CN101199556A - Recipient beta-blocker of prevention weaning piggy diarrhea and piggy oedematous disease, preparing and application - Google Patents
Recipient beta-blocker of prevention weaning piggy diarrhea and piggy oedematous disease, preparing and application Download PDFInfo
- Publication number
- CN101199556A CN101199556A CNA2007101914679A CN200710191467A CN101199556A CN 101199556 A CN101199556 A CN 101199556A CN A2007101914679 A CNA2007101914679 A CN A2007101914679A CN 200710191467 A CN200710191467 A CN 200710191467A CN 101199556 A CN101199556 A CN 101199556A
- Authority
- CN
- China
- Prior art keywords
- asd
- probiotics
- gene
- fedf
- escherichia coli
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a receptor antagonist for preventing diarrhea occurring on the weaned piglets and edema disease occurring on the piglets, a preparation method and an application. The receptor antagonist of the invention is an assemblage series of recombinant strains of probiotics, and is a MisL-Beta recombinant vector which expresses FedF formed by F18 or Fed adhering to prime subunit gene, and FaeG formed by K88 or F4 or Fae adhering to the prime subunit gene, based on the recombinant strain EP2 of the probiotics of escherichia coli with pig origin advantage and strain LP2 of probiotics of lactobacillus with pig origin advantage. The surface of the cell is good to express antigen technology, so after the piglets orally administrate the cell, the cell can colonize, sedentarily live and proliferate in the intestine. Furthermore, the cell on the surface of the epithelium of the intestine functionally shows secretory expression of highly conservative and cofunctional adhesins FedF and FaeG, that is, the receptor binding domain of F18 and K88 adhesins, which is preferentially bound with the surface of the epithelium of the intestine of the piglets, and thus the diarrhea occurring on the weaned piglets and the edema disease occurring on the piglets are controlled. The invention overcomes the defects that the present antibiotics are almost ineffective in the clinical treatment as most of piglets suffering from the edema disease die though treated by the present antibiotics, and the present antibiotics causes multiple drug resistance.
Description
Technical field
The present invention relates to a kind of biological preparation-receptor blocking agent, particularly a kind of receptor blocking agent, preparation and application that prevents diarrhea of weaned piglets and baby pig edema.
Background technology
Before the present invention, be used for the prevention of diarrhea of weaned piglets and baby pig edema in the world without any commercialized vaccine, and when just diarrhea of weaned piglets and baby pig edema case taking place clinically, same passive employing antibiotic clinical treatment, newborn piglet and diarrhea of weaned piglets rate have been reduced to a certain extent, but antibiotic clinical treatment at the baby pig edema case, substantially invalid, its piglet course of disease final result of suffering from edema disease is last dead mostly, and the use of long-term and a large amount of composite antibiotics, cause multiple drug resistance enterotoxigenic escherichia coli (ETEC) F18 and K88 bacterial strain constantly to produce and spread in the animal body horizontal transmission, and cause antibiotic poor efficiency and poultry in animal body to amass, also can cause the disorder of animal gastrointestinal tract normal flora, dyspepsia, cause gastrointestinal tract and Abwehrkraft des Koepers and descend, easily secondary or concurrent multiple disease.The The main pathogenic fungi of diarrhea of weaned piglets is two kinds of F18 ETEC and K88 ETEC escherichia coli, the The main pathogenic fungi of baby pig edema is F18 ETEC, in view of F18 ETEC is divided into F18ab and 2 serotype antigen mutation of F18ac by adhesin F18, K88 ETEC is divided into K88ab, K88ac, 3 kinds of serotype antigen mutation of K88ad by adhesin K88, the somatic antigen serotype of its clinical epidemic strain is then more, thereby effective vaccine of developing at diarrhea of weaned piglets and baby pig edema exists great difficulty so far.
Summary of the invention
Purpose of the present invention just is to overcome above-mentioned defective, develops a kind of receptor blocking agent and preparation method of preventing diarrhea of weaned piglets and baby pig edema.
Technology of the present invention is:
The receptor blocking agent of prevention diarrhea of weaned piglets and baby pig edema, its major technique is characterised in that receptor blocking agent is a probiotics reorganization bacterium combination series, this probiotics reorganization fungus strain is classified 2 boar source advantage probiotics bacterial strains as, wherein a kind is pig source advantage escherichia coli probiotics reorganization bacterium EP2, the 2nd kind is pig source advantage lactobacillus probiotics bacterial strain LP2, pig source advantage escherichia coli probiotics EP2 reorganization bacterium includes surperficial secreting, expressing F18 or Fed adhesin subunit gene FedF, the MisL β recombinant vector of K88 or F4 or Fae adhesin subunit gene FaeG, wherein adhesin F18 is divided into F18ab and 2 serotype antigen mutation of F18ac, be to encode by F18 enterotoxigenic escherichia coli wild type large plasmid DNA, F18ab plasmid size 48 and 98Mu between, and the big plasmid of F18ac is about 98Mu.5 gene fellowships of one group of operon are finished in the expression of adhesin Fed and the big plasmon of synthetic needs, they are respectively FedA, B, C, E and F, wherein the FedF subunit is a F18 escherichia coli adhesin receptor binding domains, F18ab FedF and F18ab FedF are the common functionality adhesin of high conservative in conjunction with identical piglet enterocyte surface macromole receptor; Adhesin K88 is divided into K88ab, K88ac, 3 kinds of serotype antigen mutation of K88ad, be to encode by K88 enterotoxigenic escherichia coli wild type large plasmid DNA, by FaeA to FaeJ totally 10 genes participate in the biosynthesis of its pili adhesin, wherein the FaeG subunit is for being K88 escherichia coli adhesin receptor binding domains, in conjunction with piglet enterocyte surface macromole receptor, be the functional adhesin of high conservative.
