strains were recovered using a centrifuge and resuspended in lysis buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris-Cl, 8 M urea [pH 8.0]) and shaken at room temperature for 30 minutes. The completely dissolved bacteria were centrifuged (10,000 × g, 20 minutes), and 4 ml of the supernatant and 1 ml of Ni-NTA resin (nickel-nitrilotriacetic acid agarose, Qiagen Inc.) were mixed at room temperature in a water bath and packed in a column. Wash buffer _{(100mM NaH 2 PO 4, 10mMTris-} Cl, 8M urea [pH 6.3]) and elution buffer _{(100mM NaH 2 PO 4, 10mMTris} -Cl, 8M urea [pH 5.9]) to the washing and elution with a spectrophotometer the resin It was confirmed by the absorbance (A ₂₈₀ ) using. Fractionated proteins were dialyzed in 6M, 4M, 2M urea buffer (100 mM NaH ₂ PO ₄ , 10 mMMTris-Cl [pH 8.0]) stepwise, and protein concentration was determined using BCA kit (Pierce CO.) And SDS denaturation was performed. Specific proteins of IntC were identified on the run (FIG. 2, Lane 1: Cell protein before induction, Lane 2: Induced cell protein, Lane 3: Cell lysate, Lane 4: Purified (His) ₆ -IntC protein) Rabbit antiserum was prepared using the same. The prepared rabbit antiserum was used as an antibody of Western blot.

Example 5

Immunogenicity Test of Recombinant Protein IntC

Immunogenicity testing of the recombinant protein IntC was performed according to the method of Actman (1983), et al. The cells of enteral hemorrhagic E. coli cultured overnight in pennasay broth were dissolved in lysis buffer [50 mM Tris (pH 7.5), The cells were resuspended with 50 mM NaCl, 1 mM phenymethylsulfonyl fluoride], and the cells were disrupted, and centrifuged at low speed to remove unbroken bacteria. Then, a solution containing 1.7% N- lauroylsarcosine (Sigma Chemical Co.) in 11.1 mM Tris (pH 7.6) was added at a ratio of 1: 8 (vol / vol) and shaken for 30 minutes at room temperature. It was dissolved in a sample buffer and used as a sample of SDS-PAGE After the electrophoresis, Western blot was performed using the antiserum prepared in Example 4, and as a result, the reaction at 97kDa was confirmed ( 3).

Example 6

Immune Induction of Laying Hens Using Recombinant Protein (His) ₆ -IntC

Animals used for induction of laying hens' eggs were used with 15 eggs of 36-week-old ISA-Brown hens. Recombinant protein (His) ₆ -IntC 200 ㎍ / ㎖ isolated in Example 4 and Freund's complete adjuvant (Sigma Chemical Co.) were each equally mixed and emulsified. 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 every two weeks after the first inoculation, and the immunogen was emulsified with Freund's incomplete adjuvant (Gibco) in the same manner as the first inoculation. Eggs were collected daily and stored at 8 ° C. to be used for the experiment.

Example 7

Isolation of Yolk Antibodies

Eggs were collected from the immunized laying hens in Example 5 to separate egg yolk and diluted 10-fold with distilled water (pH 5.0). The diluted yolk solution was adjusted to pH 5.0 and frozen at -20 ° C overnight. The frozen yolk was thawed at room temperature and then centrifuged (10,000 × g) at 15 ° C. for 30 minutes to collect only the supernatant. The supernatant was filtered through filter paper (Whatman No. 1) to separate the yolk protein fraction.

Example 8

Antibodies measured

Using the yolk protein fraction obtained in Example 6, the degree of antibody formation was measured by enzyme-linked immunosorbent assay (ELISA) and microtiter method. Recombinant protein (His) _6- IntC 1.8mg / ml was diluted to carbonate-bicarbonate buffer solution (pH 9.6) to 5µg / ml, and the microplate (MicrotestIII flexible Assay plate, Falcon 3991) was 4 ° C. Coated overnight. The coated plate was washed three times with washing solution (0.02M phosphate buffer, 0.13M NaCl, pH 7.2, 0.05% Tween20) and left at room temperature for 2 hours with 5% skim milk solution (pH 7.3, Difco). . Egg yolk and serum were diluted in 3x multiples 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 the same amount and then reacted at 37 ° C for 2 hours. The secondary antibody was diluted 5,000-fold in conjugated Affini Pure rabbit anti-chicken IgY (IgG, Jackson, USA) with alkaline phosphate and reacted at 37 ° C for 2 hours. . As a substrate solution, a solution of Phosphate substrate tablets (ρ-nitrophenyl phosphate, Sigma-104) dissolved in 10% diethanolamine (dieethanolamine containing 0.5 mM MgCl ₂ pH 9.8) was used. The enzyme was reacted for 15 minutes. The plate was washed six times during each procedure. The reaction inhibitor was 5M sodium hydroxide and the antibody value was measured at 405nm using a microplate reader (Molecular Devices; E Max).

As a result, the change in antibody titer was maintained at around 20,000 antibody titer in 2 weeks, and showed double antibody titer after 4 weeks and rapidly increased after 6 weeks (Fig. 4).

Antibodies isolated from eggs and egg yolk prepared according to the present invention contain a large amount of antibodies against intimin, which acts as an important pathogenic factor of enterocoliform Escherichia coli O157: H7, and can effectively prevent diseases caused by enterococci.

1 is a construction of a recombinant vector for expressing the adhesion protein IntC of intestinal hemorrhagic E. coli causing human diarrhea.

2 is a SDS denaturation electrophoresis picture of recombinant protein (His) ₆ -IntC expressed from a transformant (lane 1: cell protein before induction, lane 2: cell protein after induction, lane 3: cell lysate, Lane 4: purified (His) ₆ -IntC protein).

Figure 3 is the result of Western blot using the antiserum of the extracellular membrane protein (OMP) and recombinant protein IntC isolated from intestinal hemorrhagic Escherichia coli causing human diarrhea.

Figure 4 is a graph showing the results of changes in the antibody of ELISA against egg yolk of the present invention.

Claims (14)

delete delete delete delete delete delete delete (a) to a vector comprising one or more expression control sequences operatively linked to the DNA sequence encoding intimin of E. coli hemorrhagic to regulate 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) Crushing the converter and recovering substantially pure intimin from the lysate, (e) immunizing the laying hens with recovered intimin, and (f) spawning eggs from the immunized laying hens A method of making an egg comprising an antibody useful for preventing and treating Escherichia coli. delete The method of claim 8, wherein said recombinant expression vector is recombinant plasmid pQE: (His) ₆ -IntC. (a) to a vector comprising one or more expression control sequences operatively linked to the DNA sequence encoding intimin of E. coli hemorrhagic to regulate 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) Disrupting the transformants and recovering substantially pure intimin from the lysate, (e) immunizing the laying hens with recovered intimin, (f) spawning eggs from the immunized laying hens, and (e) And separating the antibody, wherein the antibody is useful for preventing and treating intestinal hemorrhagic E. coli. delete The method of claim 11, wherein said recombinant expression vector is recombinant plasmid pQE: (His) ₆ -IntC. The method of claim 11, wherein the separating the antibody from the egg yolk is performed 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 to centrifugation. And separating the formed supernatant by filtration.