CN111471107B - Anti-coccidiosis multivalent recombinant protein yolk antibody and preparation method and application thereof - Google Patents

Abstract

The invention discloses an anti-coccidiosis multivalent recombinant protein yolk antibody and a preparation method and application thereof. The preparation method of the chicken coccidium resisting multivalent recombinant protein yolk antibody comprises the following steps: respectively connecting encoding genes of Eimeria tenella rod-shaped body protein 41, eimeria tenella gametophyte protein 22, eimeria maxima gametophyte protein 56 and Eimeria acervulina merozoite protein Ea3-1E with an Escherichia coli expression vector to obtain a recombinant expression vector, expressing the recombinant protein, mixing different recombinant proteins with an adjuvant according to a certain proportion, immunizing a hen, collecting eggs and preparing the egg yolk antibody. Compared with a control group, the egg yolk antibody can improve the survival rate and the relative weight gain rate of the chicks and reduce the output of oocysts, and shows good coccidian resisting effect and the prospect of the multivalent recombinant protein egg yolk antibody for treating and preventing the coccidiosis of the chickens.

Description

Anti-coccidiosis multivalent recombinant protein yolk antibody and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological veterinary drugs, in particular to an anti-coccidiosis multivalent recombinant protein yolk antibody and a preparation method and application thereof.

Background

Chicken Coccidiosis (Coccidiosis) is a disease of the intestinal tract caused by infection with one or several species of Eimeria, and is also the most economically significant parasitic infection in poultry farming worldwide. Worldwide economic losses due to coccidiosis are in the order of billions of dollars each year, mainly including losses due to reduced costs for prevention and performance (e.g., low feed conversion, slow growth, death). At present, the prevention and treatment of coccidiosis mainly depends on anticoccidial drugs and live vaccines, but the use of anticoccidial drugs is limited due to the problems of drug-resistant strains, drug residues and the like; problems with the storage, transport, application and management of live vaccines have limited their widespread use. Therefore, the development of a new control method for chicken coccidiosis is urgently needed, and the development of a new method for preventing and treating chicken coccidiosis independent of drug control is widely concerned by people.

Avian egg yolk antibodies (IgY) are immunoglobulins of hen serum antibodies transported in egg yolk and capable of conferring immune protection to offspring chicks. The yolk antibody is widely concerned due to the characteristics of non-invasiveness, low cost and the like.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

The invention aims to provide an anti-coccidiosis multivalent recombinant protein egg yolk antibody, which aims to solve the problem of drug resistance of the current coccidiosis drug prevention and control.

In order to realize the aim, the invention provides a preparation method of an anti-coccidium multivalent recombinant protein yolk antibody, which comprises the following steps: preparation of mixed antigens: selecting 4 chicken coccidian proteins: connecting encoding genes of the 4 coccidian proteins with an escherichia coli expression vector to obtain a recombinant expression vector, expressing the 4 recombinant proteins, and then combining the 4 recombinant proteins into a mixed antigen by equal mass, wherein the 4 coccidian proteins sequentially have amino acid sequences shown as SEQ ID NO. 1-4; immunization: further mixing the mixed antigen with any adjuvant in equal mass, and immunizing the laying hens according to an immunization program; acquisition of yolk antibody: the eggs laid by the immunized laying hens are disinfected, egg white and egg yolk are separated, the egg white and egg yolk serial membranes are sufficiently removed, the egg yolk and acetate buffer solution (6.89 g of anhydrous sodium acetate/L and 2ml of glacial acetic acid/L) are mixed into first mixed solution, the supernatant of the first mixed solution is sucked, 0.8% of caprylic acid is added at the temperature of 18 ℃ to mix into second mixed solution, and the second mixed solution is further filtered to obtain the anti-coccidiosis multivalent recombinant protein egg yolk antibody.

The four proteins are obtained by screening in the following modes: the protein Et.ROP41 and Ea3-1E are obtained by screening proteins with high expression in a sporozoite stage from a ToxoDB database through New sera-Genes-Transcriptomics-RNA Seq evaluation, predicting the molecular weight, signal peptide, B cell and T cell epitope of the proteins, taking secreted proteins with the molecular weight of 20-100kDa, signal peptide, large expression amount in each stage and many antigen epitope as screening basis, and verifying and screening through a chicken immune protection test. Ea3-1E is an antigen gene which is frequently researched at present and is a surface antigen of E.acervulina sporozoite and merozoite stages. The Ea3-1E sequence has high homology among e.tenella, e.necatrix and e.acervulina, and can be used as a cross antigen of a candidate vaccine, while the et.rop41 protein is a newly discovered and identified protein; the Etgam22 gene is a multicopy gene specifically expressed by e.tenella at the gametophyte stage, and its expression product is associated with the oocyst wall formation. Immune protection results show that rEtgam22 can improve weight gain to a certain extent, reduce lesion scores and reduce oocyst yield; emGAM56 protein is commercialized abroad

The main components of the coccidian vaccine are verified by laboratories and fields in a plurality of countries and regions, so that the coccidian vaccine has a good immune effect, but the coccidian vaccine has not been popularized and used in China due to the problems of complex production process, high cost and the like. The 4 chicken coccidiosis recombinant protein is mixed with an adjuvant according to a certain proportion, a hen is immunized, eggs are collected to prepare the egg yolk antibody, and the result shows that the excellent coccidiosis resistant effect is achieved.

In one embodiment of the invention, the encoding nucleotide sequence of the 4 chicken coccidian proteins is shown in SEQ ID NO.5-8 in sequence.

The invention also provides a primer for amplifying the nucleotide sequence, which has the following sequence: amplification of EtROP41 upstream primer: 5' TTCCAGGGGCCCCTGGATCCGAAACCTCCCGAGTCAACCT-: 5; amplification of EtGAM22 upstream primer: 5 'CAAGGCCATGGTGATATCGGCACCTGAGTATCCTTCTTCAGCTTG-3' (SEQ ID NO. 11), amplification of the downstream primer of EtGAM 22: 5-; amplification of EmGAM56 upstream primer: 5 'TTCCAGGGGCCCCTGGATCCCAGGGTTCACCCTTACAGGAG-3' (SEQ ID NO. 13), downstream primer for amplifying EmGAM 56: 5-; amplifying Ea3-1E upstream primer: 5-: 5 'TCGAGTGCGGCGGCCGCAAGCTTGAAGCCGCCCTGGTTACAGGT-3' (SEQ ID NO. 16).

In one embodiment of the present invention, the method for preparing the mixed antigen comprises the following steps: gene amplification: amplifying gene sequences of 4 coccidian proteins by respectively taking cDNA sequences of Eimeria tenella rod-shaped protein 41 (EtROP 41), eimeria tenella gametophyte protein 22 (EtGAM 22), eimeria maxima gametophyte protein 56 (EmGAM 56) and Eimeria acervulina merozoite protein Ea3-1E as templates; construction of a recombinant vector: constructing a recombinant vector by using the amplified EtROP41 gene sequence and pGEX-6p-1 skeleton, constructing a recombinant vector by using the amplified EtGAM22 gene sequence and pET-28a skeleton, constructing a recombinant vector by using the amplified EmGAM56 gene sequence and pGEX-6p-1 skeleton, and constructing a recombinant vector by using the amplified Ea3-1E gene sequence and pET-28a skeleton; expression of the recombinant protein: respectively transforming the 4 recombinant vectors into expression bacteria, inoculating the expression bacteria into 1L of fresh LB (containing AMP 100 mu g/mL) culture solution until the OD600nm value of the bacteria solution is about 1, adding an inducer IPTG (0.8 mmol/L), and respectively carrying out induced expression and purification; and mixing: and (4) mixing the 4 chicken coccidian recombinant proteins subjected to induction expression with equal mass to obtain the chicken coccidian recombinant protein.