Another technical scheme of the present invention is:
The preparation method of described receptor blocking agent, its major technique step is as follows:
(1) pig source advantage escherichia coli probiotics strain vector bacterium EP2 separates from the normal mucous membrane of small intestine of piglet with pig source advantage lactobacillus probiotics bacterium LP2, the antibacterial method is separated, is identified routinely, meet escherichia coli and lactobacillus form, cultivation and biological general characteristic, further detect by the intestine of young pigs field planting of oral back, proliferation test bacterial population in the external adherence test of piglet intestinal epithelial cell and the body, filter out to the intestinal epithelial cell adhesive capacity by force, easily can be in the intestine of young pigs field planting, settle down and EP2 and two kinds of antibacterials of LP2 of breeding and preserve;
(2) choose pig source advantage escherichia coli probiotics strain vector bacterium EP2,, choose pig source advantage lactobacillus probiotics bacterium LP2, press conventional cultivation of lactobacillus and propagation by conventional antibacterial culturing of escherichia coli and propagation;
(3) amplification, evaluation FedF and FaeG subunit gene, according to FedF and the known sequence of delivering of FaeG gene, with F18ab, F18ac and K88ac advantage serotype escherichia coli large plasmid DNA is template, adopt high-fidelity PCR Kit increase above-mentioned subunit gene FedF and FaeG, the PCR fragment inserting clone carrier pGEM-T that further 3 ' end is had A, preparation transforms engineering bacteria DH5 à competence antibacterial, through blue white macula screening, from the cultivation bacterium liquid of screening, extract and contain the segmental recombiant plasmid pGEM-T of purpose DNA, the sequencing checking;
(4) the V-type excretory system MisL β recombinant vector system constructing of surperficial secreting, expressing FedF and FaeG subunit gene, DNA recombinant technique operation routinely, FedF will encode, the secretion expression carrier MisL β of FaeG subunit gene and V-type excretory system digests through restricted enzyme NheI enzyme action respectively, after the connection, transform engineering bacteria DH5 à competence antibacterial respectively, from the cultivation bacterium liquid of screening, extract and contain the segmental positive plasmid DNA of purpose, PCR detects, in conjunction with the plain agglutination positive of bacterial adhesion and sequencing checking result, screen the V-type excretory system MisL β recombinant vector of energy functional surface secreting, expressing FedF and FaeG subunit;
(5) the aspartic acid semialdehyde dehydrogenase house-keeping gene on evaluation, the clones coding escherichia coli chromosome is the complete encoding gene of ASD: according to the known sequence of delivering of said gene, with engineering bacteria DH5 à chromosomal DNA is template, adopt long range PCR method amplification ASD complete genome, making nucleic acid molecular hybridization detects, obtain the complete encoding gene of ASD at last, about 2Kb size through specific restricted enzyme PstI enzyme action digestion binding nucleotide sequence analysis;
(6) the MisL β recombinant vector system constructing of surperficial secreting, expressing FedF, FaeG adhesin subunit gene and ASD encoding gene.The MisL β recombinant vector of surperficial secreting, expressing FedF and FaeG adhesin subunit gene in the step 5, operate through recombinant DNA technology, insert the multiple clone site zone of ASD encoding gene MisL β recombinant vector in step 5, inactivation the resistant gene activity on the former MisL β carrier, in engineering bacteria DH5 à ASD deletion mutation strain DH5 à Δ ASD indicator cells, filter out to contain and express ASD encoding gene and surperficial secreting, expressing FedF, the MisL β recombinant vector of FaeG adhesin subunit gene, exist for the screening sign with ASD and substitute the antibiotics resistant gene screening sign, and the penbritin resistant gene screening of having dispeled on the MisL β original vector indicates;
(7) pig source advantage escherichia coli probiotics EP2 reorganization bacterium makes up and screening, extraction contains surperficial secreting, expressing FedF, the MisL β recombinant plasmid vector DNA of FaeG adhesin subunit gene and ASD encoding gene, in the DNA conversion method imports pig source advantage escherichia coli probiotics carrier bacterium EP2 respectively, from the cultivation bacterium liquid of screening, extract and contain the segmental positive recombinant plasmid dna of purpose, in conjunction with the plain agglutination of bacterial adhesion, the external adherence test of piglet intestinal epithelial cell and intestine of young pigs field planting test bacterial population detects positive findings, filter out FedF and the effective and functional exhibition of two kinds of adhesin subunits of FaeG can be with secreting, expressing on the epithelial EP2 of the intestine of young pigs bacterium surface of recombinating;
(8) receptor blocking agent preparation of product: pig source advantage escherichia coli probiotics EP2 reorganization bacterium and the single bacterium colony of pig source advantage lactobacillus LP2 in the picking step (7), at 18 hours bacterium liquid of 37 ℃ of incubated overnight as seed liquor, distinguish amplification culture, propagation 16～18 hours again, through conventional bacterial colony count, with colony forming units cfu tolerance, concentration reaches 2～6 * 10
9Cfu, mixed configuration forms probiotics reorganization fungus strain row after reorganization bacterium EP2 and the LP2 amplification culture.
Another technical scheme of the present invention is:
Described receptor blocking agent is as the application of prevention diarrhea of weaned piglets and edema disease of piglets medicine, and its major technique is characterised in that:
1) pig source advantage escherichia coli probiotics strain vector bacterium EP2, through the DNA genetic modification, promptly utilize bacteriophage lambda Red recombinase system and homologous recombination technique, the aspartic acid semialdehyde dehydrogenase house-keeping gene that lacks on its strain chromosome is the ASD encoding gene, constructs pig source advantage escherichia coli probiotics strain vector bacterium ASD deletion mutation strain EP2 Δ ASD;
2) surperficial secreting, expressing FedF, the MisL β recombinant vector of FaeG adhesin subunit gene and ASD encoding gene, operate through recombinant DNA technology, insert the ASD encoding gene in the multiple clone site zone of the MisL β recombinant vector of surperficial secreting, expressing FedF and FaeG adhesin subunit gene, inactivation the resistant gene activity on the former MisL β carrier, in engineering bacteria DH5 à ASD deletion mutation strain DH5 à Δ ASD indicator cells, filter out to contain and express ASD encoding gene and surperficial secreting, expressing FedF, the MisL β recombinant vector of FaeG adhesin subunit gene, exist for the screening sign with ASD and substitute the antibiotics resistant gene screening sign, and the penbritin resistant gene screening of having dispeled on the MisL β original vector indicates;
3) pig source advantage escherichia coli probiotics EP2 reorganization bacterium is the chromosome of selection pressure based on ASD non-antibiotic resistance---the carrier strain construction of plasmid balanced lethal system, and its system itself does not have any antibiotics resistant gene screening sign;
4) pig source advantage lactobacillus probiotics bacterial strain LP2 can effectively promote pig source advantage escherichia coli probiotics EP2 reorganization bacterium and its ASD deletion mutation strain EP2 Δ ASD reorganization bacterium in the field planting of intestine of young pigs, settle down and breed;
5) the receptor blocking agent product is used for an age in days piglet, and oral 2ml can be in the intestine of young pigs field planting after 4 hours, settle down, breed.
