CN107118275B

CN107118275B - Fish IL-1 β -resistant egg yolk antibody and preparation method thereof

Info

Publication number: CN107118275B
Application number: CN201710530673.1A
Authority: CN
Inventors: 宋学宏; 孙丙耀; 郭培红; 徐乐; 吴康; 肖兰莹; 汤健
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2017-07-03
Filing date: 2017-07-03
Publication date: 2020-06-12
Anticipated expiration: 2037-07-03
Also published as: CN107118275A

Abstract

The invention provides a fish IL-1 β egg yolk antibody capable of effectively preventing and treating fish inflammation and a preparation method thereof, wherein grass carp IL-1 β recombinant protein is added into sterilized medical white oil containing span 80 accounting for 2-6% of volume percentage and aluminum stearate accounting for 1-4% of mass percentage, and is subjected to high-speed shearing emulsification for more than 10min to prepare grass carp IL-1 β immune antigen with 0.5-2 mg/ml of antigenic protein.

Description

Fish IL-1 β -resistant egg yolk antibody and preparation method thereof

Technical Field

The invention belongs to the technical field of animal feed additives and preparation methods thereof, and particularly relates to a fish IL-1 β -resistant egg yolk antibody and a preparation method thereof.

Background

Under the current intensive high-density culture mode, cultured fishes are very easily infected by bacteria, viruses and parasites, generally cause inflammatory reaction, generate various diseases, cause mass death and cause huge economic loss to fishery in China. Meanwhile, in the present day where food safety is concerned by people, many problems such as drug-resistant strains, drug residues, environmental pollution and the like caused by disease control means mainly comprising drugs such as chemical agents, antibiotics and the like are more and more attracting attention of people. Therefore, the search for disease control measures which accord with the strategies of environmental friendliness, aquatic product quality safety and sustainable development becomes the main direction of the development of the aquaculture industry.

The egg yolk antibody is also called egg yolk immunoglobulin (IgY), is a specific antibody formed by transferring chicken serum IgG into egg yolk, has the immunological activity similar to that of mammal IgG, and can effectively prevent and treat specific diseases. The IgY has the advantages of stable chemical property, strong specificity, simple preparation, low cost, greenness, safety and the like, and is widely applied to the aspects of food, medicine, biological products and the like.

However, in the research reports at home and abroad, most of the antibodies are prepared by taking inactivated particle antigens such as viruses, bacteria and cells as immunogens, and the selection of specific strains or viruses becomes a restrictive problem under the condition of uncertain etiology, thus presenting great hysteresis; and pathogenic bacteria are easy to generate variation, so that the treatment and prevention effects of the yolk antibody are easy to reduce, and the phenomenon is particularly obvious in the current livestock and poultry aquaculture practice. Therefore, endogenous factors or active substances causing diseases of the cultured animals are searched, and the endogenous factors or the active substances are used as antigens to produce the yolk antibody or the compound preparation, which is more effective.

IL-1 β is an endogenous pyrogen source in the body and can modulate many inflammatory responses by triggering and boosting immune and inflammatory responses, playing an important role in cell proliferation, differentiation and some immune processes specific to immune cells (Smith DE, Renshaw BR, Ketchem RR, et al. four new cell expansion of the interconnected cell-1 superior [ J ] Biol Chem,2000,275(2): 1169-75.).

The inventor discloses that grass carp IL-1 β (BoYX, Song XH, Wu K, et. Characterisation of intercleukin-1 β as a protiflamation cytokinase in grasscarp) is cloned and prokaryotically expressed at the early stage of the subject group, the cDNA full length of grass carp IL-1 β gene is 1260bp (GenBank accession number: JN705663.2), the gene comprises a 813bp open reading frame, encodes a polypeptide with the length of 270 amino acids, the predicted molecular weight is 30.1kDa, the amino acid sequence has an IL-1 family characteristic sequence, but the gene lacks a typical IL-1 convertase cleavage site conserved in mammalians, IL-1 β is expressed in grass carp bodies, the expression of IL-1 family characteristic sequences, but the gene lacks a mammalians IL-1 convertase cleavage site is highly effective, the gene has a gene sequence inhibiting effect in the bacterial growth of other Fish IL-1 proteins, the gene of the protein is highly effective in the research of bacterial growth of other Fish, the research of the Fish, the bacterial resistance of the bacterial growth of grass carp is shown by comparison of the research of the amino acid sequence of the gene of the grass carp IL-1 gene of the rat, the gene of the rat IL-1 gene is similar to the rat, the gene of the rat, the rat IL-1 gene is marked polypeptide, the rat is marked by the rat, the rat is the rat, the rat is the rat, the rat is the rat, the rat is the rat, the rat is the rat, the rat is the rat, the rat is the rat.

Disclosure of Invention

The invention provides an anti-fish IL-1 β egg yolk antibody, a preparation method and application thereof, aiming at the current situation that cultured fishes are easily infected by bacteria, viruses, parasites and the like to cause inflammatory reaction and generate various diseases, thereby reducing the growth performance of the fishes and the reward of feed.

