An oral recombinant yeast expressing the apical membrane antigen of Eimeria tenella, a multi-component vaccine and its application

Abstract

The present invention provides a novel oral recombinant yeast vaccine expressing homologous molecules of Eimeria tenella apical membrane antigen, wherein the homologous molecules of Eimeria tenella apical membrane antigen (EtAMA1, EtAMA2, EtAMA3, EtAMA4) are expressed in the yeast vaccine, which can significantly inhibit the invasion of sporozoites. The oral recombinant yeast vaccine realizes the efficient expression of EtAMA1-4 in the saccharomyces cerevisiae expression system, has a good immune protection effect, can be used for the immune prevention of Eimeria tenella infection, and can be used for the preparation of drugs and vaccines for the treatment of chicken coccidiosis.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to an oral recombinant yeast expressing an Eimeria tenella apical membrane antigen, a monoclonal antibody against the Eimeria tenella apical membrane antigen, a multi-component vaccine containing the two active ingredients, and applications thereof. Background Art

[0002] Chicken coccidiosis is a parasitic protozoan disease caused by one or more Eimeria spp. coccidia parasitizing the intestinal epithelial cells of chickens. It is one of the most harmful epidemics in intensive farming, causing huge economic losses to the poultry industry worldwide and seriously restricting the healthy development of the industry. Globally, the cost of chicken coccidiosis prevention and control has increased from US$800 million in 2002 to US$3 billion in 2006. By 2020, the global loss has been re-estimated to be about 10.4 billion pounds per year. Practice has proved that vaccination can effectively control coccidiosis and is the development trend of coccidiosis prevention and control under the background of banning and limiting antibiotics. However, the commercial coccidiosis vaccines currently widely used are all live vaccines, composed of 3-8 live coccidia oocysts, which affect production performance after immunization, and have shortcomings such as toxicity dispersion, cumbersome production process, and high price. Therefore, it is urgent to develop a new oral coccidiosis vaccine.

[0003] Apical membrane antigens (AMAs) are a class of highly conserved micronemes in apicomplexan protozoa. They can interact with rhoptry neck proteins 2 (RON2s) secreted by rhoptry bodies to form a complex (AMAs-RON2s). The complex is distributed in the moving juncture (MJ), which is a key structure for the parasite to invade cells. In recent years, studies have found that Eimeria tenella (Et) has four AMA homologous molecules (AMA1-4). In previous studies, our laboratory found that EtAMA1 and EtAMA3 are regulated in the sporozoite stage, and their antibodies have a significant inhibitory effect on the invasion of sporozoites; EtAMA2 is expressed in the merozoite stage, and the inhibitory effect on the invasion of sporozoites is not obvious; EtAMA4 is expressed in both the merozoite and sporozoite stages, and its antibodies have a significant inhibitory effect on the invasion of sporozoites. EtAMAs can interact with corresponding RON2s: EtRON2 interacts with EtAMA1, EtRON2L1-1 interacts with EtAMA4, EtRON2L1-2 interacts with EtAMA4, and EtRON2L2 interacts with EtAMA3. Studies have shown that EtAMAs are key proteins for coccidia invasion and can be used as candidate antigen molecules for vaccines.

[0004] Yeasts such as Saccharomyces cerevisiae have long been used for the production of recombinant soluble proteins for use in vaccination and other applications. Recently, Saccharomyces cerevisiae has been used as an oral vaccine delivery vehicle in the form of fully recombinant yeast (live or dead), which can achieve multiple purposes such as antigen expression, transportation, and as an adjuvant. In addition to the convenient immunization method, oral immunization with recombinant Saccharomyces cerevisiae can also promote direct interaction between the vaccine and the intestinal mucosal surface at the site of coccidia invasion, stimulate immune responses through pattern recognition receptors (PRRs), and do not rely on adjuvants. In addition, Saccharomyces cerevisiae and its cell wall derivatives have been used as feed additives for poultry. Even without the addition of vaccine antigens, Saccharomyces cerevisiae and its derivatives alone can significantly reduce the production of coccidia oocysts and improve production performance during coccidia infection. Existing studies have shown that oral immunization of chickens with live Saccharomyces cerevisiae expressing coccidia micronematode proteins can effectively reduce the amount of oocysts discharged and the degree of cecal lesions. Finally, recombinant yeast can be inactivated, which is generally recognized as safe (GRAS), and inactivation can enhance the antigenicity of yeast lines by altering the distribution and exposure of yeast cell surface structures such as β-1,3 glucan.

[0005] In short, yeast vector vaccines have attracted attention in recent years due to their significant advantages in terms of safety, immunization methods, and production costs. However, yeast vaccines also have problems such as low titer and poor stability. When immunizing with subunit vaccines and DNA vaccines in the prior art, the reproduction of parasites and the degree of intestinal lesions can be significantly reduced. For example, after three times of subcutaneous or intramuscular injection of recombinant protein, the production of oocysts (OPG) after low-dose attack can be significantly reduced. However, the delivery method of recombinant vaccines, which is intramuscular injection or other immunization methods for each chicken, is not suitable for large-scale vaccination of chickens on site, and is therefore not conducive to commercialization. Therefore, there is an urgent need for a multi-component vaccine that combines the above-mentioned multiple immune substances, which can be used for immunization delivery from multiple angles and levels, and has strong potential application value. Summary of the invention

[0006] The purpose of the present invention is to provide an application of four AMA homologous molecules (EtAMA1, EtAMA2, EtAMA3, EtAMA4) of Eimeria tenella, which is used to inhibit the invasion of sporozoites. Among them, EtAMA1 and EtAMA3 are regulated and expressed in the sporozoite stage, and their antibodies have a significant inhibitory effect on the invasion of sporozoites; EtAMA4 is expressed in both the merozoite and sporozoite stages, and its antibodies have a significant inhibitory effect on the invasion of sporozoites. The above-mentioned EtAMAs molecules can interact with the corresponding RON2s molecules as a whole yeast vaccine.

[0007] Furthermore, the present invention provides an oral recombinant yeast vaccine, which expresses four AMA homologous molecules (EtAMA1, EtAMA2, EtAMA3, and EtAMA4) of Eimeria tenella.

[0008] Furthermore, the present invention provides a method for preparing the above-mentioned oral recombinant yeast vaccine, which is as follows: (1) amplification of four AMA homologous molecules (EtAMA1, EtAMA2, EtAMA3, and EtAMA4) of Eimeria tenella: primers are designed according to the cDNA sequence of EtAMA, and when designing the EtAMA target fragment, homologous arm sequences of the pYD1 vector are introduced at both ends of the primers; while amplifying the target fragment, the vector PYD1 is double-digested and then recovered and connected, and the correctly connected plasmid is screened; (2) EBY100 competent cells are prepared; (3) yeast transformation and identification, through yeast genomic DNA extraction, PCR identification, and 1% agarose gel electrophoresis detection, to determine the positive surface-displayed brewer's yeast vector; (4) expression of surface-displayed brewer's yeast protein; (5) preparation of oral vaccine.