Advantage of the present invention and effect are to make up bacterial chromosomal dna macromole recombinant technique, pathogen receptor and its adhesin combination and detection technique, adhesin receptor blockade/interrupter technique, surface secreting, expressing adhesin technology and poultry probiotics years of researches working foundation, a kind of receptor blocking agent of developing, it is based on the advantage probiotics carrier bacterium recombinant bacterial strain combination of pig source, can be after piglet is oral in the very fast field planting of intestinal, settle down, propagation, and the common functionality adhesin FedF and the FaeG that are the high conservative of secreting, expressing in the exhibition of intestinal epithelial cell surface-functional, be F18 and K88 adhesin receptor binding domains, preferential and intestine of young pigs surface epithelial cell receptors bind, thereby sealed intestinal epithelial cell and F18 and the mediation of K88 adhesin the ETEC pathogen combine target site, even outside contamination has a large amount of pathogen, owing to lost in conjunction with the epithelial ability of intestine of young pigs, and can not infect the susceptible piglet.Great advantage of the present invention is that this kind receptor blocking agent is different fully with the efficacy effect stage of conventional antibiotic medicine and vaccine; the latter is opposing; suppress; in and attacked the ETEC pathogen of infection in the body; the protection piglet does not fall ill; the former stops extraneous pathogen can not enter in the animal body; both relatively; the latter can be the most direct; combining of the enterotoxigenic escherichia coli pathogen of sealing/prevention intestine of young pigs epithelial receptor target site and F18 and K88 adhesin mediation most effectively is used for the prevention that anti-F18 and K88 enterotoxigenic escherichia coli infect clinically.In fact; we have been used for this receptor blocking agent product to take place usually diarrhea of weaned piglets and some pig farms of baby pig edema case; produce obviously protection effect; preventive effect to F18 and K88 enterotoxigenic escherichia coli pathogenic bacterial infection is good; using method is that piglet is oral; very simple, be easy to apply very much.It is the high-tech biological preparation of a kind of brand-new, broad-spectrum, non-antibiotic prevention diarrhea of weaned piglets and baby pig edema.This receptor blocker high-tech biological preparation itself does not have any antibiotics resistant gene screening sign, thereby can not cause existence and the propagation of any antibiotics resistance gene at pathogen and animal body.
Other advantage of the present invention and effect will continue to describe below.
Description of drawings
The V-type excretory system MisL β recombinant vector system constructing figure of Fig. 1---functional surface secreting, expressing FedF and FaeG subunit gene.
The FedF of Fig. 2---pig source advantage probiotics strain vector bacterium EP2 surface secreting, expressing and FaeG subunit system constructing and functional exhibition thereof are detection of expression figure.A. recombinant DNA technology relates to ColE
The ori replication origin, PnirB promoter, LTB signal peptide, clone, the evaluation of V-type excretory system MisL α linker+ β element.B. be template with F18ab/F18ac and K88ac escherichia coli large plasmid DNA, adopt high-fidelity PCR Kit amplification FedF or FaeG, clone and sequencing.
The design of graphics of Fig. 3---V-type excretory system MisL β-FedF and FaeG recombiant plasmid MisL β-FedF and FaeG.
Fig. 4---the pig source functional exhibition of advantage probiotics strain vector bacterium EP2 bacterial strain is the F18FedF subunit (a) of surperficial secreting, expressing and the combination figure of K88FaeG subunit (b) and intestine of young pigs surface epithelial cell receptor.
The specific embodiment
As Fig. 1, Fig. 2, Fig. 3, shown in Figure 4:
Receptor blocking agent is effective and functional surface secreting, expressing F18FedF subunit and K88FaeG subunit, i.e. the probiotics of adhesin F18 and K88 receptor binding domains reorganization bacterium combination series, and it is prepared as:
1. at first, from the normal mucous membrane of small intestine material separation of piglet, evaluation pig source escherichia coli probiotics strain and pig source advantage lactobacillus probiotics bacterium, further detect by the intestine of young pigs field planting of oral back, proliferation test bacterial population in external adherence test of piglet intestinal epithelial cell and the body, filter out strong to the intestinal epithelial cell adhesion property, easily in intestine of young pigs field planting, the pig source advantage escherichia coli probiotics carrier bacterium of settling down and breeding, code name is EP2, with pig source advantage lactobacillus probiotics bacterial strain, code name is LP2, and preserves respectively;
2. choose pig source advantage escherichia coli probiotics strain vector bacterium EP2, by conventional antibacterial culturing of escherichia coli and propagation; Choosing the advantage lactobacillus probiotics strain of pig source is LP2, presses conventional cultivation of lactobacillus and propagation;
3. pig source advantage escherichia coli probiotics strain vector bacterium EP2 strain chromosome ASD deletion mutation strain makes up.Through the DNA genetic modification, promptly utilize bacteriophage lambda Red recombinase system and homologous recombination technique, the aspartic acid semialdehyde dehydrogenase house-keeping gene that lacks on its strain chromosome is the ASD encoding gene, construct the advantage escherichia coli probiotics strain vector bacterium ASD deletion mutation strain of pig source, code name is EP2 Δ ASD, through the growth in vitro characteristic, the intestine of young pigs field planting, settle down and the proliferation test bacterial population detects and relatively, EP2 Δ ASD deletion mutation strain and EP2 be in the growth in vitro characteristic, the intestine of young pigs field planting, settle down with multiplication characteristic on basically identical;