In one embodiment of the present invention, the expressing bacterium is an e.coli transetta (DE 3) strain; the induction expression conditions of the Eimeria tenella rod-shaped body protein 41 (EtROP 41), the Eimeria tenella gametophyte protein 22 (EtGAM 22) and the Eimeria acervulina merozoite protein Ea3-1E are all 37 ℃ for 12h (160 r/min), and the induction expression condition of the Eimeria maxima gametophyte protein 56 (EmGAM 56) is 18 ℃ for 18h.

In one embodiment of the present invention, the immunization program specifically comprises the following steps: firstly, the method avoids: mixing the mixed antigen with Freund's complete adjuvant in equal mass, and performing subcutaneous injection on the breast of a hen aged 24-26 weeks; II, exemption: performing secondary immunization two weeks after the primary immunization, mixing the mixed antigen with Freund incomplete adjuvant in equal mass, and performing subcutaneous injection on the breast of the primary immunized hen; and exempt from three: and performing tertiary immunization four weeks after the secondary immunization, mixing the mixed antigen with Freund incomplete adjuvant in equal mass, and performing subcutaneous injection on the breast of the secondary immunized hen.

In one embodiment of the present invention, in the step of the immunization procedure, the immunization dose of the 4 recombinant proteins in the mixed antigen is 100 μ g/feather.

In an embodiment of the present invention, after the laying hen has undergone primary immunization, secondary immunization and tertiary immunization, titers of yolk antibodies in eggs laid by the laying hen need to be detected respectively, where the detection conditions are as follows: the mixed antigen is coated, the dilution ratio of the yolk antibody is 1 (15-25), the dilution ratio of the enzyme-labeled antibody is 1 10000, and preferably, the mixed antigen is coated after being diluted to 8 mu g/mL.

In one embodiment of the present invention, in the step of obtaining the yolk antibody, the step of sterilizing the eggs includes: sterilizing ovum gallus Domesticus in 0.1% benzalkonium bromide solution at 42 deg.C for 12-18min, sterilizing with sterile water at 95 deg.C for 3-7s, taking out, and wiping with 95% ethanol under sterile condition.

In one embodiment of the present invention, in the step of obtaining the yolk antibody, the pH of the acetate buffer is 4.5, and preferably, the volume ratio of the yolk to the acetate buffer is 1; further preferably, the first mixed solution is required to be placed at 4 ℃ overnight and then supernatant liquid of the first mixed solution is sucked; further preferably, the second mixture is allowed to stand at 4 ℃ overnight, and then the solution is coarsely filtered with clean neutral filter paper, adjusted to pH 7, sterile-filtered with a 0.22um filter, and the filtered egg yolk antibody is stored at 4 ℃ for later use.

The invention also provides the yolk antibody prepared by the preparation method of the anti-chicken coccidiosis multivalent recombinant protein yolk antibody.

The invention also provides application of the egg yolk antibody in preparing a medicine or feed for treating or preventing chicken coccidiosis.

In one embodiment of the present invention, the pharmaceutical preparation is an injection or an oral preparation containing the egg yolk antibody.

Compared with the prior art, the invention has the following beneficial effects:

the invention relates to a chicken coccidium resisting multivalent recombinant protein yolk antibody, which is prepared by mixing 4 chicken coccidium recombinant proteins with an adjuvant in a certain proportion. Immunizing hen, collecting egg to prepare yolk antibody. Compared with a control group, the egg yolk antibody can improve the survival rate and the relative weight gain rate of the chicks and reduce the output of oocysts, and shows good coccidian resisting effect and the prospect of the multivalent recombinant protein egg yolk antibody for treating and preventing the coccidiosis of the chickens.

Drawings

FIG. 1A is an electrophoretic identification chart of an amplification product of EtROP41 gene according to an embodiment of the present invention;

FIG. 1B is an electrophoretic identification chart of the amplification product of the EtGAM22 gene according to one embodiment of the present invention;

FIG. 1C is a view showing the electrophoretic identification of the Ea3-1E gene amplification product according to an embodiment of the present invention;

FIG. 1D is an electrophoretic identification chart of an amplification product of EmGAM56 gene according to an embodiment of the present invention;

FIG. 2A is a SDS-PAGE pattern of the expression and purification of the recombinant chicken coccidia protein EtROP41 according to one embodiment of the invention;

FIG. 2B is a SDS-PAGE graph showing Ea3-1E expression and purification of recombinant chicken coccidian protein according to one embodiment of the invention;

FIG. 2C is a SDS-PAGE graph of expression and purification of the chicken coccidian recombinant protein EmGAM56 according to one embodiment of the invention;

FIG. 2D is a SDS-PAGE pattern of expression and purification of the recombinant chicken coccidian protein EtGAM22 according to one embodiment of the invention;

FIG. 3 is a graph showing the variation of the IgY level of specific yolk antibodies after immunization of laying hens according to an embodiment of the present invention.

Description of the main reference numbers:

m, protein Marker; * Representing the immunization time node.

Detailed Description

Specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.

Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.

The experimental methods involved in the invention are all conventional methods unless otherwise specified.

Coli transetta (DE 3) strain: purchased from Beijing Quanji Biotechnology, inc.;

coli DH5 α: purchased from Beijing Congress Biotechnology Ltd;

high fidelity enzyme, taq enzyme: purchased from biotechnology, inc. Of nuozokenza, nanjing;

multi-fragment ligation kit, reverse transcription kit: purchased from Beijing Quanji Biotechnology, inc.;

nickel affinity chromatography packing: purchased from novagen, usa;

expression vectors pET-28a (+) and pGEX-6p-1 (+): storing in a laboratory;

freund's adjuvant (complete, incomplete): sigma-aldrich, USA;

24-26 weeks old helenium brown laying hens: purchased from a farm of Jinassist breeders, tianjin City;

eimeria tenella, eimeria maxima and eimeria acervulina: isolated, identified and stored by the national animal parasitic protozoan laboratory of university of agriculture, china.

1 day old chicks: purchased from Jia's poultry hatchery in the Fengshan district, tangshan City, shandong province.

EXAMPLE 1 expression and purification of recombinant proteins

The chicken Eimeria tenella antigen is an accession number ETH _00005405 of EtROP in a ToxoDB database (https:// ToxoDB. Org/toxo/app/record/gene/ETH _ 00005405), and the length of a gene fragment for coding the protein is 1527bp; the accession number of the EtGAM22 in the ToxoDB database is ETH _00035480 (https:// ToxoDB. Org/toxo/app/record/gene/ETH _ 00035480), and the length of a gene fragment for encoding the protein is 597bp; the accession number of the eWEY _00026710 of the EmGAM56 in the ToxoDB database (https:// ToxoDB. Org/toxo/app/record/gene/EMWEY _ 00026710), the length of the gene fragment encoding the protein is 1422bp; ea3-1E has an accession number of EAH _00057690 (https:// ToxoDB. Org/toxo/app/record/gene/EAH _ 00057690) in the ToxoDB database, and the length of the gene fragment encoding the protein is 1737bp.