[0009] Preferably, the primers in step (1) are specifically:

[0010] AMA1 5'-GGTGGTGGTGGGTTCTGCTAGCCACAAGCTGCAGCACAGGC-3'(NheⅠ)(SEQ ID

[0011] NO.1)

[0012] 5'-TGCTGGATATCTGCAGAATTCTTAGACACCGCCCCCTTTAGACT-3'(EcoRⅠ)(SEQ

[0013] ID NO.2)

[0014] AMA2 5'-GGTGGTGGTGGTTCTGCTAGCCAAAAGTTCAACATTCCATACACTCA-3'(NheⅠ)

[0015] (SEQ ID NO.3)

[0016] 5'-TGCTGGATATCTGCAGAATTCTTACTGCTGCAGTTGCAGTCGTCG-3'(EcoRⅠ)(SEQ

[0017] ID NO.4)

[0018] AMA3 5'-GGTGGTGGTGGTTCTGCTAGCGCAGCGCTAGAGATGCTGCA-3'(NheⅠ)(SEQ ID

[0019] NO.5)

[0020] 5'-GCCCTCTAGACTCGAGCGGCCGCTTACCAGTTGTTATTTTCTCCTTCTTGTT-3'

[0021] (Not Ⅰ) (SEQ ID NO.6)

[0022] AMA4 5'-GGTGGTGGTGGTTCTGCTAGCAACCGTTTTAATATACCTGCAAATCA-3'(NheⅠ)

[0023] (SEQ ID NO.7)

[0024] 5'-GCCCTCTAGACTCGAGCGGCCGCTTATGCAGCATTGCTGCTTCCT-3'(NotⅠ)(SEQ

[0025] ID NO.8)

[0026] Preferably, the step (3) is specifically as follows: add Y3 and recombinant plasmid to yeast competent cells respectively, mix thoroughly, water bath, centrifuge and take supernatant. Resuspend bacterial precipitate, apply screening plate (SD / Trp) and culture at 30°C until typical colonies are formed. Pick a single yeast colony and inoculate it with an inoculation loop into YNB-CAA liquid culture medium containing 2% glucose, and increase the bacteria at 30°C and 250rpm for 13 h. Yeast genomic DNA was extracted, PCR identified, and 1% agarose gel electrophoresis was used to detect the positive surface display type Saccharomyces cerevisiae vector, and named EBY100 / pYD1-EtAMA1, EBY100 / pYD1-EtAMA2, EBY100 / pYD1-EtAMA3, and EBY100 / pYD1-EtAMA4.

[0027] Preferably, the step (4) is to take the correctly identified monoclonal colonies of EBY100 / pYD1-EtAMA1, EBY100 / pYD1-EtAMA2, EBY100 / pYD1-EtAMA3, and EBY100 / pYD1-EtAMA4, inoculate them in a YNB-CAA liquid medium containing 2% glucose, and culture them at 30°C and 250 rpm until the OD600nm is between 2.0 and 5.0. According to the formula, the amount of bacterial liquid transferred is calculated and inoculated in a YNB-CAA liquid medium containing 2% galactose, and induced at 20°C and 250 rpm for 72 h.

[0028] The formula is: Calculation method of transfer volume of 2% glucose YNB-CAA bacterial liquid: Assuming that 10 mL of YNB-CAA liquid culture medium containing 2% galactose is newly used, then: transfer volume×OD600nm value=(10+transfer volume)×0.75.

[0029] Preferably, the step (5) is:

[0030] Preparation of live vaccine: EBY100 / pYD1-EtAMA1, EBY100 / pYD1-EtAMA2, EBY100 / pYD1-EtAMA3, and EBY100 / pYD1-EtAMA4 were induced with YNB-CAA solution containing 2% galactose. After 72 h of induction, the Saccharomyces cerevisiae was counted using a hemocytometer and the number of cells was calculated based on 1×10 8 / tube, centrifuge to remove supernatant, wash three times with PBS, and finally resuspend with 1mL sterile PBS for later use. Live vector vaccine should be used immediately after induction.

[0031] Alternatively, the preparation method of the inactivated vaccine is as follows: inactivate part of the above-mentioned packaged yeast in a 95°C water bath for 20 minutes, and resuspend it with an appropriate amount of 1 mL of sterile PBS. In the clean bench, use an inoculation loop to take a small amount of bacterial liquid and streak it on the YPD solid culture medium, and place it in a 30°C constant temperature incubator for culture to detect the inactivation effect. After centrifugation of the successfully inactivated Saccharomyces cerevisiae carrier bacterial liquid, the bacterial precipitate is stored at -80°C for use, thawed and resuspended with sterile PBS before use.

[0032] Furthermore, the present invention provides a monoclonal antibody 1G5 that can specifically recognize the EtAMA4 protein, and the amino acid sequence of the light chain variable region of the monoclonal antibody is:

[0033]

[0034] The amino acid sequence of the heavy chain variable region is:

[0035]

[0036] The bold underlined ones are CDR1-3.

[0037] Furthermore, the present invention also provides an application of the above protein, antibody or oral recombinant yeast vaccine for preparing a vaccine or medicine for preventing or treating chicken coccidiosis.

[0038] The vaccine or drug works by inhibiting the sporozoites of Eimeria tenella from invading host cells and blocking the life cycle of coccidia.

[0039] Beneficial Effects

[0040] The present invention provides an oral recombinant yeast expressing the apical membrane antigen of Eimeria tenella, a monoclonal antibody against the apical membrane antigen of Eimeria tenella, a multi-component vaccine containing the above two effective ingredients and their application. The oral recombinant yeast expresses four AMA homologous molecules (EtAMA1, EtAMA2, EtAMA3, EtAMA4) of Eimeria tenella, causing changes in the morphology of sporulated oocysts and a reduction in toxicity. At the same time, the monoclonal antibody against the above AMA provided in the present application can effectively recognize and bind to the above AMA protein, and has a significant inhibitory effect on the invasion of sporozoites. The two vaccine forms cooperate with each other, can simultaneously exert their advantages, overcome corresponding defects, have good immune protection effects through synergistic effects, can be used for immune prevention of Eimeria tenella infection, and can be used to prepare drugs and vaccines for the treatment of chicken coccidiosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 For target gene amplification, A: AMA1; B: AMA2; C: AMA3; D: AMA4;

[0042] Figure 2 The electrophoresis diagram of double enzyme digestion of recombinant plasmids, where A: AMA1; B: AMA2; C: AMA3; D: AMA4;

[0043] Figure 3 This is the growth curve of the surface display Saccharomyces cerevisiae expression vector;

[0044] Figure 4 PCR identification of the target gene integrated into the EBY100 genome. Among them, A.pYD1; B.AMA1; C.AMA2; C.AMA3; D.AMA4;

[0045] Figure 5 Immunofluorescence analysis of surface-displayed Saccharomyces cerevisiae, where A: negative control; B: EtAMA1; C: EtAMA2; D: EtAMA3; E: EtAMA4;

[0046] Figure 6Flow cytometric analysis of surface-displayed Saccharomyces cerevisiae, where A: negative control; B: EtAMA1; C: EtAMA2; D: EtAMA3; E: EtAMA4.

