CN113980102A - Refined yolk antibody of Escherichia coli K99 strain and preparation method thereof - Google Patents
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Abstract
A refined yolk antibody of Escherichia coli K99 strain and its preparation method are provided. The invention relates to a method for purifying cilium antigen of Escherichia coli K99 strain and reducing endotoxin, a preparation method of refined yolk antibody of Escherichia coli K99 strain, and the administration dose and the administration method of the refined yolk antibody of Escherichia coli K99 strain for treating calf diarrhea.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of a low-toxin cilia antigen of an escherichia coli K99 strain and a preparation method of a refined yolk antibody of an escherichia coli K99 strain, in particular to a dosing dose and a dosing method of calf diarrhea of the refined yolk antibody of an escherichia coli K99 strain.
Background
Escherichia coli (Escherichia coli) exists in various forms. One is normal bacteria parasitizing in animal intestinal tract and coexisting with organism, and the other is pathogenic colibacillus showing certain pathogenicity to cause various diseases. Pathogenic E.coli generally causes diarrhea in animals, sepsis occurs when the infection is severe, and the damage to young animals is large in particular. In a large-scale cow or beef cattle farm, colibacillosis of calves is a common bacterial infectious disease, symptoms such as diarrhea and emaciation mainly appear after calves are attacked, and if severe septicemia occurs, the mortality rate is high.
Pathogenic Escherichia coli infects animal body, releases enterotoxin in intestinal tract to cause calf diarrhea, and Escherichia coli producing enterotoxin is classified into K88, K99, 987P and F18 according to cilium serum type. Pathogenic escherichia coli breaks through the natural immune barrier of an animal body through dense cilia on the surface of the thallus, enters the intestinal tract, is combined with a cilia receptor of an intestinal mucosa epithelial cell, and infects a host. After the escherichia coli is infected, intestinal peristalsis resistance and content cleaning are generated, nutrient substances of organisms are utilized for reproduction, enterotoxin is released, and balance imbalance of intracellular water and electrolyte is caused, so that watery diarrhea is generated. It can be seen that cilia are important infection vectors for pathogenic processes and are also important bacterial antigen parts in traditional vaccine research. The cilium antigen has good immunogenicity, can generate corresponding cilium antibodies after being immunized by an organism, and has good immune protection effect on enterotoxigenic pathogenic escherichia coli.
Pathogenic escherichia coli has many serotypes, which brings difficulty to the prevention of the disease, antibiotics are generally selected for treatment, and drug resistance is easy to occur in the treatment process of the antibiotics, or ecological disturbance of intestinal beneficial flora of calves is caused to aggravate diarrhea. Under the current conditions that antibiotic use is severely limited, the prevention and treatment of colibacillosis in calves requires alternative approaches to find antibiotics. For example, Chinese patents CN101450206A and CN103509838 are used for preventing yellow scour of piglets by preparing ciliated subunit vaccines of Escherichia coli K88, K99 and 987P. Chinese patent CN104017060A provides an extraction method of Escherichia coli pilus antigen, so that the method can be used for preparing yolk antibody, and provides a thought for preventing and treating related diseases. The oral yolk antibody is a main development trend, and has good application in treating diarrhea caused by escherichia coli of poultry and piglets at present. Originally, colibacillosis in poultry and livestock is obvious in morbidity, with the gradual increase and scale of culture scale, intensive captive breeding still occupies the dominant position, calf diarrhea caused by escherichia coli is increased year by year, the prevention and control of the calf diarrhea needs to be carried out with targeted prevention and control, whether the egg yolk antibody of the escherichia coli can be applied to the treatment of the calf diarrhea or not is discussed at present, but the method cannot be industrialized, and the treatment effect is not obvious as that of poultry and other livestock.
Disclosure of Invention
The invention provides a preparation method of a high-titer refined yolk antibody aiming at Escherichia coli K99 strain, further provides a drug administration dosage and a treatment and use method for treating calf diarrhea caused by Escherichia coli, and obtains favorable industrialized clinical curative effect and stable titer of the preparation.