4. amplification, evaluation FedF and FaeG subunit gene.According to FedF and the known sequence of delivering of FaeG gene, with F18ab, F18ac and K88ac advantage serotype escherichia coli large plasmid DNA is template, adopt high-fidelity PCR Kit increase above-mentioned subunit gene FedF and FaeG, the PCR fragment inserting clone carrier pGEM-T that further 3 ' end is had A, preparation transforms engineering bacteria DH5 à competence antibacterial, through blue white macula screening, extraction contains the segmental recombiant plasmid pGEM-T of purpose DNA from the cultivation bacterium liquid of screening, the sequencing checking;
5. the V-type excretory system MisL β recombinant vector system constructing of surperficial secreting, expressing FedF and FaeG subunit gene.DNA recombinant technique operation routinely, with the secretion expression carrier MisL β of coding FedF and FaeG subunit gene and V-type excretory system respectively after the digestion of restricted enzyme NheI enzyme action, being connected, transform engineering bacteria DH5 à competence antibacterial respectively, from the cultivation bacterium liquid of screening, extract and contain the segmental positive plasmid DNA of purpose, PCR detects, in conjunction with the plain agglutination positive of bacterial adhesion and sequencing checking result, screen the V-type excretory system MisL β recombinant vector of energy functional surface secreting, expressing FedF and FaeG subunit;
6. the aspartic acid semialdehyde dehydrogenase house-keeping gene on evaluation, the clones coding escherichia coli chromosome is the complete encoding gene of ASD.According to the known sequence of delivering of ASD gene, with engineering bacteria DH5 à chromosomal DNA is template, adopt long template PCR method amplification ASD complete genome, making nucleic acid molecular hybridization detects, obtain the complete encoding gene of ASD at last, about 2Kb through specific restricted enzyme PstI enzyme action digestion binding nucleotide sequence analysis;
7. the MisL β recombinant vector system constructing of surperficial secreting, expressing FedF and FaeG adhesin subunit gene and ASD encoding gene.The MisL β recombinant vector of surperficial secreting, expressing FedF and FaeG adhesin subunit gene in the step 5, operate through recombinant DNA technology, insert the multiple clone site zone of ASD encoding gene MisL β recombinant vector in step 5, inactivation the resistant gene activity on the former MisL β carrier, in engineering bacteria DH5 à ASD deletion mutation strain DH5 à Δ ASD indicator cells, filter out to contain and express ASD encoding gene and surperficial secreting, expressing FedF, the MisL β recombinant vector of FaeG adhesin subunit gene, exist for the screening sign with ASD and substitute the antibiotics resistant gene screening sign, and the penbritin resistant gene screening of having dispeled on the MisL β original vector indicates;
8. pig source advantage escherichia coli probiotics strain vector bacterium ASD deletion mutation strain is that EP2 Δ ASD makes up.Through the DNA genetic modification, promptly utilize bacteriophage lambda Red recombinase system and homologous recombination technique, aspartic acid semialdehyde dehydrogenase house-keeping gene on the disappearance EP2 strain chromosome is the ASD encoding gene, constructing the advantage escherichia coli probiotics strain vector bacterium ASD deletion mutation strain of pig source is EP2 Δ ASD, through the growth in vitro characteristic, the intestine of young pigs field planting, settle down and proliferation test detects and relatively, EP2 Δ ASD deletion mutation strain and pig source advantage escherichia coli probiotics strain EP2 bacterium be in the growth in vitro characteristic, the intestine of young pigs field planting, settle down with multiplication characteristic on basically identical;
9. escherichia coli advantage probiotics EP2 Δ ASD reorganization bacterium makes up and screening.Surperficial secreting, expressing FedF in the extraction step 7, the MisL β recombinant plasmid vector DNA of FaeG adhesin subunit gene and ASD encoding gene, in the DNA conversion method imports pig source advantage escherichia coli probiotics carrier bacterium ASD deletion mutation strain EP2 Δ ASD respectively, from the cultivation bacterium liquid of screening, extract and contain the segmental positive recombinant plasmid dna of purpose, in conjunction with the plain agglutination of bacterial adhesion, external adherence test of piglet intestinal epithelial cell and intestine of young pigs field planting test bacterial population detect positive findings, filter out FedF and the effective and functional exhibition of two kinds of adhesin subunits of FaeG to be surperficial secreting, expressing the epithelial EP2 Δ of intestine of young pigs ASD reorganization bacterium;
Pig source advantage lactobacillus probiotics bacterial strain can effectively promote pig source advantage escherichia coli probiotics reorganization bacterium and EP2 Δ ASD reorganization bacterium in the field planting of intestine of young pigs, settle down and proliferation test.Choose EP2 respectively, EP2 Δ ASD, EP2 and LP2, four groups of probiotics reorganization of EP2 Δ ASD and LP2 bacterium, it is oral to be used for an age in days piglet, detect relatively through intestine of young pigs field planting test and bacterial population, pig source advantage lactobacillus probiotics bacterial strain LP2 can effectively promote pig source advantage escherichia coli probiotics carrier bacterium EP2 or EP2 Δ ASD reorganization bacterium in the field planting of intestine of young pigs, settle down and breed;
11. receptor blocking agent preparation of product.In the picking step 9 in pig source advantage escherichia coli probiotics reorganization bacterium EP2 Δ ASD and the step 1 the single bacterium colony of pig source advantage lactobacillus LP2 at common LB meat soup or 18 hours bacterium liquid of 37 ℃ of incubated overnight of conventional M17 culture medium as seed liquor, distinguish amplification culture, propagation 16～18 hours again, through conventional bacterial colony count, with colony forming units cfu tolerance, concentration reaches 2～6 * 10
9Cfu, reorganization bacterium EP2 Δ ASD, reorganization bacterium EP2 Δ ASD and LP2 amplification culture bacterium liquid mixed configuration form probiotics ASD deletion mutation strain reorganization fungus strain row.