[0047] Figure 7 These are the antibody levels during vaccination, including: A: EtAMA1 serum IgG; B: EtAMA4 serum IgG; C: EtAMA1 intestinal IgG; D: EtAMA4 intestinal IgG; E: EtAMA1 intestinal sIgA; F: EtAMA4 intestinal sIgA.

[0048] Figure 8 It is the level of CD4+ and CD8+ T lymphocytes during vaccine immunization, where A: CD4; B: CD8;

[0049] Fig. 9 Cytokine levels during vaccine immunization, including: A: IL-6; B: IL-17; C: INF-γ; D: IL-18. DETAILED DESCRIPTION

[0050] The present invention is further described by way of the following examples, which are not intended to limit the present invention in any way. The following examples are used to further illustrate the essential content of the present invention, but the content of the present invention is not limited thereto.

[0051] Example 1 Construction and identification of surface display Saccharomyces cerevisiae expression vector

[0052] 1. Amplification of target gene

[0053] Primers were designed according to the selected target gene sequence and the restriction site characteristics of the pYD1 expression vector. NheⅠ restriction site was designed in the upstream primers of AMA1 and AMA2 genes, and EcoRⅠ restriction site and stop codon TAA were designed in the downstream primers; NheⅠ restriction site was designed in the upstream primers of AMA3 and AMA4 genes, and NotⅠ restriction site and stop codon TAA were designed in the downstream primers. The primer design is shown in Table 1 (underlined are restriction sites):

[0054] Table 1 List of PCR primers

[0055]

[0056] Using the purified first strand of E. tenella sporulated oocyst cDNA as a template, EtAMAs1 / 2 / 3 / 4 were amplified by PCR. The PCR reaction system was as follows:

[0057]

[0058]

[0059] Use the following PCR procedure to amplify the target gene:

[0060]

[0061] The sizes of the target fragment PCR products of EtAMA1, EtAMA2, EtAMA3, and EtAMA4 were 1266 bp (SEQ ID NO.11), 1173 bp (SEQ ID NO.12), 1416 bp (SEQ ID NO.13), and 2511 bp (SEQ ID NO.14), respectively. Figure 1 shown.

[0062] 2. Extraction and double restriction enzyme digestion of pYD1 plasmid

[0063] Extract and purify the pYD1 plasmid according to the instructions of the plasmid extraction kit. Double digest the extracted pYD1 plasmid with NheⅠ and EcoRⅠ, and double digest with NheⅠ and NotⅠ. The enzyme digestion system is as follows:

[0064]

[0065] The enzyme digestion products were detected by 1% agarose gel electrophoresis, and the linearized vector was recovered by gel excision using a gel recovery kit.

[0066] 3. Construction and identification of recombinant plasmid pYD1-AMAs

[0067] The pYD1 plasmid double enzyme digestion gel recovery product and the target gene fragment were connected using T4 DNA ligase. The connection system is as follows:

[0068]

[0069] The ligation product was transformed into E. coli Trans1-T1 competent cells, heat-shocked, cooled, added to LB-enriched bacteria without resistance, coated with AMP-resistant LB plates, and inverted and cultured at 37°C overnight. A single colony was inoculated in LB+Amp liquid culture medium and cultured for 6 hours, and the bacterial liquid was identified by PCR. The PCR reaction system is as follows:

[0070]

[0071] After the above system is fully mixed, the PCR reaction is carried out according to the following PCR amplification procedure:

[0072]

[0073] The obtained PCR products were detected by 1% agarose gel electrophoresis, and the bacterial solution identified by PCR was cultured overnight for plasmid extraction and purification. The obtained recombinant plasmids were named pYD1-EtAMA1, pYD1-EtAMA2, pYD1-EtAMA3, and pYD1-EtAMA4. The four recombinant plasmids were identified by double enzyme digestion, and the double enzyme digestion system was the same as above. The plasmids identified by double enzyme digestion were sent to Sangon Biotech Co., Ltd. for sequencing, and the pYD1 expression vector universal primers were used for sequencing.

[0074] The results of double restriction digestion of recombinant plasmid are as follows Figure 2 As shown, two bands appeared, which was consistent with the expected pYD1 plasmid and the length of EtAMA1, EtAMA2, EtAMA3, and EtAMA4 genes. Double restriction enzyme digestion identification of the recombinant plasmid indicated that the target gene was successfully cloned into the surface display plasmid pYD1.

[0075] 4. Preparation of Saccharomyces cerevisiae EBY100 competent cells

[0076] 1) Activate the bacteria: Streak the bacteria stored at -80°C on a YPDA medium plate and culture at 30°C for 2-4 days.

[0077] 2) Pick a single yeast colony and streak a 3-5 mm short line on a YPDA medium plate, and culture at 30°C for 2-4 days.

[0078] 3) When the diameter of a single yeast colony grows to 2 mm, inoculate the yeast cells into 3 mL of liquid YPDA medium and culture at 30°C overnight.

[0079] 4) The next day, transfer the cells to a flask containing 50 mL of YPDA medium and continue culturing until OD 600 When the concentration reaches 0.4-0.5, centrifuge at 5000 rpm for 5 min and discard the supernatant.

[0080] 5) Resuspend the precipitate with 10 mL of Y1 solution, centrifuge at 5000 rpm for 5 min, and discard the supernatant.

[0081] 6) Add 1 mL of Y2 solution to resuspend, and dispense into 1.5 mL sterile cryovials at 50 μL / tube. The competent cells are now ready and stored at -80°C for later use.

[0082] 5. Construction of recombinant yeast EBY100 / pYD1-AMAs

[0083] Transform the above sequenced plasmids into Saccharomyces cerevisiae EBY100 competent cells, and transform the pYD1 plasmid as a negative control. Add 360 μL of transformation premix (350 μL Y3 solution, 2 μL recombinant plasmid, 8 μL ddH2O) to 50 μL yeast competent cells, incubate at 30°C water bath for 1 h, centrifuge at 12000 rpm for 15 s, and discard the supernatant. Resuspend the bacterial pellet with 400 μL of saline, apply yeast screening medium (SD-Trp), and culture at 30°C for 2-4 days. Pick a single colony on the culture medium and culture it overnight at 30°C and 250 rpm in 5 mL of 2% glucose YNB-CAA liquid medium. The obtained bacterial suspensions were named EBY100 / pYD1-EtAMA1, EBY100 / pYD1-EtAMA2, EBY100 / pYD1-EtAMA3, EBY100 / pYD1-EtAMA4 and EBY100 / pYD1, respectively.