Firstly, the invention provides a preparation method of low-toxin cilia antigen of Escherichia coli K99 strain.
In the process of preparing cilium antigen, breaking thalli, extracting cilium and reducing endotoxin released by thalli are key factors for preparing the cilium antigen.
In order to solve the problems, the invention adopts the following technical scheme:
the preparation method of the low-toxin cilia antigen of the Escherichia coli K99 strain comprises the following steps: (1) obtaining a primary cilium antigen by a thermal cracking method after culturing by using an improved Minca culture medium, (2) removing endotoxin by multiple treatments in a way of triton and centrifugation, wherein the content of the endotoxin is not higher than 1000EU/ml (limulus reagent detection method), and the titer of the cilium antigen is kept at 1:1280 or more, thus obtaining the cilium antigen.
The dosage of the triton is 0.5-2.5%, preferably 1.0-1.5%. The centrifugation rotation speed is preferably 8000rpm to 20000rpm, more preferably 8000rpm to 15000rpm, still more preferably 8000rpm to 10000rpm for 20 to 60 minutes, preferably 20 to 30 minutes. If the treatment times are not more than 4 times, for example, the treatment times are more than 4 times, the macromolecular protein is destroyed after the treatment, so that the molecular structure of the antigen is inactivated, and the titer is reduced.
Further, the invention provides a method for inoculating the cilium antigen immunogen of the Escherichia coli K99 strain, which comprises the following steps: (1) inactivating cilium antigen, and adding a white oil adjuvant to prepare an immunogen; (2) inoculating 200-240 days old commercial laying hens in a high-yield period with the immunogen, carrying out secondary immunization 28-30 days after the primary immunization, (3) carrying out boosting immunization of escherichia coli K99 cilium antigen (without adjuvant) 14-21 days after the secondary immunization, and (4) collecting eggs 10-14 days after the boosting immunization to prepare the yolk antibody.
In the immunization scheme of the invention, the immunogen for strengthening immunity is changed into cilium antigen solution from immunogen of the oil adjuvant, so that the immunostimulation reaction of the immunologic adjuvant can be reduced, and the response reaction of the organism for the immunological memory of the immunogen is enhanced.
Furthermore, the invention provides a preparation method of the refined yolk antibody of the Escherichia coli K99 strain, which comprises the following steps:
(1) collecting yolk, adding an acetate buffer solution according to 6-10 times of the volume of the yolk, and carrying out acidification treatment;
(2) centrifuging the acidified yolk mixed solution, collecting supernatant, and adding caprylic acid for defatting overnight;
(3) collecting supernatant of the yolk mixed solution subjected to degreasing overnight, concentrating, and filtering and sterilizing by using a filter membrane.
(4) Adding adjuvant such as 20% skimmed milk at a ratio of 1:1, and lyophilizing to obtain yolk antibody of Escherichia coli K99 strain. The antibody titer is 1: 256.
Compared with the prior art, the invention has the beneficial effects that:
the cilium antigen of Escherichia coli K99 strain provided by the invention has endotoxin content of 1000EU/ml or below, and the immunogen prepared by using the cilium antigen does not influence the laying rate after being inoculated to a commercial laying hen in a high-yield period. Provides qualified immunogen for subsequent multiple immunization of commercial laying hens.
The invention provides a dosing dose and a dosing method for treating calf diarrhea caused by escherichia coli by using an escherichia coli K99 strain yolk antibody.
The therapeutic effect of the refined yolk antibody is equivalent to that of antibiotics, the injection way is changed into a feeding way which is more convenient to operate, and the dosage is low (the dosage is 1:64 of the yolk antibody titer of Escherichia coli K99, the yolk antibody is fed by spraying through an oral way and is continuously used for 3 days); and compared with the yolk antibody prepared by the existing Escherichia coli mixed strain, the yolk antibody has obvious cure advantages. The stronger the relevance to the strains causing calf diarrhea is, the better the treatment effect is, and the use of the mixed strains reduces the treatment pertinence instead.