The IL-1 β recombinant protein selected by the invention is a protein obtained by assembling a nucleotide sequence of 813bp open reading frame of grass carp IL-1 β gene (GenBank accession number: JN705663.2) into pET32a (+) -IL-1 β plasmid which is expressed in BL21 strain and is induced and expressed by IPTG (isopropyl thiogalactoside), the sequence table of the used grass carp IL-1 β open reading frame is shown in figure 1, and the preparation and purification method of the grass carp IL-1 β recombinant protein is as follows:

transferring 2uL pET32a (+) -IL-1 β plasmid into Escherichia coli BL21 expression bacteria, culturing overnight at 37 ℃ in a biochemical incubator, respectively picking up a single colony to 100mL LB liquid medium (containing 100. mu.g/mL ampicillin sodium, MESGEN, CAS: 69-52-3), transferring into a fermenter (containing 75. mu.g/mL ampicillin sodium) the next day according to the inoculation amount of 1:100 (inoculating 4 h), adding IPTG (isopropyl thiogalactoside, MESGEN, CAS: 367-93-1) to the final concentration of 0.6mmol/L, inducing overnight at 28 ℃, centrifuging to collect bacterial liquid after induction, washing 2 times with PBS (pH7.4), removing the surface culture medium of bacteria, detecting the expression of recombinant protein by CAS: 367-93-1, dissolving BL21 induced to express recombinant protein of grass carp IL-1 β in a high pressure cell crusher (PBS, suspending the cells for 2 times, crushing proteins by a Mildy pressure instrument, precipitating proteins by a limited PBS-PAGE, precipitating at 12000 min, precipitating proteins by a high pressure precipitation system (30 ℃ and 120-13 ℃ C.) to obtain a purified protein.

On the basis, the invention further provides an anti-fish IL-1 β yolk antibody capable of effectively preventing and treating fish inflammation and a preparation method thereof, and concretely comprises,

the invention adopts the technical scheme that an anti-fish IL-1 β yolk antibody and a preparation method thereof comprise the following steps:

preferably, the anti-fish IL-1 β yolk antibody and the preparation method thereof comprise the following steps of (1) adding grass carp inflammatory cytokine IL-1 β recombinant protein into medicinal white oil containing span 80 and aluminum stearate, and preparing grass carp IL-1 β immune antigen by high-speed shearing and emulsification, (2) immunizing laying hens with the prepared grass carp IL-1 β immune antigen, and then collecting eggs, (3) washing, micro-coating, pasteurizing and spray drying the eggs to prepare the anti-inflammatory yolk antibody of the anti-fish IL-1 β.

Further, specifically, the anti-fish IL-1 β yolk antibody capable of effectively preventing and treating fish inflammation and the preparation method thereof of the invention comprise:

preferably, in the preparation method, the volume percentage of span 80 (also called span 80 or span 80, as sorbitan fatty acid ester) in the pharmaceutical white oil is 2-6% based on the volume of the raw white oil, and the mass percentage of aluminum stearate is 1-4% based on the mass of the raw white oil, and preferably, in the preparation method, the volume percentage of tween 80 in the purified grass carp IL-1 β recombinant protein is 4-8% based on the total volume of the recombinant protein and tween 80.

Preferably, in the preparation method, the IL-1 β recombinant protein and the white oil adjuvant are sufficiently sheared and emulsified at high speed according to the volume part of 1: 1-2 to prepare a water-in-oil emulsion vaccine for the immunization of laying hens.

Preferably, in the preparation method, the content of the prepared water-in-oil emulsion grass carp IL-1 β recombinant protein is 0.5-2 mg/ml.

Preferably, the recombinant IL-1 β water-in-oil emulsion vaccine is used for immunizing laying hens twice, and eggs laid two weeks after the second immunization are collected.

Preferably, in the preparation method, the coating agent in the wall material of the micro-coating comprises modified starch, β -cyclodextrin, sodium alginate, gelatin, chitosan and carboxymethyl cellulose, and the coating agent is used singly or compositely, and the addition amount of the coating agent is 3-10% of the weight of the fresh egg.

Preferably, in the preparation method, the micro-occlusion film comprises the following 7 steps: (1) washing eggs: and (3) removing deteriorated bad eggs, putting the fresh qualified eggs on an egg washer, and washing with normal-temperature water for 1-2 min. (2) And (3) disinfection: preparing 2.5X 10 by normal temp. running water^-5Sterilizing cleaned eggs for 1-2 min, (3) spraying sterilized eggs with clean tap water for 1-2 min, removing disinfectant, (4) beating eggs, breaking the cleaned eggs with a beating machine, putting whole eggs or egg liquid containing broken eggshells into a clean stainless steel emulsification tank, (5) mixing and emulsifying with a coating agent, namely, taking modified starch which accounts for 3-10% of fresh weight of the eggs, or β -cyclodextrin, or sodium alginate, or gelatin, or chitosan, or carboxypropyl methyl cellulose, or a compound of the above substances, slowly putting the mixture into clean water of 40-60 ℃ for stirring and homogenizing, wherein the weight of the clean water is 10-20% of the weight of the egg liquid, continuously heating to be semitransparent, fully mixing the coating liquid with the egg liquid, emulsifying for 5-20 min, (6) sterilizing, pouring the above substances into a sterilization tank, sterilizing at 60-65 ℃ for 15-30 min, drying (7), spraying the sterilized egg liquid with a spray pump, spraying an anti-inflammatory agent at a temperature of 3500-60 ℃ or a centrifugal drying tower, and drying the anti-inflammatory agent at a temperature of 3500-60 ℃ to obtain anti-IL antibody particles.

The invention also provides application of the fish IL-1 β resistant egg yolk antibody, and the inventor discovers through a large number of experiments that the fish IL-1 β resistant egg yolk antibody can be used as a feed additive, the working efficiency of the antibody is high after the fish is fed with the antibody, and the antibody can prevent fish inflammatory diseases in a targeted manner and promote growth at an oral dosage of 2.0-4.0 g/kg.