[0084] Example 2 Screening and preparation of monoclonal antibodies that specifically recognize EtAMAs proteins

[0085] Based on step 1 in Example 1, the product was improved by using the primers to carry out PCR amplification and connected to the pENTR-4 prokaryotic expression vector to express EtAMA1, EtAMA2, EtAMA3, and EtAMA4 proteins respectively. The constructed recombinant plasmid was induced by IPTG to express the recombinant protein, which is summarized as follows:

[0086] (1) Take the correctly identified recombinant plasmids pColdI-EtAMA1, pColdI-EtAMA2, pColdI-EtAMA3, and pColdI-EtAMA4 (the same below, no further description) and transform them into 100 μL Rosseta competent cells. Pick a single colony and inoculate it in 4 mL LB liquid medium (containing Amp 100 μg / mL) and culture it at 37°C, 225 rpm overnight.

[0087] (2) The overnight culture solution was inoculated into 3 mL LB liquid medium (Amp 100 μg / mL) at a ratio of 1:100 and cultured at 37°C, 225 rpm for 3 h. When the OD value reached 0.4-0.6, IPTG was added at a final concentration of 0.5 mM and cultured at 16°C, 225 rpm for 8 h.

[0088] (3) Transfer the bacterial solution to a centrifuge tube and centrifuge at 10,000 rpm / min for 5 min. Discard the supernatant and wash twice with sterile PBS. Resuspend the bacterial pellet in an appropriate amount of PBS and disrupt it by ultrasonication on ice at 30 Hz for 7 min. Centrifuge at 10,000 rpm / min for 5 min, collect the supernatant, and resuspend the pellet in 80 μL of sterile PBS.

[0089] (4) Take the supernatant and precipitate of the induced recombinant protein for SDS-PAGE analysis, add an equal amount of 2× Loading Buffer, mix thoroughly, cook the sample at 95°C for 5 min, ice bath for 2 min, load 20 μL into each well, and keep constant voltage at 80 V to separate the gel, and keep constant voltage at 120 V for 1.5 hours.

[0090] (5) After electrophoresis, the electrophoresis gel was carefully peeled off and stained with Coomassie Brilliant Blue for 1 h. The color was fully destained until the protein bands were clear and the results were observed. The results showed that the above four recombinant proteins were successfully expressed.

[0091] (5) According to the "Molecular Biology", the above four recombinant proteins were induced to express in large quantities using conventional expression conditions. The expression products were purified by HisTrapF column chromatography and concentrated by dialysis. An appropriate amount of purified protein was taken and analyzed for purification effect by SDS-PAGE. The remaining protein was measured for concentration and stored at -20°C for future use.

[0092] (6) Select 6-week-old BALB / c female mice and immunize them with purified recombinant protein. First immunization: Mix the protein with Freund's complete adjuvant in a 1:1 ratio, emulsify it with a syringe to a state of "oil-in-water, water-in-oil", and inject 0.2 mL subcutaneously to each mouse. Second immunization: Immunize again 2 weeks later, mix it with Freund's incomplete adjuvant in a 1:1 ratio, emulsify it with a syringe to a state of "oil-in-water, water-in-oil", and inject 0.2 mL subcutaneously to each mouse. Third immunization: Perform the third immunization 2 weeks after the second immunization, using the same method as the second immunization; 10 days after the third immunization, the mice were tail-cut and blood was collected, and the serum was separated and the antibody level in the serum was determined by indirect ELISA. After the serum antibody titer reached the ideal titer, prepare for cell fusion. 3 days before fusion, perform booster immunization. The protein solution is injected directly into the peritoneum without adding any adjuvant, 0.2 mL / mouse.

[0093] (7) Three days after booster immunization, spleen cells were taken and cell fusion was performed with mouse myeloma cells SP2 / 0 according to conventional experimental methods. When the cells in the culture plate grew to 1 / 3 of the bottom area, the cell culture supernatant was aspirated and positive hybridoma cells were screened using the established ELISA method. After three screenings, the cell wells with positive ELISA readings were selected for expansion culture. The positive hybridoma cells were subcloned using the limiting dilution method until all the subcloned cells screened were positive and monoclonal. Finally, a hybridoma cell line 1G5 that could stably secrete monoclonal antibodies was obtained, which could specifically recognize the EtAMA4 protein, and the cells were expanded and frozen in time.

[0094] (8) BALB / c mice aged 10-12 weeks were selected to prepare monoclonal antibody ascites, and the ascites was purified using a Protein G affinity chromatography column medium. The purity of the monoclonal antibody was identified by SDS-PAGE electrophoresis. The purified monoclonal antibody was sent to BGI for antibody sequence sequencing, and the final sequence obtained was:

[0095] The amino acid sequence of the light chain variable region is:

[0096]

[0097] The amino acid sequence of the heavy chain variable region is:

[0098]

[0099] (9) Monoclonal antibody titer determination

[0100] Using EtAMA4 protein as the coating source, the established ELISA method was used to detect the serum antibody titer of hybridoma cell supernatant and mouse ascites. Negative and positive mouse serum control wells were set up, and the hybridoma cell supernatant was diluted with PBS from 1:100, and the ascites first well was diluted with PBS from 1:1 000. Finally, the cell supernatant titer of 1G5 monoclonal antibody was 1:25600, the ascites titer before purification was 1:4 096 000, and the ascites titer after purification was 1:512 000.

[0101] (10) Specificity detection of monoclonal antibodies

[0102] Take EtAMA4 protein, ultrasonically disrupted gametocyte protein samples of Eimeria toxicophila, Eimeria tenella, Eimeria acervulina and Eimeria maxima, add 6× loading buffer and mix well, boil in boiling water on an induction cooker for 10-15 minutes, centrifuge at 12000rpm for 5 minutes, take the sample supernatant and add it to the lane for SDS-PAGE and transfer it to a nitrocellulose membrane. After the transfer, place it in 3% BSA at 4°C overnight for blocking, use the hybridoma cell supernatant as the primary antibody, incubate at room temperature for 50 minutes, wash 4 times with PBST, 5 minutes per time; the secondary antibody is HRP-labeled goat anti-mouse IgG, incubate at room temperature for 40 minutes, wash 4 times with PBST, 5 minutes per time; after ECL color development, use the Tanon5200 fully automatic chemiluminescence imaging analysis system to observe the results and scan and take pictures. The results showed that the 1G5 monoclonal antibody specifically recognized the EtAMA4 protein recombinant protein, and only specifically recognized the gametocyte protein of Eimeria tenella, but not the natural gametocyte protein of Eimeria toxicophila, Eimeria acervulina, and Eimeria maxima. The results showed that it had good specificity.