Detailed Description
The invention is further illustrated by the following examples.
Example 1 preparation of E.coli K99 cilium antigen
1.1 propagation and identification of first-class seeds of Escherichia coli K99 strain: respectively streaking and inoculating the preserved freeze-dried strains to an improved Minca agar plate, culturing at 37 ℃ for 24h, selecting a plurality of smooth and round single bacterial colonies, respectively carrying out plate agglutination reaction by using K99 factor serum, selecting 10 bacterial colonies with agglutination reaction '+++++' from each bacterial strain, inoculating to an improved Minca liquid culture medium, carrying out shake culture at 37 ℃ for 24h, respectively sampling for pure inspection, and using corresponding cilium antigen single factor serum as plate agglutination reaction. And after pure inspection is qualified, adding 10% of glycerol, uniformly mixing, and subpackaging in small sterilized tubes to serve as first-class seeds.
1.2 propagation and identification of secondary seeds of Escherichia coli K99 strain: taking first-class seeds of the large-field bacilli, inoculating improved Minca agar plates, culturing at 37 ℃ for 24h, selecting a plurality of smooth and round single colonies, respectively using K99 factor serum to perform plate agglutination reaction, selecting 10 colonies with agglutination reaction '++++++' from each strain, inoculating improved Minca liquid culture medium, performing shaking culture at 37 ℃ for 18h, respectively sampling and purely detecting, using corresponding cilium antigen single factor serum as plate agglutination reaction, taking the obtained product as second-class seeds after purely detecting is qualified, and storing at 2-8 ℃.
1.3 culture of fermentation tank: escherichia coli K99 was cultured in a fermenter with aeration. Adding 60% modified Minca liquid culture medium into fermentation tank, adding defoaming agent according to 0.1% (V/V) of culture medium volume, pressurizing at 115 deg.C for 30min, cooling to 37 deg.C,inoculating the secondary seed liquid according to the proportion of 5 percent of the total amount of the culture medium, culturing at 37 ℃ at 400r/min with automatic aeration and dissolved oxygen set to be 50 percent and continuously aerating; NaOH solution is automatically added to maintain the pH value to be 7.0; glucose 15g/L (70g) and tryptone 10g/L (56g) are supplemented according to the oxygen consumption speed, and the culture is carried out for 8h for harvest. The bacterial concentration can reach 2.0 × 1010CFU/ml, while cilium Reverse Indirect Hemagglutination (RIHA) unit can reach 2560-5120, sampling for purity and agglutination test, and requiring that the purity test is qualified and the agglutination should reach strong positive reaction (++++).
1.4 cilia antigen preparation (thermal cracking) the harvested antigen was centrifuged at 8000rpm for 20min to harvest bacterial sludge. Suspending with 0.01mol/L pH7.2PBS according to 10 times volume of bacterial sludge, placing in triangular flask containing glass beads, heating in water bath at 62 deg.C and 180rpm for 30min to release cilium antigen in liquid, and passing through 10000rpm and 30min to obtain supernatant as cilium antigen.
1.5 removing endotoxin, adding 1% by volume of triton into the supernatant, stirring overnight at 2-8 ℃, putting in a 39-DEG water bath for temperature return, and centrifuging for 30min at 12000 rpm; carefully sucking the upper liquid layer, and repeating the operation for 4 times; the treated protein solution is aseptically filtered through a 0.22 μm filter membrane, and the endotoxin content is not higher than 1000EU/ml and not lower than 1280 (cilia Reverse Indirect Hemagglutination (RIHA) unit) in detection.