1. Cloning of genes and construction of recombinant plasmids

Primers were designed based on the respective gene sequences and are shown in the following table:

primer sequences for genes in Table 1

The RNA of Eimeria tenella in sporozoite stage (ROP 41) and gametophyte stage (GAM 22), eimeria maxima in gametophyte stage (GAM 56) and Eimeria acervulina in merozoite stage (3-1E) were extracted separately by the following steps:

adding 1ml TRIzol into the extracted coccidium, shaking thoroughly, repeatedly beating until no visible lump exists, and standing in a refrigerator at-80 deg.C for about 10min; taking out after 10min, adding 0.2ml of precooled chloroform after melting, placing at room temperature for 10min,4 ℃ and 12000rpm/15min after vigorously shaking for 15 s; centrifuging, absorbing upper water phase, transferring to new EP tube, adding equal volume of precooled isopropanol, mixing, standing at-20 deg.C for 10min, standing at 4 deg.C for 12000rpm/10min; centrifuging, removing supernatant, adding 1ml of 75% ethanol, 12000rpm/5min, washing precipitate, repeating twice, removing supernatant until ethanol is completely volatilized. After the tube wall is slightly dried, 25-100 μ l of DEPC water without RNase is added according to the amount of RNA observed by naked eyes, and the tube is sucked and beaten by a gun head for several times to dissolve the RNA, so that the total RNA is obtained. Meanwhile, the extracted RNA was reverse transcribed with a cDNA reverse transcription kit, and the reverse transcription system is shown in Table 2. The cDNA obtained by reverse transcription was stored at-20 ℃ for future use.

TABLE 2 RNA reverse transcription System

* The amount of RNA added and the amount of water added were determined based on the RNA concentration.

Using cDNA of corresponding stage coccidia as a template to amplify the gene, wherein the reaction system is as follows:

TABLE 3 PCR amplification System

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 10min, 30 cycles were carried out, including denaturation at 94 ℃ for 30sec, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 90s, and final extension at 72 ℃ for 10min. The PCR product was sampled and detected by agarose gel electrophoresis (see FIG. 1 for the results). And recovering the PCR product with the size corresponding to the target fragment by using an agarose gel DNA recovery kit.

The fragments and the backbone were ligated using a multi-fragment seamless ligation Kit (Clonexpress. MultiS One Step Cloning Kit) from Vazyme, in the following reaction system:

TABLE 4 Multi-segment connection System

2. Expression of recombinant proteins

Transferring the recombinant plasmid obtained by connection into expression competence Transetta (DE 3), and carrying out ice bath for 30min; heat shock at 42 deg.C for 1min, ice bath for 3-5min; adding 500 μ l of non-resistant LB liquid culture medium, and shaking the strain at 37 ℃ for 1h; 100-200. Mu.l of the bacterial solution was spread on LB solid medium containing kanamycin resistance and cultured overnight at 37 ℃. The next day, a single colony was picked, added to LB liquid medium containing kanamycin and cultured at 37 ℃ to logarithmic phase (OD) _600nm Value about 1), IPTG was added to a final concentration of 0.8mM. Continuously culturing for 12h, centrifuging the bacterial liquid at 8000rpm for 20min at 4 ℃, collecting bacterial precipitates, and ultrasonically crushing the bacterial; centrifuging the crushed thallus lysate for 10min (12,000rpm) at 4 ℃, and separating precipitate and supernatant; the precipitate was dissolved by adding an appropriate amount of 8M urea. mu.L of each of the supernatant and the precipitate was taken, 10. Mu.L of 5 XSDS gel loading buffer was added thereto, boiled for 10min, centrifuged for 10min (12,000rpm), and subjected to SDS-PAGE to determine whether the expression was soluble. As a result, the results show that the EtROP and Ea3-1E proteins have large expression amount, are soluble expression, are expressed in supernatant, have low requirements on induced expression conditions, and are added with IPTG OD _600nm Between 0.6 and 1.5 or IPTG concentration between 0.2 and 1.6mMCan express a large amount of protein, the induction temperature is 37 ℃, and the induction time is 5h to 24 h. The EmGAM56 protein is soluble expressed in the supernatant, and added with IPTG OD _600nm Can express a large amount between 0.8 and 1.0 or between 0.2 and 1.6mM of IPTG, the induction temperature is 18 ℃, and the induction time is 15 to 18 hours. EtROP-GST, ea3-1E-His and EmGAM56-GST were expressed in the supernatant, and the proteins in the supernatant were purified with a nickel affinity chromatography packing, and the results are shown in FIGS. 2A, 2B and 2C. EtGAM22-His (expression conditions were as above) was mainly expressed in inclusion bodies, and the inclusion bodies were renatured, and the results are shown in FIG. 2D.4, the low requirement of protein expression conditions, large expression quantity and the like, so that the protein is easy to produce in large quantities.

Example 2 detection of specific yolk antibody levels

1. Immunization procedure

24-26 weeks old Hainan brown stock hens were purchased and randomly divided into 3 groups of 15 chickens. The breeding hens around the hens before the laying are started are subjected to priming, and each 100ug (/ feather) of the four recombinant proteins is mixed with Freund's complete adjuvant according to the proportion of 1; two weeks later, two immunizations were performed, and 100ug (/ feather) of each of the four recombinant proteins was mixed with Freund's incomplete adjuvant 1 and injected subcutaneously into the chest; three immunizations were performed four weeks after the second immunization, and 100ug (/ feather) of each of the four recombinant proteins was mixed with Freund's incomplete adjuvant 1 and then injected subcutaneously into the chest. The control group was treated in the same manner as the protein-immunized group. See table 5.

TABLE 5 hen immunization dose and immunologic adjuvant

2. Preparation of yolk antibody:

sterilizing eggs of each group in 0.1% benzalkonium bromide solution at 42 deg.C for 15min, sterilizing with sterile water at above 95 deg.C for 5s, taking out, drying with 95% ethanol under sterile condition, separating egg white and yolk, removing egg white and yolk mesentery, and collecting the obtained yolk in a beaker and stirring; fully mixing the egg yolk with a prepared acetate buffer solution (6.89 g of anhydrous sodium acetate/L, 2ml of glacial acetic acid/L) with the pH value of 4.5 according to the volume ratio of 1; sucking supernatant, adding 0.8% n-octanoic acid at 18 deg.C, stirring, standing at 4 deg.C overnight, coarse-filtering the solution with clean neutral filter paper, adjusting pH to 7, sterilizing with 0.22um filter, and storing the filtered yolk antibody at 4 deg.C.