The invention has the technical advantages that:

(1) the anti-fish IL-1 β egg yolk antibody provided by the invention can be used for inhibiting the inflammation of fish caused by various stimuli (pathogenic organisms such as bacteria, toxic and harmful substances, environmental factors and the like) in a targeted manner, effectively improving the immunity and disease resistance of the fish and improving the feed return rate.

(2) Compared with a serum antibody preparation method, the anti-fish IL-1 β egg yolk antibody has high antibody yield, maintains the antibody activity for a long time at normal temperature, and realizes the effect which is difficult to realize in the prior art.

(3) The micro-envelope technology adopted by the invention, namely the egg yolk antibody obtained by adding wall materials, homogenizing and emulsifying and spray drying, not only maintains the original flavor of eggs, but also has a certain food calling effect, and overcomes the problem that the traditional oral antibody usually has a special taste and has poor flavor; the micro-envelope yolk antibody passes through the stomach from the oral cavity and then enters the small intestine to play a role of slow release, thereby protecting the activity of the antibody and other components in the egg and overcoming the limitation that other antibodies in the prior art need to be administrated in a special way; compared with the existing yolk antibody, the invention overcomes the technical problem that the antibody is lost by gastrointestinal tracts, and the efficiency of orally taking the antibody is obviously improved.

(4) The micro-envelope wall material adopted by the invention is a food-grade wall material, and the micro-envelope wall material has the functions of nutrition and promotion of animal immune function.

(5) The fish IL-1 β -resistant egg yolk antibody is prepared by micro-coating and spray drying, and the antibody protein is wrapped in the membrane, so that the moisture content is only 2-5%, and various reactions caused by direct contact with air and other components can be effectively avoided, thereby overcoming the defects that self nutrition and active substances in the antibody and eggs are easy to oxidize, deteriorate and inactivate, and prolonging the quality guarantee period.

Drawings

FIG. 1 shows the sequence of open reading frame of grass carp IL-1 β gene, the open reading frame is 813 base sequence.

FIG. 2 shows the titer of anti-IL-1 β yolk antibody in fish, in which the A-H columns are yolk antibody groups with dilution times of 1:800,1:1600,1:3200,1:6400,1:12800,1:25600, 1:51200 and 1:102400, the I column is blank control group, the J-L groups are common egg powder groups with dilution times of 1:80,1:40 and 1: 20.

FIG. 3 is a Western blot for identifying the specificity of the anti-grass carp IL-1 β egg yolk antibody and the homology of grass carp and loach IL-1 β genes.

The method comprises the steps of A, treating only with a yolk antibody against grass carp IL-1 β, B, pre-combining recombinant grass carp IL-1 β protein with a yolk antibody against grass carp IL-1 β, treating, wherein

lanes

1 and 3 are loach intestinal cell lysates,

lanes

2 and 4 are grass carp intestinal cell lysates, ① is 1 splicer of loaches and grass carp IL-1 β, ② is the other splicer of loaches and grass carp IL-1 β, and ③ is β -actin of loaches and grass carps.

FIGS. 4 to 11 are schematic views showing the intestinal structure of grass carp observed by scanning electron microscope after feeding grass carp with anti-fish IL-1 β yolk antibody of the present invention, wherein FIGS. 4 to 5 are blank control groups showing X100 times and X20000 times of intestinal structure electron microscope images, FIGS. 6 to 7 are negative control groups showing X100 times and X20000 times of intestinal structure electron microscope images, respectively, FIGS. 8 to 9 are 0.15% addition groups showing X100 times and X20000 times of intestinal structure electron microscope images, respectively, and FIGS. 10 to 11 are 0.3% addition groups showing X100 times and X20000 times of intestinal structure electron microscope images, respectively.

FIGS. 12 to 21 show intestinal microvilli structures of Paramisgurnus dabryanus observed by a scanning electron microscope after Paramisgurnus dabryanus is fed with the anti-fish IL-1 β egg yolk antibody of the present invention, wherein FIGS. 12 to 13 are control groups showing X20000 times and X30000 times of structural electron micrographs, FIGS. 14 to 15 are 1.0g/kg addition groups showing X20000 times and X30000 times of structural electron micrographs, FIGS. 16 to 17 are 2.0g/kg addition groups showing X20000 times and X30000 times of structural electron micrographs, FIGS. 18 to 19 are 4.0g/kg addition groups showing X20000 times and X30000 times of structural electron micrographs, respectively, and FIGS. 20 to 21 are 6.0g/kg addition groups showing X20000 times and X30000 times of structural micrographs, respectively.

Detailed Description

It is specifically noted that all such similar substitutes and modifications which would be apparent to those skilled in the art are deemed to be incorporated herein by reference for the purpose of describing the invention, are deemed to be encompassed by the present invention.

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

EXAMPLE 1 preparation of grass carp recombinant IL-1 β Immunity vaccine

(1) Preparing an adjuvant: taking 500ml adjuvant preparation as an example, firstly adding a certain amount of imported pharmaceutical grade white oil (Acros, Belgium, CAS: 8012-95-1) into a beaker, adding 20ml of span 80 (national pharmaceutical group chemical reagent Co., Ltd., chemical pure CP, CAS: 1338-43-8) and 20g of aluminum stearate (national pharmaceutical group chemical reagent Co., Ltd., chemical pure CP, CAS: 300-92-5), stirring properly, heating to no bubble, fixing the volume to 500ml with heated white oil (electric furnace heating in the beaker until no bubble is formed), sterilizing at 121 ℃ for 30min, and then using. The prepared adjuvant is brownish yellow, clear and bright.