[0103] Example 3 Expression and identification of surface displayed Saccharomyces cerevisiae proteins

[0104] 1. Induced expression and growth curve determination

[0105] 1) Pick a single clone colony on the yeast screening medium and inoculate it into 5 mL of YNB-CAA liquid medium containing 2% glucose. Cultivate it at 30°C and 250 rpm for 16-18 h. Detect the OD by spectrophotometer. 600nm is 2.0.

[0106] 2) Take 3mL of the above OD 600nm The bacterial liquid with a concentration of 2.0 was inoculated into 5 mL of YNB-CAA liquid culture medium containing 2% galactose, and induced culture was carried out at 20°C and 250 rpm for 72 h.

[0107] Calculation method for transferring 2% glucose YNB-CAA culture medium: Assuming that 10 mL of 2% galactose YNB-CAA liquid medium is used, then: Transfer volume × OD 600nm Value = (10 + transfer amount) × 0.75

[0108] 3) During the induction culture, the OD values ​​at 0h, 12h, 24h, 36h, 48h, 60h, and 72h were measured by a visible light spectrophotometer. 600nm Values, 3 replicates for each sample.

[0109] 4) Draw the growth curve of brewer’s yeast in different time periods.

[0110] The results are as follows Figure 3 As shown, the results showed that the growth curves of different strains were similar and there was no difference in doubling time, indicating that the display of exogenous proteins (EtAMA1, EtAMA2, EtAMA3, EtAMA4) did not change the growth characteristics of yeast cells and had no effect on their growth.

[0111] 2. Genomic PCR Identification

[0112] Take 1 mL of bacterial solution after 72 hours of induction, extract yeast recombinant plasmid genomic DNA according to the instructions of the yeast genome extraction kit, use the extracted genomic DNA as a template, and perform PCR identification with pYD1 forward and reverse sequencing primers and target gene forward and reverse primers respectively.

[0113] The results are as follows Figure 4As shown in the figure, when the pYD1 forward and reverse primers were used for PCR, the DNA bands were 1587bp, 1494bp, 1711bp, and 2806bp respectively; while when the specific primers of the EtAMA1-4 genes were used for PCR, the DNA bands were 1266bp, 1176bp, 1416bp, and 2511bp respectively. The target bands amplified by the two primers EtAMA1 and EtAMA2 differed by exactly 321bp, and the target bands amplified by the two primers EtAMA3 and EtAMA4 differed by exactly 295bp, which indicated that the recombinant plasmids pYD1-EtAMA1, pYD1-EtAMA2, pYD1-EtAMA3, and pYD1-EtAMA4 had been successfully integrated into the genome of S.cerevisiae EBY100.

[0114] 3. Indirect Immunofluorescence and Flow Cytometry Analysis

[0115] Take 50 μL of EBY100 / pYD1 (negative control), EBY100 / pYD1-EtAMA1, EBY100 / pYD1-EtAMA2, EBY100 / pYD1-EtAMA3, and EBY100 / pYD1-EtAMA4 after 72 h of induction. 600nm Place the bacterial solution of 2.0 in a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 5 min, discard the supernatant, wash the precipitate with PBS three times, centrifuge at 4°C, 6000 rpm for 5 min each time. Dilute rabbit anti-EtAMAs polyclonal antibody with PBS at 1:100 and incubate overnight. Centrifuge at 12000 rpm for 5 min, discard the supernatant, wash the precipitate with PBS, centrifuge at 4°C, 6000 rpm for 5 min. Add 500 μL of FITC-labeled goat anti-rabbit IgG diluted 1:800 with PBS, and incubate at 37°C in the dark for 1 h. After washing the secondary antibody, resuspend the precipitate with 50 μL PBS, take 5 μL of bacterial suspension and drop it on a clean slide, cover with a coverslip, and observe under a fluorescence microscope.

[0116] The results are as follows Figure 5 As shown, EBY100 / pYD1 yeast cells did not show specific green fluorescence, while EBY100 / pYD1-EtAMA1, EBY100 / pYD1-EtAMA2, EBY100 / pYD1-EtAMA3, and EBY100 / pYD1-EtAMA4 yeast cells all showed specific fluorescence, indicating that each target protein was successfully displayed on the surface of Saccharomyces cerevisiae EBY100.

[0117] Add 300 μL PBS to the remaining bacterial solution and resuspend evenly before analyzing by flow cytometry. Figure 6As shown, EBY100 / pYD1 was used as a negative control, and the expression efficiency of EtAMA1, EtAMA2, EtAMA3, and EtAMA4 proteins on the surface of Saccharomyces cerevisiae EBY100 was 28.28%, 53.62%, 72.23%, and 29.92%, respectively. Since the antibody cannot enter the yeast cells, the results confirmed that EtAMA1, EtAMA2, EtAMA3, and EtAMA4 proteins were displayed on the surface of yeast cells.

[0118] Example 4 Evaluation of the immune effect of oral recombinant Saccharomyces cerevisiae vaccine

[0119] 1. Preparation of Oral Vaccine

[0120] Live vaccine: EBY100 / pYD1 (negative control), EBY100 / pYD1-EtAMA1, EBY100 / pYD1-EtAMA2, EBY100 / pYD1-EtAMA3, and EBY100 / pYD1-EtAMA4 were induced with YNB-CAA solution containing 2% galactose. After 72 h of induction, the Saccharomyces cerevisiae was counted using a hemocytometer and the number of cells was calculated based on 1×10 8 / tube, centrifuge to remove supernatant, wash three times with PBS, and finally resuspend with 1 mL sterile PBS for later use. Live vector vaccine should be used immediately after induction.

[0121] Inactivated vaccine: Inactivate part of the above-mentioned yeast in a 95℃ water bath for 20 min, and resuspend it with an appropriate amount of 1mL sterile PBS. Use an inoculation loop to take a small amount of bacterial liquid in the clean bench and streak it on the YPD solid medium, and then place it in a 30℃ constant temperature incubator to culture to detect the inactivation effect. After centrifugation, the bacterial precipitate of the successfully inactivated Saccharomyces cerevisiae carrier was stored at -80℃ for future use. Thaw it before use and resuspend it with sterile PBS.

[0122] 2. Animal Experiment Design and Immunization Procedure

[0123] The 3-day-old chicks were weighed, and the oversized or undersized chicks were removed. The chicks with basically the same weight were selected and randomly divided into 12 groups, with 20 chickens in each group, namely the healthy control group, the infected control group, the EBY100 / pYD1-live, EBY100 / pYD1-inactivated, EBY100 / pYD1-AMA1-live, EBY100 / pYD1-AMA1-inactivated, EBY100 / pYD1-AMA2-live, EBY100 / pYD1-AMA2-inactivated, EBY100 / pYD1-AMA3-live, EBY100 / pYD1-AMA3-inactivated, EBY100 / pYD1-AMA4-live, and EBY100 / pYD1-AMA4-inactivated groups. Except for the healthy control group and the infected control group, which were orally administered with 100 μL PBS, the other groups were orally administered with 1×108 Starting from the 3rd day of age, immunization was performed every 3 days for a total of 6 times. At the 20th day of age, each chicken was inoculated with 1×10 4 Fresh E. tenella sporulated oocysts.