3. Indirect ELISA detection of yolk antibodies:

the titer of the yolk antibody was measured two weeks after the first immunization, two weeks after the second immunization, and three weeks after the third immunization, respectively. Randomly selecting 10 eggs in each group for preparing the yolk antibody, and carrying out indirect ELISA detection on the prepared yolk antibody by the specific method as follows:

1) Coating: diluting the four recombinant proteins to 8 mu g/mL by using carbonate buffer solution, mixing in equal volume, adding 100 mu L/hole into a 96-hole enzyme-labeled reaction plate, and coating for 14h at 4 ℃;

2) Washing: discard the liquid in the plate, wash 4 times with 0.5% PBST;

3) And (3) sealing: sealing with 5% skimmed milk, 100 μ L/hole, and placing in 37 deg.C incubator for 1h; washing, the same as above;

4) Primary antibody incubation: the prepared yolk antibody 1 is diluted by 20 and then added into an enzyme-labeled reaction plate, 100 mu L/hole is placed in an incubator at 37 ℃ for incubation for 1h, and a negative and positive control is set, and then the steps are washed: the same as above;

5) And (3) secondary antibody incubation: diluting HRP-labeled goat anti-chicken IgG 1 by 10000, adding the diluted HRP-labeled goat anti-chicken IgG 1 into an enzyme-labeled reaction plate, placing the diluted HRP-labeled goat anti-chicken IgG 1 into a 37 ℃ incubator per hole, and incubating for 1h; washing;

6) Color development: mixing the substrate color development liquid A and the substrate color development liquid B in equal proportion under the condition of keeping out of the sun, adding the mixture into an enzyme-labeled reaction plate, reacting at the room temperature for 10min at 100 mu L/hole;

7) And (4) terminating: 2MH2SO4 was added to stop the reaction, 50. Mu.L/well;

8) Reading OD value: the absorbance was read with a microplate reader at wavelengths 450 and 630.

Indirect ELISA results showed that recombinant protein immunized hen specific yolk antibodies (fig. 1) reached 1a week after priming ⁶ (very significant difference compared with the control group) one week after the second immunizationReaches the peak value (1 ⁸ ) Until the specific yolk antibody titer remained 1 at 12 weeks after triple immunization ⁷ 。

Example 3 evaluation of yolk antibody preventive Effect

1. Evaluation of preventive Effect of yolk antibody

1.1 design of the experiment

The experiment is divided into 15 groups, each group is 7 chicks of 1 day old, wherein the 1 st to 3 rd groups are respectively orally administered with 0.3ml, 0.5ml and 1.0ml of recombinant protein egg yolk antibody at the age of 11 to 13 days, the 4 th to 6 th groups are respectively orally administered with 0.3ml, 0.5ml and 1.0ml of blank egg yolk antibody, the 7 th to 9 th groups are respectively injected with 0.3ml, 0.5ml and 1.0ml of recombinant protein egg yolk antibody, the 10 th to 12 th groups are respectively injected with 0.3ml, 0.5ml and 1.0ml of blank egg yolk antibody, the 13 th group is a pest attacking control (7 feathers), and the 14 th group is a blank control (7 feathers). Weighing at 14 days old, and orally infecting fresh sporulated oocysts of Eimeria tenella, eimeria maxima and Eimeria acervulina at a dose of 5000/feather and 1.5 × 10 ⁵ 1.5 x 10 of character/feather ⁵ Number per feather. The mental state and death of the chickens were observed and recorded daily. Weighing at 21 days of age, scoring caecum lesion of each group of 5 chickens, collecting feces of the chickens remaining in each group after 4 days, and counting oocysts.

1.2 evaluation index of chick protection effect

1) Survival rate: the mortality of the chickens in each group after infestation was recorded. The results are shown in table 6, and after attacking, it was shown that prophylactic oral administration or injection of egg yolk antibodies could improve the survival rate of chicks.

TABLE 6 survival rate of group chicks after attack of insects

2) Weight increment: weighing the insects one by one during insect attack and killing, recording the weight change conditions before and after 7 days of insect attack, and calculating the average weight gain and the relative weight gain. The effect of the recombinant protein yolk antibody on the weight gain of the chickens after attacking the worm is expressed as the average weight gain.

Average weight gain = slaughter weight-attack weight

Weight gain (%) = (weight at slaughter-weight at insect attack)/weight at insect attack × 100;

relative weight gain (%) = (weight gain in experimental group/weight gain in non-immune non-offensive group) × 100.

The average and relative weight gain for each group is shown in table 7. The results show that the average weight gain of the chicks of the oral recombinant yolk antibody group of 0.3ml, 0.5ml and 1.0ml is not different from that of the control group (P is more than 0.05). The relative weight gains of the groups injected with 0.3ml, 0.5ml and 1.0ml of recombinant egg yolk antibodies were 28.1%, 25.4% and 24.6%, respectively, while the relative weight gains of the blank control group were 17.0%, 18.4% and 13.3%, and the relative weight gain of the attack control group was 3.5%, with significant difference (P < 0.05), indicating that the injection of egg yolk antibodies can prevent the weight loss of offspring chicks infected with coccidia.

TABLE 7 average weight gain and weight gain ratio after prevention of insect attack on chicks

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3) And (3) lesion scoring: on day 7 after attack, weighing, performing autopsy, and judging the variable values of the cecum, duodenum and small intestine according to the Johnson method, wherein the standard is as follows:

scoring of cecal lesions:

0, no lesion is found;

+1, the cecum wall is scattered in a few punctate bleeding spots, the intestinal wall is not thickened, and the content is normal;

+2, the cecum content is mixed with a small amount of blood, the intestinal wall is small and fat, and a plurality of bleeding focuses can be seen;

+ 3. Large amount of blood or caecum clot (coagulopathy or grey-white plug), pachynsis of the caecum wall, marked deformation or atrophy of the caecum;

+4: the cecum shrinks significantly and the lesions extend to the rectum. The cecal wall is extremely hypertrophied and the cecal contents are coagulated blood or cored.

If the lesions of the cecum on both sides are inconsistent, the side with serious lesions is taken as the standard.

Small bowel disease scoring:

0 point without macroscopic lesions

+1 minute of small intestine middle section mucosa surface can see small bleeding point, and small amount of orange mucus is in the intestinal cavity;

slight flatulence appears in the middle section of small intestine in +2 minutes, a plurality of bleeding points can be seen on the serosal surface and extend to duodenum, and a large amount of orange mucus can be seen in the intestinal cavity;

+3 minute intestinal canal dilatation, no normal intestinal contents, mixed blood clots and orange mucus in the intestinal contents, thinned intestinal wall and rough mucosal surface;

+4 minutes intestinal canal swollen, the content of the intestine is viscous and brownish red, mixed with a lot of blood clots, in light meat wash, congestion of intestinal mucosa, bleeding and sloughing.

Scoring of duodenal lesions:

0 point without macroscopic lesions

+1 minute duodenum serosal surface and mucosal surface are both seen with transverse striatal white spots scattered, arranged transversely and in trapezoid appearance;

+2, the lesions are dense but not fused, the duodenum mucosa is covered with transverse striatal white spots which are transversely arranged, the appearance is in a ladder shape, the lesions extend to the jejunum, the content of the digestive tract is thin, and the intestinal mucosa is thickened;

+3 minute duodenal mucosal surface can see dense gray focus, lesion extends to the luteal base of the follicle, the intestinal tract is pale, and the intestinal lumen contains a large amount of water, and the content is water-like liquid.

+4 th duodenal mucosa surface is light gray, transverse striated leukoplakia is completely fused, the intestinal lumen is filled with creamy exudates, and the intestinal wall is thickened.