(2) Pretreatment of grass carp IL-1 β recombinant protein, adding 4% Tween 80 (chemical purity CP, CAS number: 9005-65-6) into purified grass carp IL-1 β recombinant protein, and mixing.

(3) And (2) preparing an IL-1 β vaccine, namely slowly adding the grass carp IL-1 β recombinant protein obtained in the step (2) into the vaccine adjuvant prepared in the step (1), stirring and adding the recombinant protein, wherein the volume ratio of the protein to the adjuvant is 1:1, the concentration of the vaccine protein is 2.0mg/ml, mixing and emulsifying the recombinant protein by using a high-speed emulsifying shearing machine (Zhongshi (Shanghai) mechanical Co., Ltd., model: ZLE-A300), 7000r/min and 10min to prepare the water-in-oil emulsion vaccine for immunization of laying hens.

EXAMPLE 2 preparation of grass carp recombinant IL-1 β Immunity vaccine

(1) Preparing an adjuvant: taking 500ml adjuvant preparation as an example, firstly adding a certain amount of imported pharmaceutical grade white oil (Acros, Belgium, CAS: 8012-95-1) into a beaker, adding 10ml of span 80 (national pharmaceutical group chemical reagent Co., Ltd., chemical pure CP, CAS: 1338-43-8) and 20g of aluminum stearate (national pharmaceutical group chemical reagent Co., Ltd., chemical pure CP, CAS: 300-92-5), stirring properly, heating to no bubble, fixing the volume to 500ml with heated white oil (electric furnace heating in the beaker until no bubble is formed), sterilizing at 121 ℃ for 30min, and then using. The prepared adjuvant is brownish yellow, clear and bright.

(2) Pretreatment of grass carp IL-1 β recombinant protein, adding 8% Tween 80 (chemical purity CP, CAS: 9005-65-6) into purified grass carp IL-1 β recombinant protein, and mixing.

(3) And (2) preparing an IL-1 β vaccine, namely slowly adding the grass carp IL-1 β recombinant protein obtained in the step (2) into the vaccine adjuvant prepared in the step (1), stirring and adding the recombinant protein, wherein the volume ratio of the protein to the adjuvant is 1:2, the concentration of the vaccine protein is 1.0mg/ml, mixing and emulsifying the recombinant protein by using a high-speed emulsifying shearing machine (Zhongshi (Shanghai) mechanical Co., Ltd., model: ZLE-A300), 7000r/min and 10min to prepare the water-in-oil emulsion vaccine for immunization of laying hens.

Example 3 preparation of grass carp I L-1 β Immunity vaccine

(1) Preparing an adjuvant: taking 500ml adjuvant preparation as an example, firstly adding a certain amount of imported pharmaceutical grade white oil (Acros, Belgium, CAS: 8012-95-1) into a beaker, adding 20ml of span 80 (national pharmaceutical group chemical reagent Co., Ltd., chemical pure CP, CAS: 1338-43-8) and 10g of aluminum stearate (national pharmaceutical group chemical reagent Co., Ltd., chemical pure CP, CAS: 300-92-5), stirring properly, heating to no bubble, fixing the volume to 500ml with heated white oil (electric furnace heating in the beaker until no bubble is formed), sterilizing at 121 ℃ for 30min, and then using. The prepared adjuvant is brownish yellow, clear and bright.

(2) Pretreatment of grass carp IL-1 β recombinant protein, adding 6% Tween 80 (chemical purity CP, CAS: 9005-65-6) into purified grass carp IL-1 β recombinant protein, and mixing.

(3) And (2) preparing an IL-1 β vaccine, namely slowly adding the grass carp IL-1 β recombinant protein obtained in the step (2) into the vaccine adjuvant prepared in the step (1), stirring and adding the recombinant protein, wherein the volume ratio of the protein to the adjuvant is 1:1, the concentration of the vaccine protein is 1.0mg/ml, mixing and emulsifying the recombinant protein by using a high-speed emulsifying shearing machine (Zhongshi (Shanghai) mechanical Co., Ltd., model: ZLE-A300), 7000r/min and 10min to prepare the water-in-oil emulsion vaccine for immunization of laying hens.

Example 4 immunized laying hens

500 feathers of a healthy laying hen (a hailan hen, 110 days old) which is laying eggs and is free of specific pathogens and has high immune response capacity are selected as a test flock, and the feeding management is carried out according to the feeding management requirements of the haian hen laying period.IL-1 β vaccine prepared by the method described in example 1, example 2 and example 3 is used for immunizing the laying hen with the dose of 1.0 ml/feather, and the immunization is carried out by three needles at the subcutaneous part and two breast muscle parts of the neck, the immunization is carried out by the same dose and method after one week, and the antibody titer in the egg is spot-checked from the 10 th day after the second immunization, and then the immunization is strengthened by the same dose and method when the antibody titer is lower than 1: 6400.

Example 5 preparation of anti-Fish IL-1 β yolk antibody

(1) Collecting eggs with antibody titer of more than 1:6400 after the second immunization, removing deteriorated bad eggs, placing fresh qualified eggs on an egg washer, and washing with normal temperature water for 2 min.

(2) By 2.5X 10^-5Soaking the cleaned eggs in chlorine dioxide water solution with concentration for 1 minute; spraying sterilized egg with tap water for 2min, and removingA disinfectant.