Example 2 preparation of purified yolk antibody against cilium antigen of Escherichia coli K99
2.1 preparation of immunogens
2.1.1 inactivation of cilia antigen A cilia solution qualified in the test was taken, added with a formaldehyde solution of final concentration of 0.1%, shaken up, and inactivated hermetically at 37 ℃ for 24 hours (during which shaking up was carried out 1 time every 4-6 hours).
2.1.2 inactivated antigen test sterile test and growth were performed according to the appendix of the current "Chinese veterinary pharmacopoeia".
2.1.3 immunogen preparation
2.1.3.1 preparation of oil phase 94 parts of white oil for injection are heated, span-806 parts are added, and the mixture is fully and uniformly mixed and sterilized under high pressure for standby.
2.1.3.2 preparation of aqueous phase 96 parts of certified cilia antigen were added to 4 parts of sterile tween-80 and shaken thoroughly to dissolve tween-80 completely.
2.1.3.3 emulsifying, introducing 2 parts of oil phase into an emulsifying tank, starting a motor to stir slowly, then slowly adding 1 part of water phase, and emulsifying for 30 minutes at 3200r/min after adding. And (4) emulsifying, and quantitatively subpackaging.
2.2 production of animals for yolk antibody: selecting commercial laying hens with production performance, wherein the commercial laying hens are 200-240 days old; before inoculation, avian leukemia virus antibodies and avian reticuloendotheliosis virus antibodies are detected by 0.5 percent sampling ELISA and are all negative; the chicken is infected without pullorum disease and mycoplasma infection.
2.3 inoculation procedure
2.3.1 basic immunization neck subcutaneous injection Escherichia coli K99 cilia antigen prepared oil emulsion immunogen, 1.0 ml/.
2.2.2 boosting immunization is carried out 28-30 days after basic immunization, and 1.0ml of immunogen is injected into the neck part subcutaneously.
2.2.3 enhanced immunopotentiation was carried out 14 to 21 days after the previous immunization, and Escherichia coli K99 strain cilium antigen (without adjuvant) was injected subcutaneously into the neck at a dose of 1.0 ml/mouse.
When the titer of Escherichia coli K99 in the yolk of the high-immunity egg is lower than 1:512, the egg is qualified, and the egg can be collected and stored at 10-15 ℃.
2.4 preparation and test of refined yolk antibody of Escherichia coli K99 strain
2.4.1 preparation method of purified yolk antibody of Escherichia coli K99 strain
(1) Sterilizing the surface of the egg with 75% alcohol, manually removing egg white, blastoderm and frenulum with an egg white and yolk separator, and collecting yolk.
(2) And (3) shaking and crushing the separated yolk, adding acetate buffer solution (0.12mol/L, pH value 5.0) which is 6 times of the volume of the yolk, uniformly mixing, adjusting the pH value to 5.2, stirring for 2 hours on a magnetic stirrer, and standing at 2-8 ℃ overnight after the completion.
(3) Centrifuging the acidified egg yolk mixed solution for 30min at 10000r/min, pouring out supernatant, slowly adding n-octanoic acid until the final concentration is 1.0-1.5%, and storing at 2-8 ℃ overnight.
(4) And (3) sucking out the supernatant of the lower layer of the yolk mixed liquid after ester removal, filtering the supernatant by using gauze, filtering the filtrate by using a 100K membrane pack for 4-5 times, concentrating the filtrate, and filtering and sterilizing the concentrated filtrate by using a 0.22 micron filter membrane.
(5) And (3) freeze-drying the yolk antibody concentrated solution by using 20% skim milk as an adjuvant according to the ratio of the yolk antibody solution to the adjuvant of 1:1, wherein the titer of the yolk antibody of the escherichia coli K99 strain is not lower than 1:256 after freeze-drying.