Lesion value = mean lesion score of groups x 10

Lesion score reduction (%) = (infection control lesion score-each group lesion score)/infection control lesion score × 100%

The lesion scores for each group are shown in table 8. The blank yolk antibody group, the immune attack group and the attack control group have the most obvious lesions, and the average lesion score of the caecum of the experimental group is not significantly different from that of the control group (P is more than 0.05); the lesion scores of the yolk antibody groups injected with 0.3ml, 0.5ml and 1.0ml of recombinant proteins are 1.4, 1 and 1.6 respectively, and the differences are significant compared with the blank yolk antibody group and the attack control group (P < 0.05).

The average duodenum lesion scores of the oral blank yolk antibody group are not significantly different (P is more than 0.05) compared with those of the oral blank yolk antibody group and the attack control group; the cecal mean lesion scores of the blank yolk antibody injection groups were 2.2, 2.2 and 1.8, respectively, and were significantly different from those of the oral blank yolk antibody group and the attack control group (P < 0.05).

The lesion scores of the 0.3ml, 0.5ml and 1.0ml oral recombinant protein yolk antibody groups are all 0.8, and the difference is significant compared with the oral blank yolk antibody group and the attack control group (P is less than 0.05); the cecal mean lesion scores of the blank yolk antibody injection groups were 1.8, 1.8 and 1.6, respectively, and were significantly different from those of the oral blank yolk antibody group and the attack control group (P < 0.05).

The results show that the recombinant protein yolk antibody has a good protection effect on chicken coccidiosis and can reduce the damage of coccidia to chicken intestinal tracts.

TABLE 8 mean lesion score after prevention of infestation by chicks in groups

4) Counting oocysts: oocyst counts were performed according to the macbect method: collecting chicken manure 7-11 days after insect attack, adding a proper amount of 2.5% potassium dichromate, uniformly mixing the manure, weighing the total weight of the manure, respectively taking three manure samples at different three positions by using a 50mL centrifuge tube, uniformly mixing the obtained three manure samples again, weighing 2g of each manure sample, putting the weighed manure sample into a 100 mL beaker, firstly adding 10mL of saturated saline solution, uniformly mixing, then adding 50mL of saturated saline solution, immediately taking the manure solution after uniform mixing, filling two counting chambers, standing for 2min, and counting by microscopic examination. Since the volume of the counting chamber was 0.15ml, the content of 0.15ml, and the volume of the two counting chambers was 0.01g, OPG = oocyst number × 100.

Oocyst output = OPG x total fecal weight

Oocyst reduction rate (%) = (control group oocyst output-adjuvant/protein immunization group oocyst output)/control group oocyst output × 100

The results of the oocyst output and the oocyst reduction rate of each group are shown in tables 9 to 12, and it can be seen from the results that the total oocyst reduction rate of the groups orally administered with 0.3ml, 0.5ml and 1.0ml of recombinant protein is 31.7%, 41.2% and 40.8%, respectively (P < 0.01), and the difference is significant compared with the blank yolk antibody group and the control group (P < 0.05). The total oocyst reduction rates of the groups injected with 0.3ml, 0.5ml and 1.0ml of recombinant proteins are respectively 68.4%, 77.5% and 81.5% (P < 0.01), and the difference is significant compared with the blank yolk antibody group and the attack control group (P < 0.05).

TABLE 9 Total oocyst output and reduction rate after prevention of insect attack in chicks of group

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It can be seen from Table 9 that the E.tenella oocyst reduction rates of the groups orally administered with 0.3ml, 0.5ml and 1.0ml of recombinant proteins were 26.8%, 30.9% and 19.9%, respectively (P < 0.01), and were not significantly different from the control group and the blank yolk antibody group (P > 0.05). The total oocyst reduction rates of the groups injected with 0.3ml, 0.5ml and 1.0ml of recombinant proteins are 69.7%, 72.3% and 80.6% respectively (P < 0.01), and the difference is significant compared with the blank egg yolk antibody group and the attack control group (P < 0.05).

TABLE 10 prevention of Eimeria tenella oocyst output and reduction rates following challenge in broiler chickens

It can be seen from Table 10 that the oocyst reduction rates of Eimeria acervulina in the recombinant proteome of 0.3ml, 0.5ml and 1.0ml were 20.8%, 31.0% and 29.1%, respectively (P < 0.01), which were not significantly different from the control group of attack worms and the blank yolk antibody group (P > 0.05). The oocyst reduction rates of Eimeria acervulina in the groups injected with 0.3ml, 0.5ml and 1.0ml of recombinant proteins are 46.7%, 75.3% and 79.2% (P < 0.01), respectively, compared with the blank yolk antibody group, the yolk antibody groups injected with 0.3ml and 0.5ml of recombinant proteins have no significant difference (P > 0.05), but compared with the control group of attack worms (P < 0.05), the yolk antibody groups injected with 1.0ml of recombinant proteins have significant difference (P < 0.05) compared with the blank yolk antibody group and the control group of attack worms.

TABLE 11 prevention of Eimeria acervulina oocyst output and reduction rate after attack of Coprinus comatus

As can be seen from Table 11, the oocyst reduction rates of E.maxima were 44.2%, 58.3% and 69.0% (P < 0.01) for the recombinant proteomes of 0.3ml, 0.5ml and 1.0ml, respectively, which were significantly different from the control group against the parasites and the blank yolk antibody group (P < 0.05). The oocyst reduction rates of the recombinant protein groups Eimeria maxima injected with 0.3ml, 0.5ml and 1.0ml are 83.1%, 84.1% and 84.0% respectively (P < 0.01), and the difference is very significant compared with the blank egg yolk antibody group and the attack control group (P < 0.01).

TABLE 12 prevention of Eimeria maxima oocyst output and reduction rates after challenge by chicks of group

The results show that the recombinant protein yolk antibody can obviously reduce the development and reproduction of the chicken coccidian in a chicken body and the formation of oocysts, and has better effect of preventing coccidiosis.

EXAMPLE 4 evaluation of therapeutic Effect of yolk antibody

1. Design of experiments

The experiment is divided into 15 groups, each group comprises 7 chicks of 1 day old, the chicks of 14 days old are weighed and then are orally infected with fresh tender, giant and eimeria acervulina sporulated oocysts, the dosage is respectively 5000 per feather and 1.5 multiplied by 10 ⁵ 1.5 x 10 of character/feather ⁵ One seed/feather. Wherein the 1 st to 3 rd groups are orally administered with 0.3ml, 0.5ml and 1.0ml recombinant protein yolk antibody respectively at 15-20 days of age, the 4 th to 6 th groups are orally administered with 0.3ml, 0.5ml and 1.0ml blank yolk antibody respectively, wherein the 7 th to 9 th groups are injected with 0.3ml, 0.5ml and 1.0ml recombinant protein yolk antibody respectively, the 10 th to 12 th groups are injected with 0.3ml, 0.5ml and 1.0ml blank yolk antibody respectively, the 13 th group is a pest attacking control (7 feathers), and the 14 th group is a blank control (7 feathers). The mental state and death of the chickens were observed and recorded daily. Weighing at 21 days of age, and scoring cecal lesions of 5 chickens per group, collecting the chicken feces left in each group after 4 days, and counting oocysts.