(3) Breaking the disinfected and cleaned eggs, storing the eggs in a clean stainless steel container, primarily stirring the eggs, and weighing the eggs for later use.

(4) Weighing sodium alginate 1 wt%, β -cyclodextrin 2 wt% and carboxymethyl cellulose 3 wt%, adding into tap water at 60 deg.C 12 wt%, heating and stirring to obtain semitransparent solution, adding into egg liquid slowly while stirring.

(5) Transferring the above materials into an emulsification tank, homogenizing and emulsifying for 10 min.

(6) Transferring the homogenized and emulsified egg liquid into a pasteurization tank, and sterilizing at 60 deg.C for 30 min.

(7) And (3) sucking the sterilized materials into a centrifugal spray drying tower by using a pump, setting the air inlet temperature to be 100 ℃, setting the air outlet temperature to be 65 ℃, and carrying out spray drying to obtain the anti-fish IL-1 β egg yolk antibody.

Example 6 preparation of anti-Fish IL-1 β yolk antibody

(1) Collecting eggs with antibody titer of more than 1:6400 after the second immunization, removing deteriorated bad eggs, placing fresh qualified eggs on an egg washer, and washing with normal temperature water for 1 min.

(2) By 2.5X 10^-5Sterilizing cleaned egg with chlorine dioxide water solution for 1 min; spraying the sterilized eggs with tap water for 2min to remove disinfectant.

(3) Breaking the cleaned eggs, primarily stirring in a clean stainless steel container, and weighing for later use.

(4) Weighing chitosan 1 wt% of egg liquid and β -cyclodextrin 5 wt%, adding 10 wt% of egg liquid into tap water heated to 45 deg.C, stirring, heating to obtain semitransparent solution, and slowly adding into egg liquid while stirring.

(5) Transferring the above materials into an emulsification tank, homogenizing and emulsifying for 30 min.

(6) Transferring the homogenized and emulsified egg liquid into a pasteurization tank, and sterilizing at 65 deg.C for 15 min.

(7) And (3) sucking the homogenized material into a centrifugal spray drying tower by using a pump, setting the air inlet temperature to be 120 ℃, setting the air outlet temperature to be 70 ℃, and carrying out spray drying to obtain the anti-fish IL-1 β egg yolk antibody.

Example 7 preparation of anti-Fish IL-1 β yolk antibody

(4) Weighing modified starch 2% of egg liquid weight and gelatin 4%, adding tap water 18% of egg liquid weight, heating to 60 deg.C, stirring, and heating until the solution is semitransparent to obtain coating solution; and slowly adding the coating liquid into the egg liquid while stirring.

(5) Transferring the above materials into an emulsification tank, homogenizing and emulsifying for 20 min.

(6) Transferring the homogenized and emulsified egg liquid into a pasteurization tank, and sterilizing at 65 deg.C for 25 min.

(7) And (3) sucking the homogenized material into a centrifugal spray drying tower by using a pump, setting the air inlet temperature at 130 ℃ and the air outlet temperature at 60 ℃, and performing spray drying to obtain the anti-fish IL-1 β egg yolk antibody.

Example 8 antibody titer detection (Indirect ELISA method)

Taking purified grass carp IL-1 β recombinant protein (purified recombinant protein containing Tween 80 obtained by pretreatment in step (2) in examples 1-3) as antigen, taking common egg powder (common eggs laid in the same chicken farm at the same period and prepared by the same preparation method as in examples 5-7) of nonspecific IgY as negative control, and measuring titer of the prepared anti-fish IL-1 β yolk antibody by indirect ELISA method, diluting the purified grass carp IL-1 β recombinant protein to 10 μ g/ml with coating solution, adding into an enzyme-labeled reaction plate according to 100 μ l/well, coating overnight at 4 deg.C, and throwing off the coated enzyme-labeled plateAdding TBS containing 5% by volume of calf serum into TBS washing solution 300. mu.l/well, repeatedly washing 2 times, and patting dry on absorbent paper, adding TBS containing 5% by volume of calf serum 300. mu.l/well, incubating at 37 ℃ for 1.5 h.TBS repeatedly washing 2 times, patting dry, adding anti-IL-1 β yolk antibody obtained in example 3 (diluted with PBS at a ratio of 1:800,1:1600,1:3200,1:6400,1:12800,1:25600, 1:51200, 1: 102400), washing at 37 ℃ for 2h, washing at T-TBS 5 times, washing at TBS 2 times, patting dry, adding 100. mu.l/well of goat anti-horseradish peroxidase (HRP) labeled antibody (purchased from Sigma), incubating at 37 ℃ for 1.5h, washing at T-TBS 5 times, patting dry, adding o-phenylenediamine (OPD) to each well, incubating at 2M H min, and judging positive reaction according to Beijing 3 technical test of Beijing 3-Bekko₂SO4 stop solution 100 ul. is used enzyme labeling instrument (U.S. Bio-Tek) to determine the OD value of 492nm sample, each sample is 8 parallel, the average value is taken, the judgment standard is that the highest dilution with the OD value more than 2.1 times of the average value of the negative control is the titer, the result is shown in Table 1 and figure 2, the OD value when the negative control dilution multiple is more than 1:40 is more than 2.1 times of the blank control, the OD value when the anti-IL-1 β egg yolk antibody is more than 1:6400 is more than 2.1 times of the negative control, the result shows that the anti-fish IL-1 β egg yolk antibody can be specifically combined with the purified grass carp IL-1 β recombinant protein, the antibody combination titer is 1:6400, while the titer of the common egg powder antibody is almost zero, the degree difference reaches a remarkable degree, the specificity and the high efficiency of the antibody are shown, therefore, the anti-IL-1 β antibody prepared by the method has very high antibody activity.