2.4.2 detection of yolk antibody titer lyophilized samples were dissolved in 5ml of PBS, 50ul of PBS (0.01mol/L, pH7.2) was added to each well of a 96-well V-type microplate, 50ul of each of the serum to be detected, the positive control serum and the negative serum was added to each well of the first row, and the wells were diluted 2-fold to the 11 th row, 50ul of the mixture was aspirated from each well of the 11 th row and discarded. Wells in row 12 were blank without serum, and 2 replicates of each sample were made; 50ul of antigen was added to each well, mixed well with a micro-shaker, covered with a membrane, and incubated at 37 ℃ for 4 hours. And (5) determining the titer of the serum to be detected. The yolk antibody titer of the lyophilized escherichia coli K99 is 1: 256.
Example 3 minimum dose of egg yolk antibody for treatment of E.coli induced calf diarrhea
1.1 treatment protocol of diarrheal calves with yolk antibodies: the calf with obvious diarrhea symptoms is divided into 7 groups of 8-10 calves with the age of 6-21 days. The yolk antibody was dissolved in the indicated physiological saline at a titer of 1:256 in 10 ml/bottle. Wherein:
8 test cattle in 1 group are sprayed and fed for 1 bottle/head by oral route, and the use lasts for 3 days;
9 test cattle in 2 groups are fed by spraying 1/2 bottles/heads through an oral route for 3 days;
10 test cattle in 3 groups are fed into 1/4 bottles/heads by spraying through an oral route for 3 days;
4 groups of test cattle are 10 heads, and are sprayed and fed to 1/8 bottles/heads by oral route for 3 days;
9 cattle in 5 groups, commercial ceftiofur sodium (recommended dose), injected for 3 days;
6 groups of test cattle are 8 heads, and the test cattle are sprayed and fed with 1 bottle/head (mixed strain egg yolk antibody) by an oral route for 3 days;
10 control groups were kept in the same group.
3.1 determination of stress test cattle after the egg yolk antibody administration treatment, the cattle are continuously observed for 6 hours, and whether the cattle have symptoms of physical discomfort, lassitude, decreased eating willingness, tremor, syncope and the like after the administration is observed. The results showed that no clinical symptoms such as significant physical discomfort appeared after 5 groups of administration groups were observed for 6 hours, and no clinical symptoms such as significant physical discomfort appeared in the test cattle after 3 days of continuous use. The components of the yolk antibody have no influence on the test cattle. The results are shown in Table 1.
TABLE 1 stress Observation of dosed cattle at different times
3.2 therapeutic effect on calf diarrhea test the calf feces were observed daily for 14 days after oral spray treatment. The feces of each cow was scored according to the following criteria, and diarrhea was judged by a feces score of 3 or more. The scores are shown in Table 2.
TABLE 2 stool Scoring standards
After the diarrhea calves are treated by egg yolk antibody oral spray for three consecutive days, the observation is continuously carried out for 14 days, the diarrhea calves show that the watery and ointment feces are changed to be soft and normal on the 5 th day after the administration, most feces of the diarrhea calves recover to be normal from the 7 th day to the 10 th day, and the diarrhea calves which are not recovered on the 14 th day still have continuous diarrhea. The results are shown in tables 3 to 7.
TABLE 3 therapeutic Effect of Calf diarrhea (before treatment)
TABLE 4 therapeutic Effect of Calf diarrhea (5 days after administration)
TABLE 5 therapeutic Effect of Calf diarrhea (7 days after administration)
TABLE 6 therapeutic Effect of Calf diarrhea (10 days after administration)
TABLE 7 therapeutic Effect of Calf diarrhea (14 days after administration)
3.4 conclusion
3.4.1 after the egg yolk antibody is administrated to the diarrhea calves, refined egg yolk antibody with 1:64 titer (3 groups) is continuously administrated for 3 days, and after 14 continuous observation days, the calf feces of 9/10 can be recovered to be normal in 7-10 days, no obvious diarrhea symptom exists, and most of the control group ceftiofur sodium injection group is recovered to be normal. After 14 days of observation, some of the diarrheal calves were not recovered (except for the control group), possibly in relation to the dose administered, or possibly in relation to the cause of the diarrhoea, caused by non-pathogenic E.coli.