The receptor blocking agent product that obtains is a probiotics reorganization bacterium combination series, and this probiotics reorganization bacterium combination series is 2 boar source advantage probiotics bacterial strains, comprises pig source advantage escherichia coli probiotics reorganization bacterium and pig source advantage lactobacillus probiotics bacterial strain.Advantage escherichia coli probiotics reorganization bacterium includes secreting, expressing F18 or Fed adhesin subunit gene FedF in the pig source, the V-type excretory system MisL β recombinant vector of K88 or F4 or Fae adhesin subunit gene FaeG, wherein adhesin F18 is divided into F18ab and F18ac2 serotype antigen mutation, be to encode by F18 enterotoxigenic escherichia coli wild type large plasmid DNA, F18ab plasmid size 48 and 98Mu between, and the big plasmid of F18ac is about 98Mu.5 gene fellowships of one group of operon are finished in the expression of adhesin Fed and the big plasmon of synthetic needs, they are respectively FedA, B, C, E and F, wherein the FedF subunit is a F18 escherichia coli adhesin receptor binding domains, F18ab FedF and F18ac FedF are the common functionality adhesin of high conservative in conjunction with identical piglet enterocyte surface macromole receptor; Adhesin K88 is divided into K88ab, K88ac, 3 kinds of serotype antigen mutation of K88ad, be to encode by K88 enterotoxigenic escherichia coli wild type large plasmid DNA, by FaeA to FaeJ totally 10 genes participate in the biosynthesis of its pili adhesin, wherein the FaeG subunit is for being K88 escherichia coli adhesin receptor binding domains, in conjunction with piglet enterocyte surface macromole receptor, be the functional adhesin of high conservative.
Detect through the serology agglutination, the receptor blocking agent product that is developed into can be distinguished coagulation rabbit or mouse-anti F18ab, F18ac and K88 adhesin subunit FedF and FaeG antibody, macroscopic agglutination is tired and is reached more than 1: 10, detect through the external adherence test of piglet intestinal epithelial cell, receptor blocking agent has the intestinal epithelial cell adhesive capacity strong, adherence test detects in the animal body, be used for an age in days piglet the earliest, can be after piglet is oral in intestinal field planting in 4 hours the earliest, settle down, propagation is in the functional exhibition of intestine of young pigs surface epithelial cell then and expresses Fed adhesin subunit FedF and Fae adhesin subunit gene FaeG.Because adhesin subunit FedF and FaeG energy specificity are in conjunction with the macromole receptor that is present in piglet intestinal epithelial cell surface, when the receptor blocking agent product uses behind piglet, field planting, propagation is at the piglet intestinal epithelial cell and further at its surface-functional secreting, expressing FedF and Fae adhesin subunit, the at first preferential and intestine of young pigs surface epithelial cell receptors bind of its effect of the adhesin subunit of these two kinds of functional expressions, thereby sealed the bonded same target site of ETEC pathogen of intestinal epithelial cell surface macromole receptor and F18 and the mediation of K88 adhesin, thereby the first step that has lost F18 and K88 enterotoxigenic escherichia coli pathogenic bacterial infection body also is the probability of essential step the most, and it infects adhesin and the combination of the corresponding specific receptor target site of host cell surface promptly to lose pathogen.After piglet is used the receptor blocking agent product, sealed the piglet under the macromole receptor situation of intestinal epithelial cell surface, be equal to the special receptor that does not have above-mentioned two kinds of pathogen in the body, even outside contamination has a large amount of pathogen, because pathogen has lost the target site point of infection animal, can not infect again, even piglet body constitution is weak, resistance is poor, can infection morbidity yet.
Among the present invention, the preparation method of receptor blocking agent since adopted to the intestinal epithelial cell adhesive capacity by force, easily can be in intestine of young pigs field planting, the pig source advantage probiotics strain combinations series of settling down and breeding, its functional secreting, expressing FedF and Fae adhesin subunit can remain in intestine of young pigs for a long time, long-time and the intestine of young pigs surface epithelial cell receptors bind of preferential also energy, anti-infectious function is lasting.Owing to contain FedF subunit and FaeG subunit in the preparation method of receptor blocking agent; be respectively F18 escherichia coli adhesin receptor binding domains and K88 escherichia coli adhesin receptor binding domains; in conjunction with piglet enterocyte surface macromole receptor; thereby stop extraneous pathogen not attack to infect the intestinal stepping of going forward side by side to be gone in the animal body; different fully with the efficacy effect stage of conventional antibiotic medicine and vaccine; the latter is opposing; suppress; in and attacked the ETEC pathogen of infection in the body; the protection piglet does not fall ill; both relatively; receptor blocking agent can be the most direct; sealing/prevention intestine of young pigs surface epithelial cell receptor combines with the target site of the enterotoxigenic escherichia coli pathogen of F18 and the mediation of K88 adhesin most effectively, is used for the direct of anti-F18 and K88 enterotoxigenic escherichia coli infection clinically; high-efficiency prevention and control.Receptor blocking agent can be used for an age in days piglet the earliest, can be after oral in intestine of young pigs field planting in 4 hours the earliest, settle down, breed and seal the combining of enterotoxigenic escherichia coli pathogen of intestine of young pigs epithelial receptor and F18 and the mediation of K88 adhesin, make give birth to piglet in very short time owing to oral receptor blocking agent becomes non-susceptible piglet, be applied in the actual production, thereby can significantly improve prevention diarrhea of weaned piglets and baby pig edema effect.
Receptor blocking agent is as the application of prevention diarrhea of weaned piglets and edema disease of piglets medicine:
1) pig source advantage escherichia coli probiotics strain vector bacterium EP2, through the DNA genetic modification, aspartic acid semialdehyde dehydrogenase house-keeping gene (ASD encoding gene) disappearance on its strain chromosome, construct the advantage escherichia coli probiotics strain vector bacterium ASD deletion mutation strain of pig source, through the growth in vitro characteristic, the intestine of young pigs field planting, settle down and proliferation test detects and relatively, EP2 Δ ASD deletion mutation strain and pig source advantage escherichia coli probiotics strain EP2 basically identical on above-mentioned characteristic;
2) surperficial secreting, expressing FedF, the MisL β recombinant vector of FaeG adhesin subunit gene and ASD encoding gene, operate through recombinant DNA technology, insert the ASD encoding gene in the multiple clone site zone of the MisL β recombinant vector of surperficial secreting, expressing FedF and FaeG adhesin subunit gene, inactivation the resistant gene activity on the former MisL β carrier, in engineering bacteria DH5 à ASD deletion mutation strain DH5 à Δ ASD indicator cells, filter out to contain and express ASD encoding gene and surperficial secreting, expressing FedF, the MisL β recombinant vector of FaeG adhesin subunit gene, exist for the screening sign with ASD and substitute the antibiotics resistant gene screening sign, and the penbritin resistant gene screening of having dispeled on the MisL β original vector indicates;
3) above-mentioned probiotics reorganization bacterium is the chromosome of selection pressure based on non-antibiotic resistance (ASD encoding gene)---the carrier strain construction of plasmid balanced lethal system, itself do not have any antibiotics resistant gene screening sign, thereby can not cause of existence and the propagation of any antibiotics resistance gene at pathogen and animal body.