TABLE 1 titer determination results of anti-fish IL-1 β yolk antibody

Example 9 evaluation of antibody specificity

By adopting a Western blotting method and using the fish IL-1 β -resistant egg yolk antibody provided by the invention as a primary antibody, the natural IL-1 β in immune tissues of grass carp and loach is identified and detectedThe specific method comprises the steps of respectively shearing, digesting and filtering intestinal tissues of the loaches and the grass carps, then separating the intestinal lymphocytes of the loaches and the grass carps by using 40% Percoll separating medium (Solarbio, density 1.131), and taking about 10 samples of each sample¹⁰Suspending cells of the order of magnitude with 10. mu.l PBS, adding an equal volume of 2 XSDS loading buffer, boiling in boiling water for 15min, centrifuging at 12000r/min for 15min, loading 30. mu.l of each well, performing SDS-PAGE gel electrophoresis, stopping electrophoresis when bromophenol blue migrates to the lower end of the gel by about 1cm, peeling off the gel, then (1) cutting a dry film and 6 filter papers according to the size of the gel, placing the gel in a membrane transfer buffer for 5min, washing the gel with ddH2O for several seconds, (2) placing the gel in a membrane transfer buffer, (3) placing a sponge pad on an electrode plate, stacking the gel, the NC film and the filter papers in sequence from the cathode to the anode, simultaneously exhausting bubbles between the layers with a glass rod, (4) installing a transfer device, turning on the power supply, 110mA, transferring in an ice water bath for about 4h, (5) removing the membrane, observing the membrane transfer efficiency, sealing the PBST 3 times, (6) placing the membrane in 2% Casn, rinsing with CAS 2min, rinsing the water, washing solution for about 4 min, washing the same color of the PBST 2-staining, washing solution, washing line, washing the PBST 2-staining the PBST 2, washing line, washing the washing line is taken out, the washing line is taken out from the PBST 2-2 line, the PBST 2 line is taken out, the PBST 2-PAGE line, the PBST 2 line is taken out, the washing line is taken out after the washing line is taken out, the49kDa) and the sample loading amount of each lane are basically consistent, when recombinant grass carp IL-1 β protein is used as antigen, and the prepared anti-fish IL-1 β egg yolk antibody protein liquid is used as primary antibody, specific bands appear at 34kDa and 30kDa of intestinal lymphocyte liquid lanes of loaches and grass carps respectively, namely IL-1 β of the loaches and the grass carps respectively, which are consistent with the predicted size of the grass carp IL-1 β, but the molecular weight of the loaches IL-1 β is slightly larger than that of the grass carps, and meanwhile, another shearing body of IL-1 β of 20kDa is also appeared.

Example 10 grass carp culture experiment using yolk antibody against fish I L-1 β provided by the present invention

Grass carp is purchased from a certain farm in Suzhou city, is bred in the pond of the current year after being domesticated for adapting to the environment for one week, fish seeds with uniform size and strong physique and about 37.8 +/-0.2 g are selected and randomly distributed into 12 glass-reinforced plastic aquarium tanks with the size of 70cm multiplied by 80cm multiplied by 100cm, 20 fish seeds are randomly grouped, the test grouping is to add anti-IL-1 β egg yolk antibodies with the mass ratio of 1.5g/kg and 3.0g/kg into grass carp basic feed, 3.0g/kg of common egg powder is used as a negative control group, the grass carp basic feed is used as a blank control group, and 3 repeated breeding groups are arranged in each treatment group, the water quality conditions during the breeding period are that the water temperature is 25 +/-2 ℃, and DO (DO) is DO (DO) 2 DEG, and the water quality is controlled by using the common egg powder 3.0g/kg as a negative control group>5.0mg/L，pH 7.5±0.2，NH₄ ⁺-N(0.25±0.05)mg/L，NO₂ ^--N (0.04. + -. 0.01) mg/L, sulfide<0.05mg/L, and all indexes meet the requirement of the culture water body. The official tests were carried out for 8 weeks from 9/2/2015 to 11/2/2015.

The effect of the anti-fish IL-1 β egg yolk antibody on the growth performance of the grass carp is shown in Table 2, and the results show that compared with a control group, the weight gain rate and the fullness of the grass carp can be improved by adding the common egg powder and the anti-fish IL-1 β egg yolk antibody, but the negative control group has no significant difference compared with a blank control group, and after the anti-fish IL-1 β egg yolk antibody is added, the weight gain rate of the grass carp is improved by 10.6% -17.1%, the feed coefficient is reduced by 6.9% -13.4%, and the fullness is improved by 8.30% -18.1%, wherein when the anti-fish IL-1 β egg yolk antibody is added to reach 0.3% of the mass percentage of the feed, the weight gain rate, the fullness and the feed coefficient are increased and reduced to significant levels (p is less than 0.05), and the result shows that the anti-fish IL-1 β egg yolk antibody is added into the feed to achieve the purpose of promoting the growth of the grass carp, and the degree of promoting the growth of the grass carp is.

TABLE 2 Effect of anti-Fish IL-1 β yolk antibody on growth Performance of grass carp

Note: the difference of the same column of the numbers and the shoulder marks is obvious, and the following is the same.