3.4.2 after the egg yolk antibody was administered to the diarrhea calves, after 3 days of continuous administration of purified egg yolk antibodies (4 groups) having a titer of 1:32, the feces of the diarrhea calves of only 6/10 recovered to normal and did not achieve the expected therapeutic effect for 14 days of continuous observation.
Therefore, the minimum administration dose of the feed for calf diarrhea caused by pathogenic Escherichia coli is determined to be Escherichia coli K99 strain yolk antibody titer 1:64, and the feed is sprayed by an oral route and continuously used for 3 days.
3.4.3 after the egg yolk antibody is administered to the calf with diarrhea, the refined egg yolk antibody with 1:64 titer (3 groups with 90% cure rate) is used to be superior to the mixed egg yolk antibody (6 groups with 75% cure rate) in the treatment effect, and the high-titer egg yolk antibody has more obvious treatment effect along with the increase of the administration dosage, and is probably superior to the universality of the mixed strain along with the treatment specificity of the Escherichia coli K99 with a single factor to the calf diarrhea.
Claims (8)
1. A method for preparing low-toxin cilia antigen of Escherichia coli K99 strain, comprising the following steps: (1) performing thermal cracking after culturing by using an improved Minca culture medium to obtain a primary cilium antigen, (2) performing multiple treatment by using a triton and centrifugation mode to remove endotoxin of the cilium antigen in the step (1), wherein the content of the endotoxin is not higher than 1000EU/ml (limulus reagent detection method), and the titer of the cilium antigen is kept at 1:1280 or more, so as to obtain the cilium antigen.
2. The method for preparing low-toxin cilia antigen of Escherichia coli K99 strain according to claim 1, wherein the cilia antigen is treated 4 times by means of triton and centrifugation.
3. The method for preparing low-toxin cilia antigen of Escherichia coli K99 strain according to claim 1 or 2, wherein the amount of triton is 0.5% -2.5%.
4. A method for inoculating a cilium antigen immunogen of escherichia coli strain K99, comprising the steps of: (1) inactivating cilium antigen, and adding a white oil adjuvant to prepare an immunogen; (2) inoculating 200-240 days old commercial laying hens in a high-yield period with the immunogen, carrying out secondary immunization 28-30 days after the primary immunization, (3) carrying out enhanced immunization on escherichia coli K99 cilium antigen 14-21 days after the secondary immunization, and (4) collecting eggs 10-14 days after the enhanced immunization to carry out preparation of egg yolk antibodies.
5. The method for inoculating ciliated antigen immunogen of Escherichia coli K99 according to claim 4, wherein the immunogen for boosting immunity is ciliated antigen solution (without adjuvant).
6. A preparation method of a refined yolk antibody of Escherichia coli K99 strain comprises the following steps:
(1) collecting yolk, adding an acetate buffer solution according to 6-10 times of the volume of the yolk, and carrying out acidification treatment;
(2) centrifuging the acidified yolk mixed solution, collecting supernatant, and adding caprylic acid for defatting overnight;
(3) collecting supernatant of the yolk mixed solution subjected to degreasing overnight, concentrating, and filtering and sterilizing by using a filter membrane.
(4) Adding adjuvant such as 20% skimmed milk at a ratio of 1:1, and lyophilizing to obtain yolk antibody of Escherichia coli K99 strain with titer of 1: 256.
7. A purified yolk antibody of Escherichia coli K99, which is obtained by the method of claims 1-6.
8. A therapeutic administration dose and an administration method for diarrhea in calves, which are obtained by feeding a yolk antibody having a titer of 1:64 to diarrhea calves through oral spraying by the purified yolk antibody according to claim 7 for 3 consecutive days.
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- 2021-11-08 CN CN202111315054.3A patent/CN113980102A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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