4) pig source advantage lactobacillus probiotics LP2 bacterial strain can effectively promote pig source advantage escherichia coli probiotics EP2 and ASD deletion mutation strain EP2 Δ ASD reorganization bacterium in the field planting of intestine of young pigs, settle down and breed, can be in the very fast field planting of intestine of young pigs, settle down, breed.
5) above-mentioned receptor blocking agent can be used for an age in days piglet the earliest, can be behind the oral 2ml, settle down, breed in intestine of young pigs field planting in 4 hours the earliest, thereby can significantly improve prevention diarrhea of weaned piglets and baby pig edema generation effect.
10 of one age in days piglets orally use 2ml receptor blocking agent product of the present invention; the oral once more 2ml of 15 ages in days; during to 35 ages in days; with F18 and K88 enterotoxigenic escherichia coli pathogen artificial challenge; do not have a piglet generation baby pig edema and symptom of diarrhea occurs, the counteracting toxic substances protective rate reaches 100%.Select original every nest piglet that 3 pig farms of diarrhea of weaned piglets and 3 pig farms of baby pig edema all take place usually, use receptor blocking agent product of the present invention after, diarrhea of weaned piglets and baby pig edema no longer take place.Former suffer from the diarrhea of weaned piglets piglet, even adopt conventional treatments is oral or the injection composite antibiotic, 5～7 days courses of treatment, general treatment poor effect, most anti-piglet of curing that crosses, causing gastrointestinal tract and Abwehrkraft des Koepers descends, easy secondary or concurrent multiple disease, it is slower to grow, and the price of deed is higher, fertility performance also can influence, thereby productivity effect obviously reduces.Because receptor blocking agent of the present invention is a kind of brand-new, non-antibiotic prevention diarrhea of weaned piglets and baby pig edema strategy and approach, and after using receptor blocking agent product of the present invention, be expected in the treatment of diarrhea of weaned piglets and baby pig edema and prevention, to significantly reduce or do not have antibiotic to use, expecting that also receptor blocking agent of the present invention substitutes gene engineering vaccine and conventional vaccine uses.
Claims (3)
1. prevent the receptor blocking agent of diarrhea of weaned piglets and baby pig edema, it is characterized in that receptor blocking agent is a probiotics reorganization bacterium combination series, this probiotics reorganization fungus strain is classified 2 boar source advantage probiotics bacterial strains as, wherein a kind is pig source advantage escherichia coli probiotics reorganization bacterium EP2, the 2nd kind is pig source advantage lactobacillus probiotics bacterial strain LP2, pig source advantage escherichia coli probiotics EP2 reorganization bacterium includes surperficial secreting, expressing F18 or Fed adhesin subunit gene FedF, the MisL β recombinant vector of K88 or F4 or Fae adhesin subunit gene FaeG, wherein adhesin F18 is divided into F18ab and 2 serotype antigen mutation of F18ac, be to encode by F18 enterotoxigenic escherichia coli wild type large plasmid DNA, F18ab plasmid size 48 and 98Mu between, and the big plasmid of F18ac is about 98Mu.5 gene fellowships of one group of operon are finished in the expression of adhesin Fed and the big plasmon of synthetic needs, they are respectively FedA, B, C, E and F, wherein the FedF subunit is a F18 escherichia coli adhesin receptor binding domains, F18ab FedF and F18ab FedF are the common functionality adhesin of high conservative in conjunction with identical piglet enterocyte surface macromole receptor; Adhesin K88 is divided into K88ab, K88ac, 3 kinds of serotype antigen mutation of K88ad, be to encode by K88 enterotoxigenic escherichia coli wild type large plasmid DNA, by FaeA to FaeJ totally 10 genes participate in the biosynthesis of its pili adhesin, wherein the FaeG subunit is for being K88 escherichia coli adhesin receptor binding domains, in conjunction with piglet enterocyte surface macromole receptor, be the functional adhesin of high conservative.