After the growth test, the influence of the anti-fish IL-1 β egg yolk antibody on the bacterial inflammation resistance of the grass carps is examined, and the three groups of grass carps are subjected to an aeromonas hydrophila challenge test, wherein the challenge amount is 10 of crude extract of aeromonas hydrophila⁸cfu/ml (0.1 ml/tail) spinal base intramuscular injection. Respectively feeding grass carp in 30 × 30 × 100cm after counteracting toxic substance³In the plastic water tank, 10 boxes are arranged in each case, 3 parallel boxes are arranged, 24 hours of oxygenation are carried out, results are observed and recorded at any time, the test time is 7d, the death condition is recorded, the pH value in the test period is 7.5 +/-0.2, the water temperature is 28 +/-1 ℃, the DO is not less than 7.0mg/L, the calculation formula is that the death rate (%) (death number/total number of tests) is multiplied by 100%, the immune protection rate is (the death rate of a control group-the death rate of the test group)/the death rate of the control group is multiplied by 100%, the toxic attack test result is shown in table 3, the table 3 shows that the 7d cumulative death rate of the control group reaches 73.3%, the grass carp immune protection rate can be slightly improved by feeding common egg powder, but the grass carp immune protection rate is not obviously different from the control group, the death rate of the egg yolk antibody groups fed with 1.5g/kg and 3.0g/kg of anti-fish IL-1 β are respectively 50.0% and 36.7%, and the death rate<0.05); the immunity protection rate of the grass carp added with 3.0g/kg is the highest and is 49.9 percent. The inflammation and death of grass carp after toxin attack are basically concentrated in the grass carp 4 days before and 4 days after the attackTherefore, the anti-fish IL-1 β yolk antibody can enhance the resistance of grass carp to the inflammatory reaction of aeromonas hydrophila, reduce death and has very obvious enhancing effect.

TABLE 3 Effect of anti-Fish IL-1 β yolk antibody on the anti-bacterial inflammation ability of grass carp

The test simultaneously considers the influence of the anti-fish IL-1 β egg yolk antibody on the intestinal structure of the grass carp, and the results of the intestinal scanning electron microscope (Nigri S-4700 cold field emission scanning electron microscope, Japan) are shown in fig. 4-11. fig. 4-11 show that the folds of the stomach and intestine of the grass carp in the blank control group (fig. 4) and the negative control group (fig. 6) are thicker and sparse when the magnification is 100 times, and the folds of the intestinal mucosa of the grass carp become more and more compact along with the increase of the addition amount of the anti-fish IL-1 β egg yolk antibody (fig. 8 and 10), which indicates that the surface area is larger, and correspondingly, the micro villi of the intestinal tract of the grass carp in the egg yolk antibody group added when the magnification is 20000 times are orderly arranged (fig. 9 and 11), so that the intestinal micro structure of the intestinal tract of the grass carp is obviously increased and the density is obviously reduced, which indicates that the addition of the anti-fish IL-1 β egg yolk antibody.

Example 11 Paramisgurnus dabryanus breeding test using anti-fish IL-1 β yolk antibody provided by the present invention

The anti-fish IL-1 β egg yolk antibody provided by the invention is used in Suzhou university aquaculture laboratories to carry out large-scale paramisgurnus dabryanus breeding tests, the tests are grouped, the test is carried out by adding 1.0, 2.0, 4.0 and 6.0g/kg anti-IL-1 β egg yolk antibody into basic feed of the large-scale paramisgurnus dabryanus, 6.0g/kg of common egg powder is used as a control group, 3 treatment groups are arranged in each treatment group, the large-scale paramisgurnus dabryanus is purchased from a loach breeding base of a free Dongbedefu loach aquaculture farm, fish species are bred in the pond in the current year, after one week of loach domestication, individuals with normal appearance, uniform size and healthy and liveness are selected as test fish, the average weight of the test fish is 9.8 +/-0.5 g, and the test fish species are randomly distributed to 15 individuals with the size of 30 multiplied³20 tails per tank. Each test group was matched according to complete randomizationThe location distribution is used for avoiding experimental errors caused by the difference of geographic positions. The water quality conditions during the culture period are as follows: water temperature 25 + -2 deg.C, DO>5.0mg/L，pH7.5±0.2，NH₄ ⁺-N(0.25±0.05)mg/L，NO₂ ^--N (0.04. + -. 0.01) mg/L, sulfide<The weight gain rate is sequentially from high to low, 4.0g/kg addition group is more than 6.0g/kg addition group and more than 1.0g/kg addition group, 2.0g/kg addition group, 4.0g/kg addition group and 6.0g/kg addition group are all significantly higher than the control group (P is less than 0.05), the increase rate is 62.0-101.8%, the feed coefficient is sequentially from low to high, 2.0g/kg addition group is less than 4.0g/kg addition group and less than 6.0g/kg addition group (P is less than 0.05), and the feed coefficient is sequentially from low to high, 2.0g/kg addition group is less than 4.0g/kg addition group and less than 6.0g/kg addition group (P is less than 0.0.0 g/kg addition group and less than 0.0 g/kg addition group is less than 1.0g/kg addition group<0.05); the specific growth rate is from high to low in sequence, the specific growth rate is greater than 2.0g/kg addition group and greater than 6.0g/kg addition group and greater than 1.0g/kg addition group and greater than a control group, and the specific growth rate of the growth regulator is significantly higher than that of the control group (p is the ratio of the specific growth rate of the growth regulator to the specific growth regulator, and the specific growth regulator, the specific growth regulator and the specific growth regulator are all significantly higher than that of the control group<0.05) and improved by 53.6-89.3%, and the results show that the addition of the anti-fish IL-1 β yolk antibody in the feed can reduce the feed coefficient and improve the weight gain rate and the specific growth rate, and the proper addition amount of the antibody is 2.0-4.0 g/kg of feed.