2. Evaluation index of chick protection effect

1) Survival rate: the mortality of the chickens in each group after infestation was recorded. The results are shown in table 13, and after the attack of the insects, the recombinant protein egg yolk antibody improves the survival rate of the chicks, which indicates that the egg yolk antibody can effectively protect the chicks against infection of coccidian in the chicks.

TABLE 13 survival rate of group chicks after attacking insects

2) The weight gain calculation method is the same as that of example 3;

the average weight gain and relative weight gain for each group are shown in table 14. The results show that the relative weight increasing rates of the 0.3ml, 0.5ml and 1.0ml recombinant protein yolk antibody groups are 39.6%, 32.0% and 34.5% respectively, the relative weight increasing rates of the blank yolk antibody group are 16.3%, 18.5% and 28.6%, and the relative weight increasing rate of the attack worm control group is 3.5%, compared with the difference (P < 0.05). The relative weight increasing rates of the 0.3ml, 0.5ml and 1.0ml recombinant protein yolk antibody groups are 39.7%, 46.2% and 49.3% respectively, the relative weight increasing rates of the blank yolk antibody group are 26.3%, 29.6% and 24.9%, and the relative weight increasing rate of the attack worm control group is 3.5%, compared with the difference (P < 0.05). The yolk antibody can treat the reduction of weight gain of the chicken infected by coccidian.

TABLE 14 mean weight gain and weight gain rates after treatment of insect infestation in chicks

3) Lesion scoring principle: the same as in example 3;

the lesion scores for each group are shown in table 15. As seen from the table, the difference in lesion scores between the attack group and the non-attack group was significant, indicating successful attack.

The lesions of the blank egg yolk antibody group, the immune attack group and the attack control group are most obvious, the average lesion scores of the caecum of the oral blank egg yolk antibody group are respectively 2.6, 3 and 3, the attack control group is 3, and the lesion scores of the recombinant protein egg yolk antibody group of the oral blank egg yolk antibody group of the oral attack control group of the oral administration of 0.3ml, 0.5ml and 1.0ml are respectively 1.8, 1.2 and 1.4, and compared with the oral blank egg yolk antibody group and the attack control group of the attack control group, the difference is obvious (P is less than 0.05); the cecum mean lesion scores of the blank yolk antibody injection group are respectively 3, 2.4 and 2.6, the lesion scores of the insect attack control group are 3, the lesion scores of the recombinant protein yolk antibody injection groups of 0.3ml, 0.5ml and 1.0ml are respectively 1.4, 1 and 1.6, and the difference is significant compared with the blank yolk antibody injection group and the insect attack control group (P is less than 0.05).

The average duodenum lesion scores of the oral blank yolk antibody group are respectively 2, 1.8 and 1.8, the control group for attacking the worm is 2, the lesion scores of the oral recombinant protein yolk antibody group are respectively 0.8, 0.8 and 0.6, and compared with the oral blank yolk antibody group and the control group for attacking the worm, the difference is obvious (P is less than 0.05); the mean lesion scores of duodenum of the blank yolk antibody injection group are 1.8, 1.2 and 1.2 respectively, the lesion scores of the attack worm control group are 2, and the lesion scores of the recombinant protein yolk antibody injection groups of 0.3ml, 0.5ml and 1.0ml are 0.8, 0.4 and 0.4 respectively, so that the difference is obvious compared with the oral blank yolk antibody group and the attack worm control group (P is less than 0.05).

The average lesion scores of the small intestine of the oral blank yolk antibody group are respectively 1.4, 1.4 and 1.6, the lesion scores of the attack worm control group are 2, the lesion scores of the oral recombinant protein yolk antibody group of 0.3ml, 0.5ml and 1.0ml are respectively 0.6, 0.8 and 0.6, and the difference is significant compared with the oral blank yolk antibody group and the attack worm control group (P is less than 0.05); the cecum mean lesion scores of the blank yolk antibody injection group are respectively 1.6, 1.2 and 1.2, the lesion scores of the worm attacking control group are 2, the lesion scores of the recombinant protein yolk antibody injection group are respectively 0.4, and the difference is obvious compared with the oral blank yolk antibody group and the worm attacking control group (P is less than 0.05).

The results show that the recombinant protein yolk antibody has a good protective effect on chicken coccidium infection, and effectively reduces the damage of coccidium to chicken intestinal tracts.

TABLE 15 mean lesion scores after infestation of chicks in treatment groups

4) The oocyst counting method comprises the following steps: collecting chicken manure 7-11 days after insect attack, adding a proper amount of 2.5% potassium dichromate, uniformly mixing the manure, weighing the total weight of the manure, respectively taking three manure samples at different three positions by using a 50mL centrifuge tube, uniformly mixing the obtained three manure samples again, weighing 2g of each manure sample, putting the weighed manure sample into a 100 mL beaker, firstly adding 10mL of saturated saline solution, uniformly mixing, then adding 50mL of saturated saline solution, immediately taking the manure solution after uniform mixing, filling two counting chambers, standing for 2min, and counting by microscopic examination. The counting chambers had a volume of 0.15ml, a content of 0.005g in 0.15ml, and 0.01g in both counting chambers, so OPG = oocyst number × 100.

The results of the oocyst output and the oocyst reduction rate of each group are shown in Table 16, and it can be seen that the total oocyst reduction rate of the groups orally administered with 0.3ml, 0.5ml and 1.0ml of recombinant protein is 31.2%, 58.7% and 68.1% (P < 0.01), respectively, wherein the groups 0.5ml and 1.0ml are significantly different (P < 0.01) from the blank yolk antibody group and the control group. The total oocyst reduction rates of the groups injected with 0.3ml, 0.5ml and 1.0ml of recombinant proteins are respectively 70.1%, 72.4% and 74.9% (P < 0.01), and the difference is very significant compared with the blank yolk antibody group and the attack control group (P < 0.01).

TABLE 16 Total oocyst output and reduction rates following challenge by chicks of the group

It can be seen from Table 16 that the E.tenella oocyst reduction rates of the groups orally administered with 0.3ml, 0.5ml, 1.0ml of the recombinant proteins were 16.6%, 47.3%, and 68.8%, respectively (P < 0.01), wherein the difference was significant when orally administered with 1.0ml compared to the control group of attack worms and the blank yolk antibody group (P < 0.05). The oocyst reduction rates of Eimeria tenella in the groups injected with 0.3ml, 0.5ml and 1.0ml of recombinant proteins are 71.0%, 60.4% and 66.9% respectively (P < 0.01), and the difference is significant compared with the blank egg yolk antibody group and the attack control group (P < 0.05).

TABLE 17 treatment of Eimeria tenella oocyst output and reduction rates following challenge in group chicks

It can be seen from Table 17 that the oocyst reduction rates of Eimeria acervulina in the recombinant proteome of 0.3ml, 0.5ml and 1.0ml were 29.2%, 44.8% and 43.7%, respectively (P < 0.01), which were not significantly different from the control group of attack worms and the blank yolk antibody group (P > 0.05). The oocyst reduction rates of Eimeria acervulina in the recombinant protein groups of 0.3ml, 0.5ml and 1.0ml are respectively 62.1%, 64.8% and 65.8% (P < 0.01), and the difference is significant compared with the blank egg yolk antibody group and the attack control group (P < 0.05).