2. the preparation method of receptor blocking agent according to claim 1, its step is as follows:
(1) pig source advantage escherichia coli probiotics strain vector bacterium EP2 separates from the normal mucous membrane of small intestine of piglet with pig source advantage lactobacillus probiotics bacterium LP2, the antibacterial method is separated, is identified routinely, meet escherichia coli and lactobacillus form, cultivation and biological general characteristic, further detect by the intestine of young pigs field planting of oral back, proliferation test bacterial population in the external adherence test of piglet intestinal epithelial cell and the body, filter out to the intestinal epithelial cell adhesive capacity by force, easily can be in the intestine of young pigs field planting, settle down and EP2 and two kinds of antibacterials of LP2 of breeding and preserve;
(2) choose pig source advantage escherichia coli probiotics strain vector bacterium EP2,, choose pig source advantage lactobacillus probiotics bacterium LP2, press conventional cultivation of lactobacillus and propagation by conventional antibacterial culturing of escherichia coli and propagation;
(3) amplification, evaluation FedF and FaeG subunit gene, according to FedF and the known sequence of delivering of FaeG gene, with F18ab, F18ac and K88ac advantage serotype escherichia coli large plasmid DNA is template, adopt high-fidelity PCR Kit increase above-mentioned subunit gene FedF and FaeG, the PCR fragment inserting clone carrier pGEM-T that further 3 ' end is had A, preparation transforms engineering bacteria DH5 à competence antibacterial, through blue white macula screening, from the cultivation bacterium liquid of screening, extract and contain the segmental recombiant plasmid pGEM-T of purpose DNA, the sequencing checking;
(4) the V-type excretory system MisL β recombinant vector system constructing of surperficial secreting, expressing FedF and FaeG subunit gene, DNA recombinant technique operation routinely, FedF will encode, the secretion expression carrier MisL β of FaeG subunit gene and V-type excretory system digests through restricted enzyme NheI enzyme action respectively, after the connection, transform engineering bacteria DH5 à competence antibacterial respectively, from the cultivation bacterium liquid of screening, extract and close the segmental positive plasmid DNA of purpose, PCR detects, in conjunction with the plain agglutination positive of bacterial adhesion and sequencing checking result, screen the V-type excretory system MisL β recombinant vector of energy functional surface secreting, expressing FedF and FaeG subunit;
(5) the aspartic acid semialdehyde dehydrogenase house-keeping gene on evaluation, the clones coding escherichia coli chromosome is the complete encoding gene of ASD: according to the known sequence of delivering of said gene, with engineering bacteria DH5 à chromosomal DNA is template, adopt long range PCR method amplification ASD complete genome, making nucleic acid molecular hybridization detects, obtain the complete encoding gene of ASD at last, about 2Kb size through specific restricted enzyme PstI enzyme action digestion binding nucleotide sequence analysis;
(6) the MisL β recombinant vector system constructing of surperficial secreting, expressing FedF, FaeG adhesin subunit gene and ASD encoding gene.The MisL β recombinant vector of surperficial secreting, expressing FedF and FaeG adhesin subunit gene in the step 5, operate through recombinant DNA technology, insert the multiple clone site zone of ASD encoding gene MisL β recombinant vector in step 5, inactivation the resistant gene activity on the former MisL β carrier, in engineering bacteria DH5 à ASD deletion mutation strain DH5 à Δ ASD indicator cells, filter out to contain and express ASD encoding gene and surperficial secreting, expressing FedF, the MisL β recombinant vector of FaeG adhesin subunit gene, exist for the screening sign with ASD and substitute the antibiotics resistant gene screening sign, and the penbritin resistant gene screening of having dispeled on the MisL β original vector indicates;
(7) pig source advantage escherichia coli probiotics EP2 reorganization bacterium makes up and screening, extraction contains surperficial secreting, expressing FedF, the MisL β recombinant plasmid vector DNA of FaeG adhesin subunit gene and ASD encoding gene, in the DNA conversion method imports pig source advantage escherichia coli probiotics carrier bacterium EP2 respectively, from the cultivation bacterium liquid of screening, extract and contain the segmental positive recombinant plasmid dna of purpose, in conjunction with the plain agglutination of bacterial adhesion, the external adherence test of piglet intestinal epithelial cell and intestine of young pigs field planting test bacterial population detects positive findings, filter out FedF and the effective and functional exhibition of two kinds of adhesin subunits of FaeG can be with secreting, expressing on the epithelial EP2 of the intestine of young pigs bacterium surface of recombinating;
(8) receptor blocking agent preparation of product: pig source advantage escherichia coli probiotics EP2 reorganization bacterium and the single bacterium colony of pig source advantage lactobacillus LP2 in the picking step (7), at 18 hours bacterium liquid of 37 ℃ of incubated overnight as seed liquor, distinguish amplification culture, propagation 16～18 hours again, through conventional bacterial colony count, with colony forming units cfu tolerance, concentration reaches 2～6 * 10
9Cfu, reorganization bacterium EP2 and LP2 amplification culture bacterium liquid mixed configuration form probiotics reorganization fungus strain row.
3. receptor blocking agent according to claim 1 is characterized in that as the application of prevention diarrhea of weaned piglets and edema disease of piglets medicine:
1) pig source advantage escherichia coli probiotics strain vector bacterium EP2, through the DNA genetic modification, promptly utilize bacteriophage lambda Red recombinase system and homologous recombination technique, the aspartic acid semialdehyde dehydrogenase house-keeping gene that lacks on its strain chromosome is the ASD encoding gene, constructs pig source advantage escherichia coli probiotics strain vector bacterium ASD deletion mutation strain EP2 Δ ASD;
2) surperficial secreting, expressing FedF, the MisL β recombinant vector of FaeG adhesin subunit gene and ASD encoding gene, operate through recombinant DNA technology, insert the ASD encoding gene in the multiple clone site zone of the MisL β recombinant vector of surperficial secreting, expressing FedF and FaeG adhesin subunit gene, inactivation the resistant gene activity on the former MisL β carrier, in engineering bacteria DH5 à ASD deletion mutation strain DH5 à Δ ASD indicator cells, filter out to contain and express ASD encoding gene and surperficial secreting, expressing FedF, the MisL β recombinant vector of FaeG adhesin subunit gene, exist for the screening sign with ASD and substitute the antibiotics resistant gene screening sign, and the penbritin resistant gene screening of having dispeled on the MisL β original vector indicates;
3) pig source advantage escherichia coli probiotics EP2 reorganization bacterium is the chromosome of selection pressure based on ASD non-antibiotic resistance---the carrier strain construction of plasmid balanced lethal system, and its system itself does not have any antibiotics resistant gene screening sign;