[0124] 3. Weight gain effect detection

[0125] The weight of each group of chickens was weighed and recorded on the 3rd day, 20th day (before insect challenge) and 28th day (7th day after insect challenge) to calculate the average weight gain and relative weight gain rate to evaluate the weight gain effect.

[0126] Average weight gain 1 = body weight before attacking - body weight at 3 days old; Average weight gain 2 = body weight after attacking - body weight before attacking; Weight gain rate = (average body weight 8 days after attacking - average body weight at the time of attacking) / average body weight at the time of attacking × 100%.

[0127] Relative weight gain rate = (weight gain of experimental group / weight gain of healthy control group) × 100%.

[0128] The weight gain during the immunization period is shown in Table 2. It can be seen from the table that compared with the healthy control group, the weight gain of all target protein expression groups and the pYD1 empty vector group during the immunization period was slightly increased, but the difference was not significant (P>0.05). Compared with the pYD1 empty vector group, the weight gain of the AMA1 live bacteria group, the AMA3 live bacteria group and the AMA4 live bacteria group was slightly higher than the empty vector live bacteria group, and the weight gain of the AMA2 inactivated group and the AMA3 inactivated group was slightly higher than the empty vector inactivated bacteria group. The other groups were basically equivalent to the empty vector. The above results show that oral administration of live and inactivated yeast expressing or not expressing AMAs can promote the growth of chickens, among which the AMA2 inactivated group and the AMA3 inactivated group have the best effect.

[0129] Table 2 Weight gain of chickens during immunization.

[0130]

[0131] The weight gain after attacking is shown in Table 3. It can be seen from the table that the weight gain of all target protein expression groups and pYD1 empty vector groups during attacking was significantly higher than that of the infected control group (P<0.05). Compared with the empty vector group, the weight gain of AMA1, AMA2 and AMA4 live bacteria groups was significantly higher than that of the empty vector live bacteria group (P<0.05); the weight gain of AMA1 inactivated group was significantly higher than that of the empty vector inactivated group (P<0.05), and the weight gain of the other groups was equivalent to that of the corresponding empty vector (P>0.05). The above results show that oral administration of live and inactivated yeast expressing or not expressing AMAs has a certain resistance to weight loss caused by coccidia infection, among which the live and inactivated AMA1 groups have the best effect.

[0132] Table 3 Weight gain of chickens after challenge.

[0133]

[0134]

[0135] 4. Scoring of bloody stools and cecal lesions

[0136] The bloody stools were scored on the 5th day after the parasite attack, which was the sum of the number of bloody stools in the feces of each group of chickens.

[0137] Chickens were killed on the 8th day after infection, and cecal tissues were removed for cecal lesion scoring according to the method reported by Johnson and Reid (1970), with lesion value = average lesion score per group × 10.

[0138] The results are shown in Table 4. As can be seen from the table, no death occurred in all groups, and the survival rate was 100%. The AMA1 live bacteria group did not have bloody stools, while the other infection groups all had certain bloody stools. In terms of intestinal lesions, the intestinal lesions of all target protein-expressing live bacteria groups were comparable to those of the pYD1 empty vector live bacteria group (P>0.05); the lesions of the AMA1 inactivated group and the AMA2 inactivated group were significantly lower than those of the pYD1 empty vector inactivated group (P<0.05), and the lesions of the AMA3 inactivated group were extremely significantly lower than those of the pYD1 empty vector inactivated group (P<0.01).

[0139] Table 4 Survival, bloody stool, and intestinal lesion scores after challenge.

[0140]

[0141] 5. Oocysts per gram of feces (OPG)

[0142] The chicken feces of each group were collected on the 6th, 7th and 8th day after the attack, and after mixing evenly, 2 g of chicken feces was weighed from each group, and 58 mL of tap water was added. After fully mixing, the mixture was filtered through four layers of gauze, 2 mL of the filtrate was taken, and 4 mL of saturated salt water was added to the filtrate. After mixing, the mixture was placed in a McFarland counting plate, left to stand at room temperature for 10 min, and then observed and counted under an optical microscope.

[0143] Counting method: Count two counting chambers for each group of feces, and calculate the average value A of the two counting chambers. The volume of the counting chamber is 0.15mL, so 90÷0.15=600, OPG=A×600. The number of oocysts discharged by each chicken per day = (OPG×feces weight) / number of chickens. Add the number of oocysts discharged by each chicken per day to get the total number of oocysts discharged by each chicken.

[0144] The results are shown in Table 5. It can be seen from the table that the egg cyst production during the attack period of all target protein expression groups and pYD1 empty vector groups was significantly reduced (P<0.05). Compared with the pYD1 empty vector group, the pYD1-AMA1 live bacteria group was significantly less than the empty vector live bacteria group (P<0.05), the pYD1-AMA2 inactivated group, the pYD1-AMA3 inactivated group, and the pYD1-AMA4 inactivated group were significantly less than the empty vector inactivated group (P<0.05), and the remaining groups were equivalent to the corresponding vector groups (P>0.05).

[0145] Table 5 Oocyst OPG and relative oocyst production after challenge.

[0146]

[0147] 6.Anticoccidial Index (ACI)

[0148] The anticoccidial index was calculated based on the survival rate, relative weight gain, oocyst value and lesion score of each group. Anticoccidial index (ACI) = (survival rate + relative weight gain) - (lesion value + oocyst value).

[0149] The results are shown in Table 6. It can be seen from the table that the anti-coccidial index of all target protein expression groups and pYD1 empty vector groups was significantly higher than that of the infected control group (P<0.05). Compared with the pYD1 empty vector group, the pYD1-AMA1 live bacteria group was extremely significantly higher than the empty vector live bacteria group (P<0.01), achieving a good anti-coccidial effect, the pYD1-AMA2 live bacteria group and the pYD1-AMA4 live bacteria group were significantly higher than the empty vector live bacteria group (P<0.05), achieving a moderate anti-coccidial effect; the pYD1-AMA1 inactivated group, the pYD1-AMA2 inactivated group, the pYD1-AMA3 inactivated group, and the pYD1-AMA4 inactivated group were significantly higher than the empty vector inactivated group (P<0.05), achieving a moderate anti-coccidial effect.

[0150] Table 6 Anti-coccidial Index (ACI) of each group after challenge.