TABLE 18 treatment of Eimeria acervulina oocyst output and reduction rates after challenge in chicks

It can be seen from Table 18 that the oocyst reduction rates of E.maxima in the recombinant proteome of 0.3ml, 0.5ml and 1.0ml were 46.5%, 79.7% and 85.5%, respectively (P < 0.01), which was significantly different from the control group and the blank yolk antibody group (P < 0.05). The oocyst reduction rates of the Eimeria maxima in the recombinant protein groups of 0.3ml, 0.5ml and 1.0ml are respectively 75.1%, 89.3% and 89.2% (P < 0.01), and the difference is very significant compared with the blank yolk antibody group and the attack control group (P < 0.01).

TABLE 19 treatment of Eimeria maxima oocyst output and reduction rates following challenge in chicks

The results show that the recombinant protein yolk antibody can obviously reduce the development and propagation of the Eimeria in chicken bodies and the formation of oocysts, and has better effect of preventing coccidiosis.

The foregoing description of specific exemplary embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

SEQUENCE LISTING

<110> university of agriculture in China

<120> chicken coccidium resistant multivalent recombinant protein yolk antibody and preparation method and application thereof

<130> P200288DD1F

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 508

<212> PRT

<213> Eimeria tenella

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Met Glu Ser Pro Pro Val Ser Phe Pro Glu Ala Val Pro Gly Ala Tyr

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<213> Eimeria tenella

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gacatgcagt ggcggggccc gctaggccgg gagttaatgc tcgttcttgc tgagcacctc 480

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gagctgggtg aggtttacag cggtcgcttg agtgatatat tcgcagcagg gactgctgcc 960

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gctgaggggg aggaagatct cgccaaacct gtagttgacg agaagtcgga tatgtggggc 1260

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gccaaacttg ccaaagtctt tcaacccaac atatacccaa cccctcctag tccccagaca 240

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<210> 7

<211> 1422

<212> DNA

<213> Eimeria maxima

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caggagggga gtgccccggg ggctccggag gacaccacca ccaccactac gtcgtcccct 180

gtttccgatg gagccgagca gtggcttgag agctttgttc gtgctgtgca gcgccagctg 240

cagcttcagg accaaatgat gcgtcagctc atgagggaca ttcaggagta cctgagcact 300

gcgttcaact gggcagagaa ccagtctact gcctacaccc gtgttaccga gatgatggac 360

atgatctcca acagaatgaa cgctgccatg gacagctcaa acgaactcat gaccactagc 420

gacaccacag accccgagac cctccgccgt gcaactcgca agtacatgaa ggaggttcgc 480

gttcaggacg tcctggtaga tgctctctgg gcctctctcc gcggtgtaca gacagctgcc 540

tggatgaatg gagtgaccgc tattgagaag gaggagacga ctcccatggc tagccgcgct 600

gctgaggagt tcctccaccg catgtaccat aacctgaggg cagcaggtat gtctgaagaa 660

gatgttgcca agttcatccc tagagccgag tacaacccct ccgagcagtc aagaaatatg 720

ggcagaaagg gcaggagctt ctactacggc ggctatccca gctactacaa ctccccctac 780

tacagctaca gcagctaccc cagctactac aactacagct acccgtcata cagctacagc 840

agctatccca gctactaccg ctacagcagc tacccctact acaactacag ctatcccagc 900

tactacaact acggcagcta cccgtactac agttatagca gctaccccag ctggtactgg 960

cgccgtctcc gctctttggc aacagcaact tgcccagact gccctcctct caccactccc 1020

agcatgatcc caactccccc cccaatgatg aacatgatga acaccccacc ccccatggca 1080

aacatgatga ccagcatgat gatgaacact cccatggttc ctcctccccg caccctcgga 1140

actgaagcca tgagcctcgg cttggccccc atcggtatca ccggcgcccc catgacaggt 1200

ttcggtgttc ctcctgagtt cggtcccttt ggagccgaag gtatcggcct ccccaccgat 1260

gccctcggca gcacccccga aatgacacca ttcgacccaa ctacccccta cagaagtctc 1320

gcccccatgg acctcccccc catcccccct cctgtcttcc ctgaaacccc tatgaggcca 1380

cctactccct tcggcttcgg acctgcacct gttcctccgt aa 1422

<210> 8

<211> 1737

<212> DNA

<213> Eimeria acervulina

<400> 8

atgggtgaag aggctgatac tcaggcgtgg gatacctcag tgaaggaatg gctcgtggat 60

acggggaagg tatacgccgg cggcattgct agcgtaagtt gttgtttgtt tttcttcctt 120

ttttgttgct gctgcttctt agaccttgct gcagcagctg ctcttttgta cacctgctgc 180

agcgaacttc gcaagcagca aacgcctcta tcaaatacac atcctgagaa aaagaggagg 240

gaggcccaac gagccatgct gctgctgctg ctgctgctgc tgctgctgat cgccgttctc 300

ctctcccttg cacacaagcc aactatccct tgtggtgttt tcgctgtttt cttcttctgt 360

ttcgtgctgc tctttccctg accaagtggc tgcgttgctt ccctgctacg gctgctgcac 420

cggtggctgc tgcagctgct gcagcaggag gaagagagct gctgatgctt ctttttttct 480

gcatgttata tgcggcttgt gctttattaa tgcacacaag gctagctaga aagagcagct 540

cagtgtgtgt gtggtggtgt tcgcttgatg aatgctgcag tgtagagttc aaagtgttta 600

cggatctgtc tttattatta ttattgttaa ttacctcctc tttcttttat ctttccctcg 660

ttgtatgaat ggcgttgttt gacttcaccc tccctccttt atttcgtccc caccagattc 720

tgattcacaa gtatatcatc tgcatgtgtg cattcatctt gtactctcat catgcatgtg 780

tgcacacata ttttcatgta tgtctgggta ttgtgtagaa tatatataaa atgtatgagt 840

gtgtgtgtgt gtgtatatat tcgagcagag tacgatgtgt gtgtacacac gaattatgct 900

ctgctcaaat gaacaacgta tatctgcatc tgtatatgta tatatatacg cacgtaaatg 960

cataaatgta tgtatgtata tattagtttc tgaagggcgc tgtttagtgt tggtgttagt 1020

atatggtatg gatttgctct ttgttcattc ttctctctgc gtgggtgtga tgatgtgctc 1080

ttagattgca gatgggtgcc gcctgtttgg cgctgcaata gacaatgggg aggatgcgtg 1140

gagtcagttg gtgaagacag gatatcagat tgaagtgctt caagaggacg gctcttcaac 1200

tcaagaggac tgcgatgaag cggaaaccct gcggcaagca attgttgacg gccgtgcccc 1260

aaacggtgtt tatattggag gagttaaata taaactcgca gaagttgtaa gtttccttca 1320

tactctagaa gaatagcgct tgctcatcca tggtgtcgtg cagtgggatg caatcgccac 1380

gcggggtgta cagacacctc aaagttgaat ggtagtaata atagtcatgt tcttcatgat 1440

gatggaataa gtgaataatt agggtgtttt gtgacggcgt tttcgttttt tttgtcattt 1500

tcgtcgtttc tctttgttta tttcgggccg atgatgcaga aacgtgattt cacctataac 1560

gaccagaact acgacgtggc gattttgggg aagaacaagg gtggcggttt cctgattaag 1620

actccgaacg acaatgtggt gattgctctt tatgacgagg agaaggagca gaacaaagca 1680

gatgcgctga caacggcact tgccttcgct gagtacctgt accagggcgg cttctaa 1737

<210> 9

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttccaggggc ccctgggatc cgaacctccc cgagtcaacc t 41