4) pig source advantage lactobacillus probiotics bacterial strain LP2 can effectively promote pig source advantage escherichia coli probiotics EP2 reorganization bacterium and its ASD deletion mutation strain EP2 Δ ASD reorganization bacterium in the field planting of intestine of young pigs, settle down and breed;
5) the receptor blocking agent product is used for an age in days piglet, and oral 2ml can be in the intestine of young pigs field planting after 4 hours, settle down, breed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101914679A CN101199556A (en) | 2007-12-19 | 2007-12-19 | Recipient beta-blocker of prevention weaning piggy diarrhea and piggy oedematous disease, preparing and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101914679A CN101199556A (en) | 2007-12-19 | 2007-12-19 | Recipient beta-blocker of prevention weaning piggy diarrhea and piggy oedematous disease, preparing and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101199556A true CN101199556A (en) | 2008-06-18 |
Family
ID=39515036
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007101914679A Pending CN101199556A (en) | 2007-12-19 | 2007-12-19 | Recipient beta-blocker of prevention weaning piggy diarrhea and piggy oedematous disease, preparing and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101199556A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532052B (en) * | 2008-12-11 | 2010-11-24 | 江西农业大学 | Susceptible/resistant MUC13 molecular marker capable of identifying F4 weaning brash of piglets and application |
| CN102031262A (en) * | 2010-08-17 | 2011-04-27 | 浙江省农业科学院 | Construction method and application of faeG expressing gene engineering probiotic |
| CN109223888A (en) * | 2018-09-04 | 2019-01-18 | 华中农业大学 | Inhibit the fae of enterotoxigenic escherichia coli K88 pili, the Traditional Chinese medicine composition of faeG gene expression and application |
| CN109468256A (en) * | 2018-11-27 | 2019-03-15 | 扬州大学 | Probiotics clone strain, the construction method of four copy F18 pili operon gene of integration and double copy F4 pili operon genes |
| CN109628361A (en) * | 2018-11-27 | 2019-04-16 | 扬州大学 | Integrate double copy function F4 pili operon gene pig sources probiotics EP1 clone strain, construction method and application |
| CN114752531A (en) * | 2022-04-29 | 2022-07-15 | 扬州大学 | Swine-derived escherichia coli nontoxic isolate capable of simultaneously expressing F4 and F18 pili and application thereof |
-
2007
- 2007-12-19 CN CNA2007101914679A patent/CN101199556A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532052B (en) * | 2008-12-11 | 2010-11-24 | 江西农业大学 | Susceptible/resistant MUC13 molecular marker capable of identifying F4 weaning brash of piglets and application |
| CN102031262A (en) * | 2010-08-17 | 2011-04-27 | 浙江省农业科学院 | Construction method and application of faeG expressing gene engineering probiotic |
| CN109223888A (en) * | 2018-09-04 | 2019-01-18 | 华中农业大学 | Inhibit the fae of enterotoxigenic escherichia coli K88 pili, the Traditional Chinese medicine composition of faeG gene expression and application |
| CN109223888B (en) * | 2018-09-04 | 2021-09-10 | 华中农业大学 | Fae and faeG gene expression traditional Chinese medicine composition for inhibiting enterotoxigenic escherichia coli K88 fimbriae and application |
| CN109468256A (en) * | 2018-11-27 | 2019-03-15 | 扬州大学 | Probiotics clone strain, the construction method of four copy F18 pili operon gene of integration and double copy F4 pili operon genes |
| CN109628361A (en) * | 2018-11-27 | 2019-04-16 | 扬州大学 | Integrate double copy function F4 pili operon gene pig sources probiotics EP1 clone strain, construction method and application |
| CN109628361B (en) * | 2018-11-27 | 2022-08-19 | 扬州大学 | Integrated double-copy functional F4 pilus operon gene pig-derived probiotic EP1 clone strain, construction method and application |
| CN114752531A (en) * | 2022-04-29 | 2022-07-15 | 扬州大学 | Swine-derived escherichia coli nontoxic isolate capable of simultaneously expressing F4 and F18 pili and application thereof |
| CN114752531B (en) * | 2022-04-29 | 2023-05-26 | 扬州大学 | Pig-derived escherichia coli nontoxic isolate capable of simultaneously expressing F4 and F18 fimbriae and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cousin et al. | Detection and genomic characterization of motility in Lactobacillus curvatus: confirmation of motility in a species outside the Lactobacillus salivarius clade | |
| CN101199556A (en) | Recipient beta-blocker of prevention weaning piggy diarrhea and piggy oedematous disease, preparing and application | |
| CN107858317B (en) | Attenuated live vaccine for preventing and controlling aeromonas hemorrhagic disease of aquaculture animals | |
| JP2004532628A (en) | Transporting disease control in aquaculture and agriculture using microorganisms containing bioactive proteins | |
| US9775867B2 (en) | Non-pathogenic F18 E. coli strain and use thereof | |
| CN108753650B (en) | Enterococcus faecium and composite microecological preparation prepared from enterococcus faecium | |
| CN101880647B (en) | Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application | |
| CN102031262A (en) | Construction method and application of faeG expressing gene engineering probiotic | |
| CN103275228A (en) | K99-987P-F41 recombinant protein and application thereof | |
| CN109468256B (en) | Probiotic clone strain integrating four-copy F18 pilus operon gene and double-copy F4 pilus operon gene and construction method | |
| KR20110124976A (en) | Fermented white rice by clostridium butyricum idcc 9207 having antibacterial activity and immunostimulatory activity | |
| CN101204404A (en) | RA preventing and curing for piglet yellow scour, preparation and application thereof | |
| Li et al. | Evaluation of the immunogenicity of auxotrophic Lactobacillus with CRISPR-Cas9D10A system-mediated chromosomal editing to express porcine rotavirus capsid protein VP4 | |
| CN1195062C (en) | Non-antibiotics resistant shuttle plasmid expression carrier and its constructing method and uses | |
| CN106148376A (en) | A kind of K88ac+enterotoxigenic escherichia coli weakening strain construction method and application | |
| WO2014108497A2 (en) | Vaccine | |
| CN109504643B (en) | Probiotic clone strain integrating four-copy functional F18 pilus operon gene, construction method and application | |
| CN109628361B (en) | Integrated double-copy functional F4 pilus operon gene pig-derived probiotic EP1 clone strain, construction method and application | |
| CN102363762A (en) | Modified bacillus source and Alpha-amylase gene recombination lactobacillus as well as product and application thereof | |
| CN113174354A (en) | Recombinant lactobacillus reuteri, and preparation method, preparation and application thereof | |
| Jiao et al. | Immunization effect of recombinant Lactobacillus casei displaying Aeromonas veronii Aha1 with an LTB adjuvant in carp | |
| CN105524158A (en) | Fly maggot antibacterial peptide, encoding gene, carrier, engineering bacteria and application thereof | |
| CN101892189B (en) | Transformant for blocking effect of porcine somatostatin by oral immunization and application thereof | |
| CN101525589A (en) | Recombinant lactobacillus casei for expressing infectious pancreas necrosis virus (IPNV) VP3 protein and preparation method | |
| CN101812424A (en) | Thymine auxotrophic bacillus anthracis and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Open date: 20080618 |