[0151]

[0152] 7. Serum and mucosal antibody testing during immunization

[0153] Before attacking the parasites (20 days old), four chickens were randomly selected from each group for blood sampling to prepare chicken serum, which was then stored at -20°C for later use. At the same time, the cecal tissue was removed, the cecal contents were removed, the intestinal wall was rinsed with pre-cooled PBS, the intestinal mucosal tissue was scraped, and the serum was resuspended with cold PBS and mixed for 1 min. The serum was centrifuged at 5000 g for 5 min at 4°C, and the supernatant was collected and stored at -80°C.

[0154] Serum and intestinal supernatant specific IgG detection: quantitatively coat antigen according to protein concentration, incubate at 4℃ for 16-18 h; remove the liquid in the reaction well, add 250μL washing solution for washing; add 200μL blocking solution and incubate at 37℃ for 2 h; after washing, add diluted serum or intestinal supernatant and incubate at 37℃ for 2 h; after washing, add pre-diluted HRP-rabbit anti-chicken IgG and incubate at 37℃ for 2 h; after washing, add colorimetric solution and stop solution first, and measure its OD with an enzyme-labeled instrument 450nm value.

[0155] Detection of intestinal supernatant-specific IgA: AMA1, AMA2, AMA3 and AMA4 proteins were coated separately at 4°C overnight; after washing with TBST, the liquid in the wells was wiped clean, and 1% BSA blocking solution was used for incubation at 37°C for 2 h; after washing with TBST, diluted chicken intestinal supernatant was added as primary antibody and incubated at 37°C for 2 h; after washing with TBST, HRP-labeled goat anti-chicken IgA was added and incubated at 37°C for 2 h; after washing and patting dry, the color developing solution and the stop solution were added successively, and the OD was determined by an enzyme-labeled instrument. 450nm value.

[0156] The results are as follows Figure 7As shown, the serum and mucosal IgG titers of the oral EBY100 / pYD1-AMA1 group were significantly higher than those of the PBS group and the EBY100 / pYD1 group, regardless of whether it was immune live bacteria or inactivated bacteria (P<0.05) (Figure A); the serum and mucosal IgG titers of the oral EBY100 / pYD1-AMA4 group were significantly higher than those of the EBY100 / pYD1 group, regardless of whether it was immune live bacteria or inactivated bacteria (P<0.05) (Figure B); the IgG titer in the cecum of the oral EBY100 / pYD1-EtAMA1 group was extremely significantly higher than that of the PBS group and the EBY100 / pYD1 group (P<0.001), and the IgG titer in the cecum of the oral EBY100 / pYD1-EtAMA1 group was extremely significantly higher than that of the EBY100 / pYD1 group (P<0.05), which was not significantly different from the healthy control group. (P>0.05)(Figure C); For the immune inactivated bacteria, the IgG titer in the cecum of the oral EBY100 / pYD1-EtAMA4 group was significantly higher than that of the PBS group and the EBY100 / pYD1 group (P<0.001), while the IgG titer in the cecum of the immune live bacteria group was not significantly different from that of the PBS group and the EBY100 / pYD1 group (P>0.05)(Figure D); For both the immune live bacteria and the inactivated bacteria, the serum and mucosal sIgA titers of the oral EBY100 / pYD1-AMA1 group were significantly higher than those of the PBS group and the EBY100 / pYD1 group (P<0.001), (Figure E); For both the immune live bacteria and the inactivated bacteria, the serum and mucosal sIgA titers of the oral EBY100 / pYD1-AMA4 group were significantly higher than those of the PBS group and the EBY100 / pYD1 group (P<0.01), (Figure F).

[0157] CD4 and CD8 lymphocyte population detection: ELISA was used in vitro to detect CD4 in the serum of the control group and the experimental group during the vaccination period. + 、CD8 + Expression levels of T lymphocytes.

[0158] The results are as follows Figure 8 As shown, compared with the empty vector live vaccine group, the CD4 + The expression level of lymphocytes was significantly increased (P<0.001), significantly decreased in EtAMA1 and EtAMA3 groups (P<0.001), and no significant difference was found in EtAMA4 group. The CD8 + The expression level of lymphocytes was significantly decreased, while there was no significant difference in the EtAMA4 group (P>0.05).

[0159] Compared with the group immunized with the empty vector inactivated vaccine, the CD4 + The expression level of lymphocytes was significantly increased (P<0.01), and the CD4 + The expression level of lymphocytes increased but the difference was not significant (P>0.05); the CD8 + The expression level of lymphocytes was significantly increased (P<0.001), significantly decreased in the EtAMA3 group (P<0.05), and had no significant difference in the EtAMA4 group (P>0.05).

[0160] Compared with the healthy control group, the CD4 + 、CD8 + The expression levels of lymphocytes were significantly increased (P<0.001), and the CD4 + 、CD8 + There was no significant difference in the expression levels of lymphocytes (P>0.05).

[0161] Cytokine level detection: The ELISA method was used in vitro to determine the expression levels of IFN-γ, IL-6, IL-17 and IL-18 in serum during vaccine immunization.

[0162] The results are as follows Fig. 9 As shown, compared with the empty vector live vaccine immunization group, the expression level of IL-6 in the serum of the EtAMA2, EtAMA3, and EtAMA4 live vaccine immunization groups was significantly decreased (P<0.001), while there was no significant difference in the EtAMA2 group (P>0.05); the expression level of IL-17 in the serum of the EtAMA2 live vaccine immunization group was significantly increased (P<0.05), and there was no significant difference in the EtAMA1, EtAMA3, and EtAMA4 groups (P>0.05); the expression level of INF-γ in the serum of the EtAMA1, EtAMA3, and EtAMA4 live vaccine immunization groups was significantly decreased (P<0.001), while it was significantly increased in the EtAMA2 group (P<0.001); there was no significant difference in the expression level of IL-18 in the serum of the EtAMA1, EtAMA2, EtAMA3, and EtAMA4 live vaccine immunization groups (P>0.05).

[0163] Compared with the empty vector inactivated vaccine immunization group, the expression level of IL-6 in the serum of the EtAMA1, EtAMA2, EtAMA3, and EtAMA4 inactivated vaccine immunization groups was significantly increased (P<0.001); the expression level of IL-17 in the serum of the EtAMA2 and EtAMA4 inactivated vaccine immunization groups was significantly decreased (P<0.01), and there was no significant difference between the EtAMA1 and EtAMA3 groups (P>0.05); the expression level of INF-γ in the serum of the EtAMA1, EtAMA2, EtAMA3, and EtAMA4 inactivated vaccine immunization groups was significantly increased (P<0.001); while there was no significant difference in the expression level of IL-18 in the serum (P>0.05).