TABLE 4 influence of anti-fish IL-1 β yolk antibody on growth performance of Paramisgurnus dabryanus

After the test is finished, the influence of the anti-fish IL-1 β egg yolk antibody on the bacterial inflammation resistance of the paramisgurnus dabryanus is examined, the paramisgurnus dabryanus of five groups is subjected to an aeromonas hydrophila challenge test, 10 fishes in each jar have the challenge amount of 10 crude extract of aeromonas hydrophila⁸cfu/ml (0.1 ml/tail) injected intramuscularly at the base of the dorsal spine. Water temperature maintenance 2 when using heating deviceAt 8 ℃, continuously observing for 7d, and recording death, and calculating the formula: mortality (%) — mortality (number of deaths/total number of trials) × 100%, immunoprotection — (control mortality-trial mortality)/control mortality × 100%. The results are shown in Table 5. Table 5 shows that the control group 7d reached 100% cumulative mortality. The mortality rate of the groups treated by the antibodies of 1.0, 2.0, 4.0 and 6.0mg/kg is 53.3-66.7%, which is obviously different from that of the control group (p)<0.05) and 2g/kg of antibody group, the protection rate of the paramisgurnus dabryanus is the highest and is 46.7%, the inflammation symptoms and death are basically concentrated before 3d, and the experimental fish gradually recovers health after 3d, so the anti-fish IL-1 β egg yolk antibody can enhance the resistance of the paramisgurnus dabryanus to the inflammatory reaction of aeromonas hydrophila, reduce death, and the enhancement degree reaches a very remarkable degree.

TABLE 5 Effect of different levels of anti-Fish IL-1 β egg yolk antibodies on the anti-bacterial inflammation ability of Paramisgurnus dabryanus

After the culture test is finished, the intestinal tracts of the Paramisgurnus dabryanus are observed by a scanning electron microscope (Nigri S-4700 type cold field emission scanning electron microscope, Japan), and the results are shown in Table 6 and figures 12-21. Table 6 and figures 12-21 show that the intestinal microvilli of the Paramisgurnus dabryanus are obviously increased and densified and mucus is obviously reduced after the anti-fish IL-1 β egg yolk antibody is added, and the intestinal microvilli structures of the Paramisgurnus dabryanus are better in the groups added by 2.0mg/kg and 4.0mg/kg than those of other experimental groups.

TABLE 6 Effect of anti-inflammatory yolk antibodies on the height, width and thickness of the intestinal folds of Paramisgurnus dabryanus

SEQUENCE LISTING

<110> Suzhou university

<120> anti-fish IL-1 β yolk antibody and preparation method thereof

<130>2017A061-I

<160>1

<170>PatentIn version 3.5

<210>1

<211>1260

<212>DNA

<213> grass carp IL-1 β

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Claims

1. The anti-fish IL-1 β egg yolk antibody is used as a feed additive, the intestinal micro-structure of grass carp and loach is improved by oral administration at a dose of 2.0-4.0 g/kg in a feed additive mode, so that the food absorption area is increased, and the feed return rate is improved, and the preparation method of the anti-fish IL-1 β egg yolk antibody comprises the following steps:

(1) adding recombinant protein of grass carp inflammatory cytokine IL-1 β into white oil containing span 80 and aluminum stearate to prepare grass carp IL-1 β immune antigen;

(2) immunizing laying hens with the prepared grass carp IL-1 β immune antigen, and collecting immunized eggs;

(3) and (3) cleaning the immune eggs, performing micro-coating treatment, pasteurizing and spray drying to prepare the anti-fish IL-1 β yolk antibody.

2. The use as claimed in claim 1, wherein in the preparation method, the volume percentage of span 80 in the white oil is 2-6% by volume of the raw white oil, and the mass percentage of aluminum stearate in the white oil is 1-4% by mass of the raw white oil.

3. The use according to claim 2, wherein in the preparation method, the volume percentage of tween 80 in the purified grass carp IL-1 β recombinant protein is 4-8%.

4. The use according to any one of claims 1 to 3, wherein in the preparation method, the IL-1 β recombinant protein and the white oil adjuvant are sufficiently sheared and emulsified at a high speed according to the volume ratio of 1: 1-2 to prepare a water-in-oil emulsion vaccine for the immunization of laying hens.

5. The use according to any one of claims 1 to 3, wherein the water-in-oil emulsion vaccine prepared in the preparation method contains 0.5 to 2mg/ml of recombinant protein IL-1 β, which is an inflammatory cytokine of grass carp.

6. Use according to any one of claims 1 to 3, wherein the recombinant IL-1 β water-in-oil emulsion vaccine is used to immunize a layer twice, and eggs laid two weeks after the second immunization are collected.

7. The use according to claim 1, wherein the preparation method comprises, in step (3), preparing from the yolk, albumen or whole egg including eggshell thereof.

8. The use according to claim 6, wherein the preparation method comprises adding micro-coated wall material into egg, and micro-coating the active substance in egg by homogenizing, emulsifying and spray drying.

9. The use according to claim 8, wherein in the preparation method, the coating agent in the wall material of the micro-coating comprises modified starch, β -cyclodextrin, sodium alginate, gelatin, chitosan and carboxymethyl cellulose, and the coating agent is used singly or in a compound way, and the addition amount of the coating agent is 3-10% of the weight of fresh eggs.