<210> 10

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cacgatgcgg ccgctcgaga tcctggaact ccctggacac c 41

<210> 11

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

caaggccatg gctgatatcg gcacctgagt atccttctca gcttg 45

<210> 12

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttgtcgacgg agctcgaatt gttgatgtcg gtaggctgct cttcc 45

<210> 13

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ttccaggggc ccctgggatc ccaggttcac ccttacagcg ag 42

<210> 14

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cacgatgcgg ccgctcgagg ttgccaaaga gcggagacg 39

<210> 15

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

agcaaatggg tcgcggatcc atgggtgaag aggctgatac 40

<210> 16

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tcgagtgcgg ccgcaagctt gaagccgccc tggtacaggt 40

Claims (8)

1. A preparation method of an anti-coccidiosis multivalent recombinant protein yolk antibody is characterized by comprising the following steps:

preparation of mixed antigens: selecting 4 chicken coccidian proteins: eimeria tenella rod body protein 41 (EtROP 41), eimeria tenella gametophyte protein 22 (EtGAM 22), eimeria maxima gametophyte protein 56 (EmGAM 56) and Eimeria acervulina merozoite protein Ea3-1E, the coding genes of the 4 kinds of the coccidian proteins are respectively connected with an Escherichia coli expression vector to obtain a recombinant expression vector, 4 kinds of recombinant proteins are expressed, and then the 4 kinds of recombinant proteins are combined into a mixed antigen in equal mass, wherein the coding genes of the 4 kinds of the coccidian proteins are obtained by amplification by using the following primers:

amplification of EtROP41 upstream primer: 5' -

TTCCAGGGGCCCCTGGGATCCGAACCTCCCCGAGTCAACCT-3’(SEQ ID NO.9)；

Amplification of EtROP41 downstream primer: 5' -

CACGATGCGGCCGCTCGAGATCCTGGAACTCCCTGGACACC-3’(SEQ ID NO.10)；

Amplification of EtGAM22 upstream primer: 5' -

CAAGGCCATGGCTGATATCGGCACCTGAGTATCCTTCTCAGCTTG-3’(SEQ IDNO.11)；

Amplification of EtGAM22 downstream primer: 5' -

TTGTCGACGGAGCTCGAATTGTTGATGTCGGTAGGCTGCTCTTCC-3’(SEQ IDNO.12)；

Amplification of EmGAM56 upstream primer: 5' -

TTCCAGGGGCCCCTGGGATCCCAGGTTCACCCTTACAGCGAG-3’(SEQ ID NO.13)；

Downstream primer for amplification of EmGAM 56: 5' -

CACGATGCGGCCGCTCGAGGTTGCCAAAGAGCGGAGACG-3’(SEQ ID NO.14)；

Amplifying Ea3-1E upstream primer: 5' -

AGCAAATGGGTCGCGGATCCATGGGTGAAGAGGCTGATAC-3’(SEQ ID NO.15)；

And (3) amplifying Ea3-1E downstream primer: 5' -

TCGAGTGCGGCCGCAAGCTTGAAGCCGCCCTGGTACAGGT-3’(SEQ ID NO.16)；

Immunization: mixing the mixed antigen with any adjuvant in equal mass, and immunizing the laying hens according to an immunization program; and

obtaining of yolk antibody: and (2) disinfecting eggs laid by the immunized laying hens, separating egg white and egg yolk, mixing the egg yolk with acetate buffer solution to obtain first mixed solution, sucking supernatant of the first mixed solution, mixing the supernatant with n-caprylic acid to obtain second mixed solution, and further filtering the second mixed solution to obtain the anti-coccidiosis multivalent recombinant protein egg yolk antibody.

2. The method for preparing an anti-chicken coccidia multivalent recombinant protein yolk antibody of claim 1, wherein the method for preparing the mixed antigen comprises the following steps:

gene amplification: performing gene amplification by using cDNA sequences of Eimeria tenella rod-shaped protein 41, eimeria tenella gametophyte protein 22, eimeria maxima gametophyte protein 56 and Eimeria acervulina merozoite protein Ea3-1E as templates;

constructing a recombinant vector: constructing a recombinant vector by using the amplified EtROP41 gene sequence and pGEX-6p-1 skeleton, constructing a recombinant vector by using the amplified EtGAM22 gene sequence and pET-28a skeleton, constructing a recombinant vector by using the amplified EmGAM56 gene sequence and pGEX-6p-1 skeleton, and constructing a recombinant vector by using the amplified Ea3-1E gene sequence and pET-28a skeleton;

recombinant protein expression: respectively transforming the 4 recombinant vectors into expression bacteria, and respectively carrying out induction expression and purification by IPTG; and

mixing: and (4) mixing the 4 chicken coccidian recombinant proteins subjected to induction expression with equal mass to obtain the chicken coccidian recombinant protein.

3. The method for preparing a yolk antibody against a multivalent recombinant protein of chicken coccidia as claimed in claim 1, wherein the immunization program comprises the following steps:

firstly, the method avoids: mixing the mixed antigen with Freund's complete adjuvant in equal mass, and performing subcutaneous injection on the breast of a hen aged 24-26 weeks;

and (2) avoiding: performing secondary immunization two weeks after the primary immunization, mixing the mixed antigen with Freund incomplete adjuvant in equal mass, and performing subcutaneous injection on the breast of the primary immunized hen; and

and (3) three-step (I): and performing tertiary immunization four weeks after the secondary immunization, mixing the mixed antigen with Freund's incomplete adjuvant in equal mass, and performing subcutaneous injection on the breast of the secondary-immunized hen.

4. The method for preparing the anti-Coccidium and multivalent recombinant protein yolk antibody of claim 3, wherein the titer of the yolk antibody in the eggs laid by the laying hen is detected after the laying hen is subjected to primary immunization, secondary immunization and tertiary immunization, wherein the detecting conditions are as follows: coating the mixed antigen, wherein the dilution ratio of the yolk antibody is 1 (15-25), and the dilution ratio of the enzyme-labeled antibody is 1.

5. The method for preparing a yolk antibody against the multivalent recombinant protein of chicken coccidia as claimed in claim 1, wherein in the step of obtaining the yolk antibody, the step of disinfecting the eggs comprises: sterilizing ovum gallus Domesticus in 0.1% benzalkonium bromide solution at 42 deg.C for 12-18min, sterilizing with sterile water at 95 deg.C for 3-7s, taking out, and wiping with 95% ethanol under sterile condition.

6. The method for preparing an anti-Coccidium gallinarum multivalent recombinant protein yolk antibody according to claim 1, wherein in the step of obtaining the yolk antibody, the pH value of the acetate buffer solution is 4.5, and the volume ratio of the yolk to the acetate buffer solution is 1.

7. Use of the yolk antibody prepared by the method of claims 1-6 in the preparation of a pharmaceutical preparation or feed for treating or preventing chicken coccidiosis.

8. The use of claim 7, wherein the pharmaceutical preparation is an injection or oral preparation comprising the egg yolk antibody.

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