[0164] Compared with the healthy control group, the expression level of IL-6 in the serum of the empty vector live vaccine immunization group was significantly increased (P<0.001), the expression level of IL-17 had no significant difference (P>0.05), the expression level of INF-γ was significantly increased (P<0.001), while the expression level of IL-18 had no significant difference (P>0.05); the expression level of IL-6 in the serum of the empty vector inactivated vaccine immunization group was significantly decreased (P<0.05), the expression level of IL-17 was significantly increased (P<0.001), the expression level of INF-γ was significantly decreased (P<0.001), while the expression level of IL-18 had no significant difference (P>0.05).

[0165] The above results show that after oral immunization of chicks with live or inactivated bacteria, the recombinant yeast expressing the apical membrane antigens EtAMA1 / 2 / 3 / 4 of Eimeria tenella increased the weight of the experimental group, reduced the output of oocysts, alleviated the cecal lesions, and induced the production of specific serum IgG and intestinal sIgA. It has a certain anti-Eimeria tenella immune effect and is a potential oral vaccine for coccidia.

[0166] Example 4 Evaluation of the combined therapeutic effect of EtAMA4 antibody and oral recombinant Saccharomyces cerevisiae vaccine

[0167] Similar to Example 3, 40 one-day-old SPF chicks were randomly divided into 5 groups, with 10 chickens in each group. Group 1 was a non-immunized parasite control group (infection control group), Group 2 was an antibody treatment group, Group 3 was an EBY100 / pYD1-EtAMA1 live vaccine immunized parasite group, and Group 4 was a combined treatment group. Groups 1 and 2 were orally administered with 100 μL PBS, and Groups 3 and 4 were orally administered with 1×10 8 The live carrier bacteria of Saccharomyces cerevisiae were used for vaccination. The chickens were vaccinated every 3 days starting from the 3rd day of age for a total of 6 times. Each chicken in all groups was vaccinated with 1×10 4Fresh E. tenella sporulated oocysts. At the same time, on the 4th day after inoculation, the purified EtAMA4 antibody protein (0.5 mg / mL, 200 μL per chicken) was injected into the 2nd and 4th groups. The experiment was terminated on the 8th day after inoculation. The average weight gain, bloody stool, cecal lesion score, and the number of oocysts per gram of feces (OPG) were calculated as indicators to comprehensively evaluate the effect of combined treatment of chicken coccidiosis infection.

[0168] The results showed that compared with the infected control, the weight gains of the antibody treatment group, live vaccine immunization group, and combined treatment group were significantly higher than that of the infected control group (P<0.05). Among them, the antibody treatment group was 92%, the live vaccine immunization group was 95%, and the combined treatment group was 100%, which was significantly better than that of the single treatment group.

[0169] The results of bloody stool and cecal lesion scoring showed that there was no death in all groups, and the survival rate was 100%. The live vaccine immunization group and the combined treatment group did not have bloody stools, while the other infection groups all had some bloody stools. Similarly, the intestinal lesion value of the combined treatment group (14.5) was significantly lower than that of the antibody treatment group (24.3) and the live vaccine immunization group (23.4).

[0170] The results of the number of oocysts per gram of feces (OPG) showed that the oocyst production in all treatment groups was significantly reduced (P<0.05). The effect of the combined treatment group was similar to that of the antibody treatment group, and was better than that of the live vaccine immunization group.

[0171] The above results suggest that the combined treatment and immunization using the EtAMA4 antibody obtained in the present application and the oral recombinant brewer's yeast vaccine can induce chicks to effectively resist Eimeria tenella infection, improve the survival rate of chicks, increase the relative weight gain rate of chicks, and reduce the excretion of coccidia oocysts. It is a new combination vaccine with good immune protection effect and has the potential to be developed as a vaccine for the prevention and treatment of Eimeria tenella disease in chickens.

[0172] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. An oral recombinant yeast, characterized in that: The yeast expresses an AMA homologous molecule of Eimeria tenella (Et), wherein the AMA homologous molecule is EtAMA4, and its nucleotide sequence is SEQ ID NO.

14. The preparation method of the oral recombinant yeast is as follows: (1) amplification of the EtAMA4 gene: primers are designed according to the cDNA sequence of EtAMA4, and when designing the target fragment of EtAMA4, homologous arm sequences of the pYD1 vector are introduced at both ends of the primers; while amplifying the target fragment, the vector PYD1 is double-digested and then recovered and connected, and the correctly connected plasmid is screened; (2) EBY100 competent cells are prepared; (3) yeast transformation and identification, through yeast genomic DNA extraction, PCR identification, and 1% agarose gel electrophoresis detection, to determine the surface display type brewer's yeast vector that is positive; (4) expression of surface display type brewer's yeast protein; (5) preparation of oral vaccine, the primers in step (1) are as follows: The step (5) is: Preparation of live vaccine: EBY100 / pYD1-EtAMA4 was induced with YNB-CAA solution containing 2% galactose; after 72 h of induction, the Saccharomyces cerevisiae was counted using a hemocytometer and the number of cells was calculated based on 1×10 8 / tube, centrifuge to remove supernatant, wash three times with PBS, and finally resuspend with 1 mL sterile PBS for use; live vector vaccine is used immediately after induction; Alternatively, the preparation method of the inactivated vaccine is as follows: a portion of the above-packaged yeast is inactivated in a 95°C water bath for 20 min, and resuspended with an appropriate amount of 1 mL of sterile PBS. A small amount of bacterial liquid is streaked on a YPD solid culture medium with an inoculation loop in an ultra-clean bench, and the culture is placed in a 30°C constant temperature incubator to detect the inactivation effect; the successfully inactivated brewer's yeast carrier bacterial liquid is centrifuged, and the bacterial pellet is stored at -80°C for later use, and thawed and resuspended with sterile PBS before use.

2. A monoclonal antibody 1G5 that specifically recognizes the protein encoded by Eimeria tenella EtAMA4 of claim 1, characterized in that: The amino acid sequence of the light chain variable region of the monoclonal antibody is: DVVMTQTPLPLPVSLGHQASISCRSS ASLKHSGNTY LHWYLQRPGQSPKLLIY KAVS NRFSGSGVPDRFSGSGTDFTLKVEISRAEDLGVYFC SQETHIPT FGGRGTKLEIKKS; The amino acid sequence of the heavy chain variable region is: EVQLQEQPSGPGLVSQSSITLCTVS GHSLNAYG VHWVRGLQSPGKELGV IWEGNTD DYNMNAAFSRDNSKSQRLINFFKSLQTDDIYYC VDANGYVDAMDY WGQGTSVASSAK, where CDR1-3 are bold and underlined.

3. A composition, characterized in that The composition comprises the oral recombinant yeast according to claim 1, and / or the monoclonal antibody 1G5 according to claim 2.

4. Use of the composition according to claim 3, characterized in that The composition is used for preparing a vaccine or medicine for preventing chicken coccidiosis.

5. The use according to claim 4, wherein the vaccine or drug exerts its effect by inhibiting the invasion of Eimeria tenella sporozoites into host cells and thereby blocking the life cycle of the coccidia.

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